CN101981191A - Nucleotide sequences and corresponding polypeptides conferring modulated growth rate and biomass in plants grown in saline and oxidative conditions - Google Patents

Nucleotide sequences and corresponding polypeptides conferring modulated growth rate and biomass in plants grown in saline and oxidative conditions Download PDF

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CN101981191A
CN101981191A CN2007801016166A CN200780101616A CN101981191A CN 101981191 A CN101981191 A CN 101981191A CN 2007801016166 A CN2007801016166 A CN 2007801016166A CN 200780101616 A CN200780101616 A CN 200780101616A CN 101981191 A CN101981191 A CN 101981191A
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plant
polypeptide
seq id
tolerance
nucleic acid
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CN2007801016166A
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F·周
J·索萨
K·费尔德曼
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塞瑞斯公司
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Priority to PCT/US2007/078962 priority Critical patent/WO2009038581A2/en
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Abstract

The invention relates to separated nucleic acid molecules and corresponding polypeptides coded thereby, which endow following characters in saline and/or oxidative conditions, such as improved plant size, nutrition growth, growth speed, seedling activity and/or biomass. And the invention also relates to applications of transgenic plants, plant cells, plant materials or plant seeds with improved plant size, nutrition growth, growth speed, seedling activity and/or biomass in saline and/or oxidative conditions, compared to wild type plants prepared in similar conditions.

Description

赋予在盐和氧化条件下生长的植物调节的生长速率和生物量的核苷酸序列及相应多肽 Growth rate and biomass imparting plant salt and adjusting the oxidation conditions and the growth of a nucleotide sequence corresponding polypeptide

技术领域 FIELD

[0001] 本发明涉及分离的核酸分子及其编码的相应多肽,所述核酸分子和多肽能够在盐和/或氧化胁迫条件下增强植物生长。 [0001] The corresponding polypeptide relates to an isolated nucleic acid molecule of the present invention and encoding a polypeptide and a nucleic acid molecule capable of enhancing plant growth under salt and / or oxidative stress conditions. 本发明还涉及核酸分子和多肽制备与相似条件下生长的野生型植物相比,在盐和/或氧化胁迫条件下具有改进的生长速率、营养生长、幼苗活力和/或生物量的转基因植物、植物细胞、植物材料或植物种子的用途。 The present invention further relates to a wild type plant prepared polypeptide under similar growth conditions as compared to a nucleic acid molecule, having improved growth rate in salt and / or oxidative stress conditions, vegetative growth, seedling vigor and / or biomass in transgenic plants, a plant cell, a plant material or plant seed.

背景技术 Background technique

[0002] 可以使用分子技术获得用于农业、园艺、生物量转化和其他工业(例如造纸工业, 植物作为蛋白质或其他化合物的生产工厂)的明确改进的植物。 [0002] clearly improved techniques may be used to obtain molecular agriculture, horticulture, biomass conversion, and other industries (e.g. paper industry, plants as a protein or other compounds in the production plant) plant. 例如,在盐和/或氧化胁迫条件下增强植物生长可获得巨大的农业价值。 For example, enhanced salt and / or oxidative stress conditions for plant growth achieved great agricultural value.

[0003] 盐度 [0003] salinity

[0004] 多种具有农业重要性的植物物种显示对盐和/或氧化胁迫条件的显著敏感性。 [0004] more plant species of agronomic interest display significant sensitivity to salt and / or oxidative stress conditions. 在盐浓度超过相对低的阈值后,许多植物遭受矮化的生长、坏死和死亡,这导致总体矮化的外观,和植物材料、种子、果实和其它有价值产物产量的降低。 After the salt concentration exceeds a relatively low threshold, many plants suffer from stunted growth, necrosis and death, which leads to an overall stunted appearance, and plant material, seeds reduce, fruit and other valuable product yield. 就生理学而言,用盐度攻击的植物经历离子和水体内稳态的破坏,代谢的抑制,和对细胞膜的损害,这导致发育停止和细胞死亡(Huh 等(2002) Plant J, 29 (5) :649_59) On physiology, the use of plants to salinity attack ions and water homeostasis damage, inhibit the metabolism of, and damage to the cell membrane, which leads to cell death and growth stops (Huh et (2002) Plant J, 29 (5 ): 649_59)

[0005] 在世界上最重要的许多生产性农业区域中,农业活动自身导致提高的水盐度和土壤盐度,这威胁它们的持续生产力。 [0005] the most important in many of the world production of agricultural area, agricultural activities themselves lead to increased water salinity and soil salinity, which threatens their continued productivity. 一个例子是具有丰富阳光的干旱区域中的作物灌溉。 One example is crop irrigation in arid areas with abundant sunshine in. 对农田应用灌溉水后,所述灌溉水通过蒸发和蒸腾的过程被去除。 After application of agricultural irrigation water, the irrigation water is removed by evaporation and transpiration process. 尽管这些过程从土壤中去除水,但是它们留下灌溉水中带有的溶解盐。 While these processes remove water from the soil, but they leave behind dissolved salts with the irrigation water. 因此,土壤和地下水盐浓度随时间而增加,使得土地和浅表地下水变咸,进而损害作物。 Thus, soil and groundwater salt concentrations increased with time, so that the land and shallow groundwater salty, further crop damage.

[0006] 除了人活动以外,天然的地质学过程产生了大量在非盐时会非常高产的盐田。 [0006] In addition to human activities, natural geological process produces a large amount of salt in the non-salt will be very productive. 总计约20%被灌溉的田地受盐度的负面影响(Yamaguchi和Blumwald,2005,Trends in Plant Science,10 =615-620) 0由于这些原因及其他,所以鉴定下述基因具有巨大的兴趣和重要性,所述基因赋予改进的盐耐性特征,从而使得能够产生在盐条件下具有增强的生长和/ 或生产力特征的转基因植物(例如作物植物)。 About 20% of the total irrigated land negatively affected by salinity (Yamaguchi and Blumwald, 2005, Trends in Plant Science, 10 = 615-620) 0 For these reasons and others, the identification of the genes of great interest and importance salt tolerance characteristic, the gene confers improved, thereby making it possible to produce salts having enhanced under conditions of growth and / or productivity characteristics of transgenic plants (e.g. crop plants).

[0007] 尽管具有这一进步,但是目前仍然非常需要下述普遍适用的方法,所述方法促进森林或农业植物生长,以适应取决于特定环境条件的具体需要。 [0007] In spite of this progress, but there is still a great need for universal application of the following methods, the method of promoting forest or agricultural plant growth, depending on the specific environmental conditions to suit specific needs. 为此,本发明涉及有利地改造植物对盐的耐性,从而根据所搜寻的利益使多种作物的利益最大化,并且本发明的特征是在植物中表达重组的DNA分子。 To this end, the present invention relates advantageously modified plant tolerance to salt, so that the interest of many crops to maximize the benefits in accordance with the search, and features of the present invention is the expression of recombinant DNA molecules in plants. 这些分子可来自植物自身,并简单地以更高或更低的水平表达,或者所述分子可以来自不同的植物物种。 These molecules may be from the plant itself, and simply at a higher or lower expression level, or the molecules may be from different plant species.

[0008] 氧化胁迫 [0008] Oxidative Stress

[0009] 植物进行固着的生活方式,因此通常注生活于它们的种子萌发的地方。 [0009] plants are sessile way of life, it is often inject life in their local seed germination. 因此,它们可暴露于由于天气、污染和区域而导致的不良环境条件中。 Thus, they may be exposed to adverse environmental conditions due to weather, pollution and resulting in regions. 胁迫条件例如极端温度、干旱和干燥、盐度、土壤养分含量、重金属、UV辐射、污染物例如臭氧和SO2、机械胁迫、强光和病原体攻击对植物生长和发育具有巨大影响。 Stress conditions, such as extreme temperatures, drought and dried, salinity, soil nutrient content, heavy metals, UV radiation, pollutants such as ozone and SO2, mechanical stress, high light and pathogen attack has a great influence on plant growth and development. 这些类型的胁迫暴露诱导有毒氧物类(toxic oxygenspecies)形成,所述有毒氧物类在所有需氧细胞中产生,并且伴随着细胞水平的氧化损害。 These types of stress exposure induce toxic oxygen species (toxic oxygenspecies) is formed, the toxic oxygen species generated in all aerobic cells and is accompanied by oxidative damage at the cellular level. 若干最近公开的报道表征了非生物胁迫引起的有毒氧物类产生和随后的氧化损害(见Larkindale 和Knight (2002) ;Borsani 等(2001) ;Lee 等(2004) ;Aroca 等(2005); Luna 等(2005) ;and Noctor 等(2002)。 Several reports recently published characterization of toxic oxygen species abiotic stress-induced generation and subsequent oxidative damage (see Larkindale and Knight (2002); Borsani et (2001); Lee et (2004); Aroca, etc. (2005); Luna etc. (2005); and Noctor et al (2002).

[0010] 有毒氧物类被称作活性氧物类(R0S)、活性氧中间产物(ROI)或活化的氧物类(AOS),并且是时部分还原或活化的氧衍生物。 [0010] The toxic oxygen species are referred to as reactive oxygen species (R0S), reactive oxygen intermediates (ROI) or activated oxygen species (AOS), and is partially reduced or activated derivatives of oxygen. R0S/R0I/A0S包括以氧为中心的超氧化物(O2)和羟基(·0Η)自由基,以及过氧化氢(H2O2)、一氧化氮(NO)和0Λ这些氧物类作为光合作用、呼吸作用和光呼吸作用的反应副产物而产生,并且主要在叶绿体、线粒体、内质网、 微体(例如过氧化物酶体和乙醛酸循环体)、质膜和细胞壁中形成。 R0S / R0I / A0S comprising oxygen-centered superoxide (O2) and hydroxyl (· 0Η) radical, and hydrogen peroxide (by H2O2), nitric oxide (NO) and oxygen species such as 0Λ photosynthesis, respiration and photorespiration reaction by-products generated, and mainly in the chloroplasts, mitochondria, endoplasmic reticulum, microbodies (e.g. peroxisomes and glyoxysomes), plasma membranes and cell walls are formed. 尽管O2-和H2O2自身的毒性相对低,但是它们的高毒性· OH的金属依赖型转化被认为是造成与这些分子有关的大部分生物损伤的主要原因。 Although O2- and H2O2 their toxicity is relatively low, but they are highly toxic metal · OH-dependent transformation is considered to be the main cause of most of the biological damage associated with these molecules.

[0011] 氧化胁迫损伤细胞结构并影响细胞的代谢和分解代谢。 [0011] Oxidative stress and cellular structure damage cellular metabolism and catabolism. 膜脂通过R0S/R0I/A0S被氧化,导致高分子量的、交联的脂肪酸和磷脂的累积。 Lipid oxidation by R0S / R0I / A0S, resulting in crosslinked fatty acids and phospholipids accumulated high molecular weight. 对蛋白质的氧化攻击导致位点特异性氨基酸修饰、肽链的片段化、交联的反应产物累积、改变的电荷和提高的蛋白酶解易感性, 这些均常常导致酶活性的消除。 Oxidative attack on proteins results in site-specific amino acid modifications, fragmentation of the peptide chain, the reaction product of an accumulation, altered charge and increased susceptibility to proteolysis, which are often leads to elimination of enzyme activity. 产生氧自由基的R0S/R0I/A0S (例如离子化辐射)也在DNA 的糖部分和碱基部分中诱导大量损伤,所述损伤导致缺失、突变和其他致死的遗传作用,例如碱基降解、单链断裂和与蛋白质交联。 Generation of oxygen radicals R0S / R0I / A0S (e.g. ionizing radiation) the sugar moiety and base moiety also induces a large number of DNA damage, the role of genetic damage caused by deletion, mutation and other lethal, e.g., base degradation, single strand breaks and cross-linking with proteins. 从形态学上,高水平ROS累积的不良影响证实为矮化的生长和坏死损害。 From the morphology, high levels of ROS accumulation of adverse effects proved to be stunted growth and necrotic lesions.

[0012] 尽管能够诱导损伤,但是R0S/R0I/A0S也是代谢和防御通路的关键调节器,其发挥信号发放(signaling)或第二信使分子的作用。 [0012] Although capable of inducing injury, but R0S / R0I / A0S also key regulators of metabolic and defense pathways, which play a role in signaling (Signaling) or the second messenger molecule. 例如,病原体诱导的R0S/R0I/A0S产生在疾病抗性中是关键的,其中在三个不同的水平上涉及这些分子:穿透抗性、高敏感度应答(HR)和系统性获得的抗性(Levine 等(1994) ;Lamb and Dixon(1997) ;Zhou 等(2000); Aviv等(2002))。 For example, pathogen-induced R0S / R0I / A0S produced is critical in disease resistance in which these molecules are directed in three different levels: penetration resistance to anti-high sensitivity response (HR) and systemic acquired sex (Levine et (1994); Lamb and Dixon (1997); Zhou et (2000); Aviv et al (2002)). 在穿透抗性中,R0S/R0I/A0S通过多酚类交联强化细胞壁来发挥功能。 In the resistance to penetration, R0S / R0I / A0S to function by strengthening the cell wall crosslinking polyphenols. 对高敏感度应答而言,H2O2是活性信号发放分子,其作用是剂量依赖型的。 For high sensitivity to response, H2O2 is an active signaling molecule whose effect is dose dependent. 在高剂量下,H2O2 引起高敏感度细胞死亡,并因此将病原体限制于局部感染位点(Lamb和DiXOn(1997)),而低剂量则阻断细胞周期进程(Reichheld等(1999))和信号转导二级壁分化(Potikha等(1999))。 At high doses, H2O2 induced cell death high sensitivity, and thus limit the pathogen to local infection sites (Lamb and Dixon (1997)), low doses block cell cycle progression (Reichheld, et al. (1999)) and the signal transduction two wall differentiation (Potikha et al. (1999)). 最后,R0S/R0I/A0S分子通过在第一次病原体接种后系统性引发微小-HR而在广谱系统性获得的疾病抗性中起作用。 Finally, R0S / R0I / A0S minute -HR initiator molecules by systematically after the first pathogen inoculation play a role in broad-spectrum systemic acquired disease resistance in.

[0013] 在导致氧化胁迫的信号转导级联放大中,水杨酸(SA)被鉴定为在多种胁迫条件下介导R0S/R0I/A0S累积的重要信号发放分子,所述胁迫条件例如盐和渗透压胁迫(Borsani 等(2001))、干旱(Senaratna 等(2000))、热(Dat 等(1998))、冷(Scott 等(2004))、UV-光(Surplus等(1998))、百草枯(Kim等(2003))和针对不同病原体的疾病抗性(Zhou等(2004))。 [0013] In the oxidative stress leads to a signal transduction cascade, the salicylic acid (SA) has been identified that mediate R0S / R0I / A0S accumulated important signaling molecule, the stress conditions at various stress conditions e.g. salt and osmotic stress (Borsani et al. (2001)), drought (Senaratna et al (2000)), heat (Dat et al. (1998)), cold (Scott et (2004)), UV- light (Surplus et al (1998)) paraquat (Kim et al (2003)), and disease resistance against different pathogens (Zhou et al. (2004)). 高水平的SA诱导H2O2产生以及细胞死亡。 SA produces high levels of H2O2-induced cell death as well.

[0014] SA-介导的R0S/R0I/A0S累积和基因表达需要的若干信号转导组件已经表征。 Some components of signal transduction [0014] SA- mediated R0S / R0I / A0S accumulation and gene expression have been characterized required. 例如,SA诱导的I3R基因表达和疾病抗性需要NPRl (Cao等(1994))。 For example, SA-induced gene expression and disease resistance I3R need NPRl (Cao et al. (1994)). edsl和eds5中的突变阻断SA-介导的信号发放并增强疾病易感性(Rusterucci等(2001))。 edsl eds5 mutations and SA- mediated blocking signaling and enhance disease susceptibility issued (Rusterucci et al. (2001)). 多种植物物种中NahG 的过表达也阻抑SA-诱导的对非生物胁迫以及生物胁迫的应答(Delaney等(1994))。 Overexpression of a variety of plant species NahG also SA- induced inhibition of the response to abiotic stress and biological stress (Delaney et al. (1994)). 最近,Scott和同事(2004)报道了冷冻处理在拟南芥(Arabidopsis)中诱导SA的累积,通过 Recently, Scott and colleagues (2004) reported that the accumulation of freezing treatment in Arabidopsis (Arabidopsis) SA induced by

7NahG的过表达降解SA增强了转基因植物中的冷耐性。 Overexpression of 7NahG degradation SA enhanced cold tolerance of transgenic plants.

[0015] SA作为植物激素,也促进早期开花(Martinez等(2004))。 [0015] SA as a plant hormone, and promote early flowering (Martinez et al. (2004)). 多种水平的SA可在植物生长和胁迫应答中起不同的作用。 SA can grow a variety of levels and stress response plays a different role in the plant. 然而,大部分时间中,对高水平SA的提高的耐性似乎是有益的,因为其降低了SA累积的副作用,同时刺激SA介导的胁迫应答。 However, most of the time, the patience to improve the high level of SA appears to be beneficial because it reduces the SA accumulation of side effects, while stimulating the SA-mediated stress response.

[0016] 类似地,NO能够产生R0S/R0I/A0S,并且是一种植物信号发放分子,其涉及种子萌发、气孔关闭(Mata 和Lamattina (2001) ;Desikan 等(2002))、开花时间(He 等(2004))、阻抑细胞死亡的抗氧化反应(Beligni等(2002))和对生物和非生物胁迫条件的耐性(Mata 和Lamattina(2OOl))。 [0016] Similarly, NO is capable of generating R0S / R0I / A0S, and is a plant signaling molecule, which relates to seed germination, stomatal closure (Mata and Lamattina (2001); Desikan et al. (2002)), flowering time (He (2004)), inhibitory anti-oxidation (Beligni et al (2002)) cell death and tolerance to biotic and abiotic stress (Mata and Lamattina (2OOl)). 尽管可以通过应用硝普纳(sodium nitroprusside, SNP)模拟NO的作用,但是植物中的内源NO产生由一氧化氮合酶的活性引起,所述一氧化氮合酶使用L-精氨酸(Guo等(2003))以及硝酸盐还原酶介导的反应(Desikan等(2002))。 While the role of NO can be simulated by the application of sodium nitroprusside (sodium nitroprusside, SNP), but the plant endogenous NO production caused by nitric oxide synthase activity, the use of L- arginine nitric oxide synthase ( Guo et al. (2003)) as well as nitrate reductase-mediated reactions (Desikan et al. (2002)). NO可与蛋白质和膜中的氧化还原中心反应,从而引起细胞损伤并诱导细胞死亡。 NO can react with proteins and membrane redox centers, thereby causing cell damage and inducing cell death.

[0017] 为了控制R0S/R0I/A0S分子的双重性质,植物发育出复杂的调节系统,所述调节系统涉及细胞中R0S/R0I/A0S的产生以及清除。 [0017] In order to control the dual nature of R0S / R0I / A0S molecule, complex of plant development regulation system, the adjustment system involves the generation of a cell R0S / R0I / A0S and clear. 在正常的生长和发育期间,该通路监测代谢产生的R0S/R0I/A0S水平,并控制R0S/R0I/A0S清除通路的表达和活性。 During normal growth and development, R0S metabolism of the path monitoring / R0I / A0S level, and controls R0S / R0I / A0S Clear expression and activity pathways. 主要的R0S/R0I/ AOS清除机制包括超氧化物岐化酶(SOD)、抗坏血酸过氧化物酶(ascorbate perioxidase, APX)和过氧化氢酶(CAT)以及非酶组件例如抗坏血酸、α-生育酚和谷胱甘肽的作用。 The main R0S / R0I / AOS clearance mechanisms include superoxide dismutase (SOD), ascorbate peroxidase (ascorbate perioxidase, APX) and catalase (CAT) and the non-enzymatic components such as ascorbic acid, α- tocopherol and the role of glutathione.

[0018] 相信抗氧化的酶是预防氧化胁迫的关键性组件,部分因为用诱导这些酶表达的一种胁迫形式预处理植物能够提高对不同胁迫的耐性(交叉耐受MAllen (1995))。 [0018] The antioxidant enzymes are believed critical component preventing oxidative stress, in part because a form induces the expression of these enzymes can be improved plant stress tolerance pretreatment of different stress (cross- tolerance MAllen (1995)). 另外,针对通过诱导R0S/R0I/A0S而发挥作用的除草剂的抗性所选择的植物品系通常具有提高的一种或多种这些抗氧化酶水平,并且也显示交叉耐受(Gressel和Galun (1994))。 Further, herbicide resistant functioning by inducing R0S / R0I / A0S selected plant lines typically have one or more of these increased levels of antioxidant enzymes, and also shows cross-tolerance (Gressel and Galun ( 1994)).

[0019] 植物发育和产量取决于植物通过信号发放或是清除通路来操控氧化胁迫的能力。 [0019] Plant development and yield of plants depends on the path through signaling or remove the ability to manipulate the oxidative stress. 因此,改善植物抵抗氧化胁迫的能力或在经历氧化胁迫后获得更高程度交叉耐受的能力具有显著的农业价值。 Thus, the ability to improve plant resistance to oxidative stress or the ability to obtain a higher degree of cross-tolerance with significant agricultural value after experiencing oxidative stress. 本发明的序列和方法提供了下述手段,通过所述手段能够通过信号发放或者清除通路改进对氧化胁迫的耐性。 Sequences and methods of the present invention provides the following means can issue or clear passage improve resistance to oxidative stress by means of said signal.

[0020] 在人类文明历史中人,和家养动物对食物和饲料流的可用性和可持续性已具有高度优先级,并且展现于农业的起源。 [0020] In the history of human civilization, and domestic animals have a high degree of priority to the availability and sustainability of food and feed streams, and to show the origins of agriculture. 农学科学、农业、作物科学、园艺学和森林科学领域的专家和研究人员甚至在今天仍持续地争取寻找和产生具有提高的生长潜能的植物,以喂养越来越多的世界人口并保证提供可再生的原材料。 Agronomy science, agriculture, crop science, horticulture and forestry experts in the field of science and researchers even today continues to struggle to find and produce plants with improved growth potential, to feed the world's growing population and to guarantee the availability renewable raw materials. 这些科学领域中强大的研究水平表明世界上每种地理环境和气候的领导人对提供合适的食物、饲料和能源来源所施加的重视水平。 These powerful scientific field research shows that the level of importance for each level of world geography and climate leaders provide the right food, feed and energy source applied.

[0021] 作物性能的控制已经通过选择和植物育种常规地进行了数个世纪。 [0021] Control of crop performance has been performed for centuries through selection and plant breeding conventionally. 然而育种过程是既耗时又费力的。 However, the breeding process is time-consuming and laborious. 另外,对于各有关的植物物种必须特定地设计适当的育种程序。 In addition, for each related plant species must be specific to the design of appropriate breeding programs.

[0022] 另一方面,在使用分子遗传方法控制植物以提供更好的作物中获得了长足的进步。 [0022] On the other hand, the use of molecular genetic methods to control plants to provide better crops obtained considerable progress. 通过在植物中引入和表达重组的核酸分子,研究人员目前准备好了提供给社会下述植物物种,所述植物物种被改造为即使在亚最优的地理和/或气候环境下也能更有效地生长和生产更多的产物。 By introducing and expressing recombinant nucleic acid molecules in plants, researchers are ready to provide to the community following plant species, the plant species can be transformed into a more efficient even at sub-optimal geographic and / or climatic environment grow and produce more product. 这些新的方法具有不局限于一个植物物种而是可应用于多个不同植物物种的优点(Zhang 等(2004)Plant Physiol. 135 :615 ;Zhang 等(2001)Proc. Natl. Acad. Sci.USA 98 : 12832)。 These new method is not limited to one plant species but can be applied to different plant species advantages plurality (Zhang et al (2004) Plant Physiol 135:.... 615; Zhang et (2001) Proc Natl Acad Sci.USA 98: 12832).

[0023] 发明概述 [0023] Summary of the Invention

[0024] 本发明提供了与下述植物相关的方法和材料,所述植物对盐度和/或氧化胁迫具有调节的耐性水平。 [0024] The present invention provides a plant related to the methods and materials described below, the plant to salinity and / or oxidative stress tolerance level having adjustable. 例如,本发明提供了对盐度和/或氧化胁迫具有提高的耐性水平的转基因植物和植物细胞,用于产生对盐度和/或氧化胁迫具有提高的耐性水平的植物和植物细胞的核酸,和用于制备对盐度和/或氧化胁迫具有提高的耐性水平的植物和植物细胞的方法。 For example, the present invention provides transgenic plants and plant cells to salinity and / or oxidative stress tolerance levels have increased, for the nucleic acid levels of tolerance to salinity generating and / or having increased oxidative stress in plants and plant cells, and methods for plants and plant cells produced levels of tolerance to salinity and / or oxidative stress of having improved. 这类植物和植物细胞提供了在盐和/或氧化胁迫条件下产生作物或植物而无矮化生长和减少产率的机会。 Such plants and plant cells provide the opportunity to produce crops or plants under salt and / or oxidative stress conditions without stunted growth and reduction in yield. 对盐度和/或氧化胁迫的提高的耐性水平可用于在目前有较低生产力的土地上产生可转化为液体燃料或其他化学品的生物量和/或产生食物和饲料,导致可耕土地的总体扩张。 Salinity and / or oxidative stress tolerance levels can be used to increase the biomass can be converted to produce liquid fuel or other chemicals and / or produce food and feed on land there are less productive, resulting in arable land The overall expansion.

[0025] 本文提供了产生植物和/或植物组织的方法。 [0025] Provided herein are methods of producing a plant and / or plant tissues. 在一个方面中,方法包括培养包含外源核酸的植物细胞。 In one aspect, the method comprises culturing a plant cell comprising an exogenous nucleic acid. 外源核酸包含与编码多肽的核苷酸序列有效连接的调节区。 Exogenous nucleic acid comprising nucleotide sequences encoding regulatory region linked to the polypeptide. 使用由图1-6之一中所示氨基酸序列产生的HMM时,多肽氨基酸序列的隐蔽马尔科夫模型(Hidden Markov Model,HMM) 二进制值(bit score)大于约30。 When using an HMM generated from the amino acid sequence depicted in Figure 1-6, the hidden Markov model (Hidden Markov Model, HMM) binary value (bit score) the amino acid sequence of a polypeptide of greater than about 30. 植物和/或植物组织与不包含外源核酸的对照植物对盐度和/或氧化胁迫的相应耐性水平相比,对盐度和/或氧化胁迫的耐性水平有差异。 Plants and / or plant tissue with a control plant does not comprise the exogenous nucleic acid as compared to the corresponding level of salinity tolerance and / or oxidative stress, there are differences in the level of salinity tolerance and / or oxidative stress. 在一些实施方案中,使用图1中所示氨基酸序列得到的HMM时,多肽的氨基酸序列具有大于约400的HMM 二进制值。 In some embodiments, the amino acid sequence shown in FIG 1 obtained when the HMM, the amino acid sequence of the polypeptide HMM bit score greater than about 400. 在一些实施方案中,使用图2中所示氨基酸序列得到的HMM时,多肽的氨基酸序列具有大于约30的HMM 二进制值。 In some embodiments, an amino acid sequence shown in FIG. 2 HMM obtained, having the amino acid sequence of the polypeptide is greater than about 30. HMM bit score. 在一些实施方案中,使用图3中所示氨基酸序列得到的HMM时,多肽的氨基酸序列具有大于约120的HMM 二进制值。 In some embodiments, an amino acid sequence shown in FIG. 3 HMM obtained, having the amino acid sequence of the polypeptide is greater than about 120 of the HMM bit score. 在一些实施方案中,使用图4中所示氨基酸序列得到的HMM时,多肽的氨基酸序列具有大于约150的HMM 二进制值。 In some embodiments, the amino acid sequence shown in Figure 4 obtained using the HMM, the amino acid sequence of the polypeptide is greater than about 150 HMM bit scores. 在一些实施方案中,使用图5中所示氨基酸序列得到的HMM 时,多肽的氨基酸序列具有大于约425的HMM 二进制值。 In some embodiments, the amino acid sequence shown in Figure 5 obtained using the HMM, the amino acid sequence of the polypeptide HMM bit score greater than about 425. 在一些实施方案中,使用图6中所示氨基酸序列得到的HMM时,多肽的氨基酸序列具有大于约550的HMM 二进制值。 In some embodiments, an amino acid sequence shown in FIG. 6 HMM obtained, having the amino acid sequence of the polypeptide is greater than the HMM bit score of about 550.

[0026] 在另一方面中,方法包括培养包含外源核酸的植物细胞。 [0026] In another aspect, the method comprises culturing a plant cell comprising an exogenous nucleic acid. 所述外源核酸包含与编码下述多肽的核苷酸序列有效连接的调节区,所述多肽与SEQID NO :2、4、6、8、9、11、13、 14、15、17、19、20、22、23、24、25、27、29、30、31、33、35、36、37、38、39、41、42、43、44、45、47、 49、50、52、54、56、58、60、62、63、64、66、68、69、71、73、74、76、78、80、81、83、84、86、88、90、 91、93、94、96、98、100、101、102、104、106、107、109、110、112、114、116、118、119、121、122、 123、125、126、127、128、129、130、132、134、136、138、140、141、142、143、144、145、147、149、 151、153、154、156、158、160、162、163、165、166、167、168 和SEQ ID NO :140 的氨基酸等同物(coordinate) 1到135中所示的氨基酸序列具有85%或更高的序列同一性。 The exogenous nucleic acid comprising a regulatory region and a nucleotide sequence encoding a polypeptide operably linked to the polypeptide and SEQID NO: 2,4,6,8,9,11,13, 14,15,17,19 , 20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47, 49,50,52,54 , 56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90, 91,93,94,96 , 98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122, 123,125,126,127,128,129,130,132,134 , 136,138,140,141,142,143,144,145,147,149, 151,153,154,156,158,160,162,163,165,166,167,168 and SEQ ID NO: 140 amino acid equivalents (Coordinate) amino acid sequence 1-135 in 85% or more sequence identity. 由所述植物细胞产生的植物与不包含外源核酸的对照植物中的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 A plant produced from the plant cell compared to a control plant does not comprise the exogenous nucleic acid in the respective levels of tolerance, the difference of the level of tolerance to salinity or oxidative stress.

[0027] 在另一方面中,所述方法包括培养包含外源核酸的植物细胞。 [0027] In another aspect, the method comprises culturing a plant cell comprising an exogenous nucleic acid. 所述外源核酸包含与下述核苷酸序列有效连接的调节区,所述核苷酸序列至少与SEQ ID N0. 1、3、5、7、10、 12、16、18、21、26、28、32、34、40、46、48、51、53、55、57、59、61、65、67、70、72、75、77、79、82、 85、87、89、92、95、97、99、103、105、108、111、113、115、117、120、124、131、133、135、137、139、 146、148、150、152、155、157、159、161和164中所示的核苷酸序列的片段和与编码序列表中公开的任何氨基酸序列的核苷酸序列具有85%或更高的序列同一性。 The exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence, the nucleotide sequence is at least SEQ ID N0. 1,3,5,7,10, 12,16,18,21,26 , 28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82, 85,87,89,92,95 , 97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139, 146,148,150,152,155,157,159,161 and 164 a nucleotide sequence fragment of any amino acid sequence shown in the nucleotide sequence disclosed in the sequence Listing and encoding of 85% or more sequence identity. 由所述植物细胞产生的植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对盐度和/或氧化胁迫的耐性水平有差异。 A plant produced from the plant cell and / or plant tissue tolerance compared to the corresponding level in a control plant does not comprise the exogenous nucleic acid, and the difference of salinity and / or oxidative stress tolerance levels.

[0028] 本文提供了调控植物对盐度耐性和/或氧化胁迫耐性水平的方法。 [0028] Provided herein are methods of modulating a plant salinity tolerance and / or oxidative stress tolerance levels. 在一个方面中,方法包括向植物细胞中引入外源核酸,所述外源核酸包含与编码多肽的核苷酸序列有效连接的调节区。 In one aspect, a method comprises introducing exogenous nucleic acid into a plant cell, the exogenous regulatory region comprises a nucleotide sequence of a nucleic acid encoding a polypeptide operably linked. 使用由图1-6中所示氨基酸序列产生的HMM时,所述多肽的氨基酸序列的HMM 二进制值大于约30。 When using an HMM generated from the amino acid sequence shown in FIG. 1-6, HMM binary values ​​of the amino acid sequence of the polypeptide is greater than about 30. 由所述植物细胞产生的植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 A plant produced from the plant cell and / or plant tissue tolerance compared to the corresponding level in a control plant does not comprise the exogenous nucleic acid, there are differences in the level of tolerance to salinity or oxidative stress.

[0029] 在一个方面中,方法包括向植物细胞中弓丨入外源核酸,所述外源核酸包含与编码下述多肽的核苷酸序列有效连接的调节区,其中所述多肽与SEQ ID N0:2、4、6、8、9、ll、13、 14、15、17、19、20、22、23、24、25、27、29、30、31、33、35、36、37、38、39、41、42、43、44、45、47、 49、50、52、54、56、58、60、62、63、64、66、68、69、71、73、74、76、78、80、81、83、84、86、88、90、 [0029] In one aspect, the method comprises the exogenous nucleic acid into a plant cell bow Shu, the exogenous nucleic acid comprises a regulatory region and a nucleotide sequence encoding a polypeptide operably linked, wherein the polypeptide of SEQ ID N0: 2,4,6,8,9, ll, 13, 14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37, 38,39,41,42,43,44,45,47, 49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76, 78,80,81,83,84,86,88,90,

91、93、94、96、98、100、101、102、104、106、107、109、110、112、114、116、118、119、121、122、 123、125、126、127、128、129、130、132、134、136、138、140、141、142、143、144、145、147、149、 151、153、154、156、158、160、162、163、165、166、167、168 和SEQ ID NO :140 的氨基酸等同物1到135中所示的氨基酸序列具有85%或更高的序列同一性。 91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122, 123,125,126,127,128, 129,130,132,134,136,138,140,141,142,143,144,145,147,149, 151,153,154,156,158,160,162,163,165,166,167, 168 and SEQ ID NO: 1 is the amino acid equivalent to 140 amino acid sequence shown in 135 of 85% or more sequence identity. 由所述植物细胞产生的植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 A plant produced from the plant cell and / or plant tissue tolerance compared to the corresponding level in a control plant does not comprise the exogenous nucleic acid, there are differences in the level of tolerance to salinity or oxidative stress.

[0030] 在一些实施方案中,方法包括向植物细胞中引入编码下述多肽的外源核酸,所述多肽选自SEQ ID NO :43、44、45、86、140、141、142、143、144 和SEQ ID N0:140 的氨基酸等同物1到135。 [0030] In some embodiments, the method comprises introducing an exogenous nucleic acid encoding a polypeptide into a plant cell, the polypeptide is selected from SEQ ID NO: 43,44,45,86,140,141,142,143, 144 and SEQ ID N0: 140 is the amino acid equivalents 1-135. 由所述植物细胞产生的植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对盐度的耐性水平有差异。 A plant produced from the plant cell and / or plant tissue tolerance compared to the corresponding level in a control plant does not comprise the exogenous nucleic acid, there are differences in the level of salinity tolerance. 在一些实施方案中,方法包括向植物细胞中引入编码选自SEQ ID NO :136和141的多肽的外源核酸,并且由所述植物细胞产生的植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对氧化胁迫的耐性水平有差异。 In some embodiments, the method comprises introducing encoder of SEQ ID NO into plant cells: an exogenous nucleic acid 136 and 141 of the polypeptide, and plant produced from the plant cell and / or plant tissue does not comprise the exogenous nucleic acid compared to a corresponding control plant tolerance level, the difference of the level of tolerance to oxidative stress.

[0031] 在另一方面中,方法包括向植物细胞中引入外源核酸,所述外源核酸包含与下述核苷酸序列有效连接的调节区,其中所述核苷酸序列与SEQ IDN0:1、3、5、7、10、12、16、18、 21、26、28、32、34、40、46、48、51、53、55、57、59、61、65、67、70、72、75、77、79、82、85、87、89、 [0031] In another aspect, a method comprises introducing into a plant cell an exogenous nucleic acid, said exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence, wherein said nucleotide sequence SEQ IDN0: 1,3,5,7,10,12,16,18, 21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70, 72,75,77,79,82,85,87,89,

92、95、97、99、103、105、108、111、113、115、117、120、124、131、133、135、137、139、146、148、 150、152、155、157、159、161和164中所示的核苷酸序列和与编码序列表中公开的任何氨基酸序列的核苷酸序列具有85%或更高的序列同一性。 92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148, 150,152,155,157,159, 161 and the nucleotide sequence of any amino acid sequence shown in the nucleotide sequence of the coding sequence and 164 disclosed in the table having 85 percent or greater sequence identity. 由所述植物细胞产生的植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 A plant produced from the plant cell and / or plant tissue tolerance compared to the corresponding level in a control plant does not comprise the exogenous nucleic acid, there are differences in the level of tolerance to salinity or oxidative stress.

[0032] 本文提供了包含外源核酸的植物细胞。 [0032] herein provides a plant cell comprising an exogenous nucleic acid. 在一个方面中,外源核酸包含与编码多肽的核苷酸序列有效连接的调节区。 In one aspect, the exogenous nucleic acid comprising a nucleotide sequence encoding a polypeptide is operably linked regulatory region. 使用基于图1-6之一中所示氨基酸序列的HMM时,所述多肽的氨基酸序列的HMM 二进制值大于约30。 When using the HMM based on the amino acid sequence shown in one of FIGS. 1-6, the amino acid sequence of HMM binary value of the polypeptide is greater than about 30. 所述植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 The plant and / or plant tissue tolerance compared to the corresponding level in a control plant does not comprise the exogenous nucleic acid, there are differences in the level of tolerance to salinity or oxidative stress. 在另一方面中, 外源核酸包含与编码下述多肽的核苷酸序列有效连接的调节区,所述多肽与选自SEQ ID NO :2、4、6、8、9、11、13、14、15、17、19、20、22、23、24、25、27、29、30、31、33、35、36、37、38、39、 41、42、43、44、45、47、49、50、52、54、56、58、60、62、63、64、66、68、69、71、73、74、76、78、80、 81、83、84、86、88、90、91、93、94、96、98、100、101、102、104、106、107、109、110、112、114、116、 118、119、121、122、123、125、126、127、128、129、130、132、134、136、138、140、141、142、143、 144、145、147、149、151、153、154、156、158、160、162、163、165、166、167、168 和SEQ ID NO: In another aspect, the exogenous nucleic acid comprises a nucleotide sequence encoding a polypeptide and a regulatory region active connection, said polypeptide selected from SEQ ID NO: 2,4,6,8,9,11,13, 14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39, 41,42,43,44,45,47, 49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80, 81,83,84,86,88,90, 91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116, 118,119,121,122,123,125,126,127,128, 129,130,132,134,136,138,140,141,142,143, 144,145,147,149,151,153,154,156,158,160,162,163,165,166,167, 168 and SEQ ID NO:

10140的氨基酸等同物1到135的氨基酸序列具有85%或更高的序列同一性。 10140 amino acid sequence of amino acids 1 to 135 equivalents of 85% or more sequence identity. 由所述植物细胞产生的植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 A plant produced from the plant cell and / or plant tissue tolerance compared to the corresponding level in a control plant does not comprise the exogenous nucleic acid, there are differences in the level of tolerance to salinity or oxidative stress. 在一个方面中,外源核酸包含与下述核苷酸序列有效连接的调节区,所述核苷酸序列至少与选自SEQ ID NO. 1、3、5、7、10、12、16、18、21、26、28、32、 34、40、46、48、51、53、55、57、59、61、65、67、70、72、75、77、79、82、85、87、89、92、95、97、99、 103、105、108、111、113、115、117、120、124、131、133、135、137、139、146、148、150、152、155、 157、159、161和164的核苷酸序列的片段或编码序列表中公开的任何氨基酸序列的核苷酸序列具有85%或更高的序列同一性。 In one aspect, the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence of at least the nucleotide sequence selected from SEQ ID NO. 1,3,5,7,10,12,16, 18,21,26,28,32, 34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87, 89,92,95,97,99, 103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155, 157, any amino acid sequence of a nucleotide sequence or coding sequence fragment table 159, 161 and 164 of the nucleotide sequences disclosed having 85 percent or greater sequence identity. 由所述植物细胞产生的植物和/或植物组织与不包含外源核酸的对照植物的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 A plant produced from the plant cell and / or plant tissue tolerance compared to the corresponding level in a control plant does not comprise the exogenous nucleic acid, there are differences in the level of tolerance to salinity or oxidative stress. 还提供了包含这类植物细胞的转基因植物。 Also it provides a transgenic plant comprising such a plant cell. 在一些实施方案中,转基因植物是选自以下的物种成员:柳枝稷(Panicumvirgatum)、两色蜀黍(Sorghum bicolor)(高粱,双色高粱)、巨芒草(Miscanthus giganteus)(芒属)、甘蔗属物种(Saccharum sp.)(能源蔗)、香脂白杨(Populus balsamifera)(白杨)、玉蜀黍(Zea mays)(玉米)、大豆(Glycine max)(黄豆)、欧洲油菜(Brassica napus)(卡诺拉油菜)、普通小麦(Triticum aestivum)(小麦)、 ^jftiS (Gossypium hirsutum) ( M^c )、禾g (Oryza sativa) ( /Jcfg ) ^ H^ (Helianthus annuus)、紫苜猜(Medicagosativa)(苜猜)、舌甘菜(Beta vulgaris)或御谷(Pennisetum glaucum)(珍珠粟)。 In some embodiments, the transgenic plant is a species member selected from the group consisting of: switchgrass (Panicumvirgatum), Sorghum bicolor (Sorghum bicolor) (sorghum, Sorghum bicolor), giant miscanthus (Miscanthus giganteus) (miscanthus), Saccharum species ( saccharum sp.) (energy cane), balsam poplar (Populus balsamifera) (poplar), maize (Zea mays) (maize), soybean (Glycine max) (soybean), Brassica napus (Brassica napus) (canola), wheat (Triticum aestivum) (wheat), ^ jftiS (Gossypium hirsutum) (M ^ c), Wo g (Oryza sativa) (/ Jcfg) ^ H ^ (Helianthus annuus), purple alfalfa guess (Medicago) (clover guess) , Gan tongue dish (Beta vulgaris) or pearl millet (Pennisetum glaucum) (pearl millet). 一些实施方案涉及包含来自上述转基因植物的种子或营养组织的制品(product)。 Some embodiments relate to articles comprising a transgenic plant from the seed or vegetative tissue (product). 一些实施方案涉及来自上述转基因植物的食品或饲料制品。 Some embodiments relate to food or feed products from transgenic plants described above.

[0033] 在另一个方面中,分离的核酸包含编码多肽的核苷酸序列,所述多肽与SEQ ID N0. 2、4、6、22、27、29、49、52、54、56、60、62、68、76、83、88、90、96、98、104、106、112、114、132、 134、149、151或160中所示的氨基酸序列具有80%或更高的序列同一性。 [0033] In another aspect, the isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide, said polypeptide SEQ ID N0. 2,4,6,22,27,29,49,52,54,56,60 , 62,68,76,83,88,90,96,98,104,106,112,114,132, 134,149,151, or 160 amino acid sequence shown in sequence identity of 80% or more .

[0034] 在另一方面中,提供了鉴定与盐度和/或氧化胁迫耐性水平变异相关的遗传多态性的方法。 [0034] In another aspect, there is provided a method of identifying or salinity and / oxidative stress tolerance level variation associated genetic polymorphism. 所述方法包括提供植物种群,并测定植物种群中一种或多种遗传多态性是否与下述多肽的基因座遗传相关联,所述多肽选自图1-6中所示多肽及其功能性同源物。 The method includes providing a population of plants, and plant population whether one or more genetic polymorphisms in the polypeptide following genetic loci associated assay, the polypeptide is selected from polypeptides and function shown in FIGS. 1-6 homologues. 测量种群植物和/或植物组织中盐度耐性和/或氧化胁迫耐性水平的变异与种群植物中一种或多种多态性的存在之间的关联,从而允许鉴定一种或多种多态性是否与这类变异相关。 / And / variability and plant populations oxidative stress tolerance levels associated with the measurement of one or populations of plants and plant tissue or salinity tolerance or more polymorphisms exist between, thereby allowing the identification of one or more polymorphic sex is associated with this type of variation.

[0035] 在另一方面中,提供了制备植物品系的方法。 [0035] In another aspect, there is provided a method of preparing plant lines. 所述方法包括测定植物种群中一种或多种遗传多态性是否与下述多肽的基因座相关,所述多肽选自图1-6中所示多肽及其功能性同源物,鉴定种群中一种或多种植物,其中一种或多种多态性的存在与盐耐性或氧化胁迫耐性的变异相关,将一种或多种鉴定的植物的每一种与其自身或不同的植物杂交产生种子,将用所述种子培养的至少一种后代植物与其自身或不同的植物杂交,将杂交步骤再重复0-5代以制备植物品系。 Population of plants comprising determining one or more genetic polymorphisms whether the polypeptide associated with the following loci, the polypeptide is selected from polypeptides, and FIG functional homologues in Figure 1-6, to identify populations one or more plants, wherein the one or more polymorphisms associated with variation salt tolerance or oxidative stress tolerance, one or more of each of the identified plants with itself or a different plant hybridization producing seed, the seed culture with at least one progeny plant with itself or a different plant hybridization, the hybridization procedure was repeated to prepare a 0-5 substituting plant line. 所述至少一种等位基因会存在于植物品系中。 The at least one allele will be present in the plant line. 制备植物品系的方法可应用于例如柳枝稷植物种群。 Method of making a plant line may be applied e.g. switchgrass plant populations.

[0036] 除非另有定义,本文使用的所有技术和科学术语具有本发明所属领域的普通技术人员通常所理解的相同含义。 [0036] Unless defined otherwise, the same meaning all technical and scientific terms used herein have the ordinary of ordinary skill in the art that the present invention is generally understood. 尽管可以使用与本文所述方法和材料相似或等同的方法和材料,但是仍在下文中描述合适的方法和材料。 Although described herein may be used with the methods and materials similar or equivalent methods and materials described below but is still suitable methods and materials. 所有出版物、专利申请、专利和本文提到的其他参考文献通过引用整体并入本文。 All publications, other references patent applications, and patents mentioned herein are incorporated herein by reference in its entirety. 在冲突的情况下,以本说明书(包括定义)为准。 In case of conflict, the present specification (including definition). 另外,材料、方法和实例仅用于阐述而非旨在限制。 Further, the materials, methods and examples are only illustrative and not intended to be limiting.

[0037] 在附图和下文的说明中公开了本发明的一个或多个实施方案的细节。 [0037] The details of one or more of the disclosed embodiment of the invention in the drawings and described below. 从说明书和 From the description and

11附图以及权利要求书中会明白本发明的其他特征、目的和优点。 The book 11 will be apparent drawings and claims of the present invention Other features, objects and advantages.

[0038] 附图概述 [0038] BRIEF DESCRIPTION

[0039] 图1是(ME08768 ;SEQ ID NO :86)的同源物的氨基酸序列比对。 [0039] FIG. 1 is a (ME08768; SEQ ID NO: 86) amino acid sequences of homologues comparison. 在本文所示的所有比对图中,比对序列中的虚线表示缺口,即该位置上缺少氨基酸。 In all alignments shown herein, the ratio of the broken line represents a gap in the sequence, i.e. the amino acid position is missing. 比对序列间相同的氨基酸或保守氨基酸替换用框标注。 Than or the same amino acid sequence conserved between amino acid replacement are denoted by boxes. 本文提供的图1和其他比对图使用3. 52版MUSCLE程序得到。 Figure 1 provided herein and the use of other alignment procedures FIG Version 3. 52 MUSCLE obtained.

[0040] 图2是ME06748 (SEQ ID NO :41)的同源物的氨基酸序列比对。 [0040] FIG. 2 is ME06748: the amino acid sequence (SEQ ID NO 41) homologues comparison.

[0041] 图3是ME19173(SEQ ID NO : 109)的同源物的氨基酸序列比对。 [0041] FIG. 3 is ME19173 (SEQ ID NO: 109) The amino acid sequences of homologues comparison.

[0042] 图4是ME02064C(SEQ ID NO : 140)的同源物的氨基酸序列比对。 [0042] FIG. 4 is a ME02064C (SEQ ID NO: 140) The amino acid sequences of homologues comparison.

[0043] 图5是Ceres克隆ID No. 1792354 (SEQ ID NO :2)的同源物的氨基酸序列比对。 [0043] FIG. 5 is a Ceres Clone ID No. 1792354: the amino acid sequences of homologues (SEQ ID NO 2) of the comparison.

[0044] 图6是Ceres克隆ID No. 56784328 (SEQ ID NO :35)的同源物的氨基酸序列比对。 [0044] FIG. 6 is a Ceres Clone ID No. 56784328: homologue amino acid sequence (SEQ ID NO 35) of the comparison.

[0045] 发明详述 [0045] DETAILED DESCRIPTION

[0046] 本发明涉及与调控植物和/或植物组织中盐耐性和/或氧化胁迫耐性水平的方法和材料。 [0046] The present invention relates to the regulation of plant and / or plant tissues salt tolerance and / or oxidative stress tolerance levels methods and materials. 在一些实施方案中,植物也可以具有提高的生物量和/或产量。 In some embodiments, the plant may have a biomass and / or increased yield. 该方法可包括用编码盐度和/或氧化胁迫耐性调控多肽的核酸转化植物细胞,其中所述多肽的表达导致受调控的盐度耐性和/或氧化胁迫耐性。 The method may comprise a nucleic acid encoding a salinity and / or oxidative stress tolerance-modulating polypeptide in a transformed plant cell, wherein expression of the polypeptide results in a regulated salinity tolerance and / or oxidative stress tolerance. 使用这类方法产生的植物细胞能够被培养产生下述植物,所述植物与相同条件下生长的野生型植物相比具有提高的盐度耐性、氧化胁迫耐性和/ 或生物量。 The test method used to generate plant cells can be cultured to produce a wild type plant growth salinity tolerance in the plant, the plant having increased following the same conditions as compared to oxidative stress tolerance and / or biomass. 这类植物和这类植物的种子可用于生产例如用于生物燃料生产的产量和/或生物量,所述生物燃料例如但不限于乙醇和丁醇。 Such plants and seeds of such plants may be used in the production yield and / or biomass, for example for the production of biofuels, for example, the biofuel but not limited to ethanol and butanol.

[0047] I.定义 [0047] I. defined

[0048] “氨基酸”是指二十种生物学上存在的氨基酸之一和合成氨基酸,包括D/L光学异构体。 [0048] "amino acid" refers to a biologically occurring twenty kinds of one amino acid and synthetic amino acids, including D / L optical isomers.

[0049] “细胞类型优先的启动子”或“组织优先的启动子”是指下述启动子,其分别优先在目标细胞类型或组织中驱动表达,但是也同样在其他细胞类型或组织中引起一些转录。 [0049] "preferentially cell types promoter" or "tissue-preferred promoter" refers to a promoter that drives expression preferentially in the respective target tissue or cell type, but also induced in other cell types or tissues Some transcription.

[0050] “对照植物”是指不含有目的转基因植物中存在的外源核酸,但是在其他方面具有与这种转基因植物相同或相似的遗传背景的植物。 [0050] The "control plant" refers to the object does not contain the exogenous nucleic acid present in a transgenic plant, but has such a plant of the same or similar genetic background as the transgenic plants in other areas. 合适的对照植物可以是非转基因的野生型植物、来自转化实验的非转基因分离体,或含有除目的外源核酸之外的外源核酸的转基因植物。 Wild type plant suitable control plant can be a non-transgenic, gene transformation experiments isolates from non-transgenic, or exogenous nucleic acid comprising a transgenic plant other than the exogenous nucleic acid.

[0051] “结构域”是多肽中基本连续的氨基酸组,其能够用于表征蛋白质家族和/或蛋白质部分。 [0051] A "domain" is a polypeptide substantially contiguous groups of amino acids that can be used to characterize protein families and / or protein moiety. 这类结构域具有“指纹”或“标记”,所述指纹或标记可包括保守的一级序列、二级结构和/或三维构象。 Such domains have a "fingerprint" or "label", the fingerprint or mark may comprise conserved primary sequence, secondary structure, and / or three-dimensional conformation. 通常结构域与特定的体外和/或体内活性相关。 Generally domain specific in vitro and / or in vivo activities. 结构域可具有10 个氨基酸到400个氨基酸、例如10到50个氨基酸、或25到100个氨基酸、或35到65个氨基酸、或35到55个氨基酸、或45到60个氨基酸、或200到300个氨基酸、或300到400个氨基酸的长度。 Domain may have 10 amino acids to 400 amino acids, for example 10 to 50 amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400 amino acids in length.

[0052] “下调”是指相对于基底或固有状态降低表达产物(mRNA、多肽或二者)产生的调节。 [0052] "down" refers to modulating the intrinsic state relative to the substrate or decreased expression product (mRNA, polypeptide, or both) generated.

[0053] 关于核酸的“外源的”是指核酸是重组核酸构建体的部分,或者不处于其天然环境中。 [0053] For nucleic acids "exogenous" refers to a portion of the nucleic acid is a recombinant nucleic acid construct, or is not in its natural environment. 例如,外源核酸可以是来自一种物种、被引入另一物种的序列,即异源核酸。 For example, an exogenous nucleic acid can be derived from one species, the sequence is introduced into another species, i.e., a heterologous nucleic acid. 通常,这样的外源核酸通过重组核酸构建体被引入其他物种中。 Typically, such an exogenous nucleic acid by recombinant nucleic acid construct is introduced into the other species. 外源核酸也可以是生物固有并被再引入该生物细胞中的序列。 Exogenous nucleic acid may be a natural and biological sequences reintroduced into the biological cell. 包含固有序列的外源核酸通常可通过与外源核酸连接的非天然序列的存在与天然存在的序列区分,所述外源核酸例如为重组核酸构建体中固有序列侧接侧的非固有调节序列。 Non-inherent sequences comprise an exogenous nucleic acid sequence may generally be distinguished by the presence of non-naturally occurring native sequence connected to the exogenous nucleic acid, said exogenous nucleic acid, for example a recombinant nucleic acid sequence of any inherent natural flanking regulatory sequences constructed side . 另外,稳定转化的外源核酸通常被整合在除固有序列存在位置之外的其他位置上。 In addition, stably transformed exogenous nucleic acids typically are integrated at a position other than the position on the presence of natural sequences. 应当明白,可以向祖细胞中引入外源核酸,而不是向正在考虑的细胞中引入。 It should be appreciated, can be introduced exogenous nucleic acid into progenitor cells, instead of introducing into the cell being considered. 例如,含有外源核酸的转基因植物可以是稳定转化的植物与非转基因植物之间杂交的后代。 For example, a transgenic plant containing the exogenous nucleic acid may be progeny of a cross between a stably transformed plant and non-transgenic plants. 这样的后代被认为含有外源核酸。 Such progeny are considered to contain an exogenous nucleic acid.

[0054] “表达”是指通过转录将多核苷酸的遗传信息转化为RNA,并通过mRNA在核糖体上的翻译转化为蛋白质,所述转录由酶(RNA聚合酶)催化。 [0054] "Expression" refers to the transcription of genetic information of a polynucleotide into an RNA, and the translation of mRNA by ribosomes into protein, transcription is catalyzed by the enzyme (RNA polymerase).

[0055] 在本文中使用“异源多肽”是指非植物细胞中天然存在多肽的多肽,所述植物细胞例如为用来自玉蜀黍植物的氮转运蛋白多肽的编码序列转化并表达所述编码序列的转基因柳枝稷植物。 [0055] Use "heterologous polypeptide" herein refers to a polypeptide is a polypeptide naturally occurring in non-plant cells, said transformed plant cell, for example, with the expression of the coding sequence and the coding sequence of a nitrogen transporter polypeptide from maize plants transgenic switchgrass plants.

[0056] 在本文中使用“分离的核酸”包括天然存在的核酸,条件是其天然存在的基因组中所述核酸两侧紧邻的序列之一或二者被去除或不存在。 [0056] using the "isolated nucleic acid" herein includes naturally occurring nucleic acids, with the proviso that one or both of the sequences which naturally occurring genome of the nucleic acid immediately adjacent sides is removed or absent. 因此,分离的核酸包括但不限于,作为纯化的分子存在或作为掺入载体或病毒中的核酸分子存在的核酸。 Thus, an isolated nucleic acid includes, without limitation, as a purified nucleic acid molecule is present as or incorporated into a vector or nucleic acid molecule in the presence of a virus. 存在于例如cDNA文库、基因组文库、或含有基因组DNA限制性消化产物的凝胶切片中数百到数百万其他核酸中间的核酸不认为是分离的核酸。 E.g. present in cDNA libraries, genomic libraries, or gel containing a genomic DNA restriction digest was sliced ​​in the middle of hundreds to millions of other nucleic acid nucleic acid is not considered an isolated nucleic acid.

[0057] 化合物或组分水平的“调控(modulation) ”是指由于植物细胞中外源核酸的表达或转录而观察到所示化合物或组分的水平改变。 [0057] compound or component level "control (Modulation)" refers to a compound or component of altered levels of a plant cell shown since exogenous transcription or expression of nucleic acids is observed. 水平的改变相对于对照植物中的相对水平进行测量。 Changing the relative level of the level measurement control plants.

[0058] “核酸”和“多核苷酸”在本文中可互换使用,并且表示RNA以及DNA,包括cDNA、基因组DNA、合成DNA,和含有核酸类似物的DNA或RNA。 [0058] "Nucleic acid" and "polynucleotide" are used interchangeably herein and represent RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA or RNA containing nucleic acid analogs. 多核苷酸可具有任何三维结构。 The polynucleotide may have any three-dimensional structure. 核酸可以是双链或单链的(即有义链或反义链)。 Nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand). 多核苷酸的非限制性实例包括基因、基因片段、外显子、内含子、信使RNA (mRNA)、转运RNA、核糖体RNA、siRNA、微小-RNA、核酶、cDNA、重组多核苷酸、分支的多核苷酸、核酸探针和核酸引物。 Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal body RNA, siRNA, micro -RNA, ribozymes, cDNA, recombinant polynucleotides , branched polynucleotides, nucleic acid probes and nucleic acid primers. 多核苷酸可含有非常规或经修饰的核苷酸。 A polynucleotide may contain unconventional or modified nucleotides.

[0059] “有效连接”是指在核酸中排列调节区和待转录的序列,使得调节区能够有效调节所述序列的转录或翻译。 [0059] "operably linked" refers to an arrangement regulatory region and a sequence to be transcribed in a nucleic acid, such that the regulatory region can be effectively regulated transcription or translation of the sequence. 例如,为了将编码序列与调节区有效连接,通常将编码序列的翻译读码框的翻译起始位点置于调节区下游1和约50个核苷酸之间。 For example, the coding sequence is operably linked to a regulatory region, the translation of the coding sequence is typically reading frame translation initiation site disposed between the downstream regulatory region 1 and about 50 nucleotides. 然而,调节区可以被置于翻译起始位点上游多达约5,000个核苷酸处,或转录起始位点上游约2,000个核苷酸处。 However, the regulatory region may be placed upstream of the translation start site as much as about 5,000 nucleotides, upstream of the transcription start site, or about 2,000 nucleotides.

[0060] 本文中使用“多肽”是指两个或更多亚基氨基酸的化合物、氨基酸类似物或其他肽模拟物,与翻译后修饰例如磷酸化或糖基化无关。 [0060] As used herein, "polypeptide" refers to two or more multi-subunit amino acid compounds, amino acid analogs, or other peptidomimetics, and post-translational modifications such as phosphorylation or glycosylation independent. 亚基可以通过肽键或其他键例如酯键或醚键连接。 Subunits can, for example, an ester bond or an ether linkage by peptide bonds or other bonds. 该定义包括全长多肽、截短的多肽、点突变体、插入突变体、剪接变体、嵌合蛋白质及其片段。 This definition includes full-length polypeptides, truncated polypeptides, point mutants, insertion mutants, splice variants, chimeric proteins and fragments thereof.

[0061] “后代”包括具体植物或植物品系的子孙。 [0061] "offspring" includes descendants of a particular plant or plant line. 现有时(instant)植物的后代包括F1, F2、F3、F4、F5、F6和随后世代植物上形成的种子,或BCp BC2、BC3和随后世代植物上形成的种子,或F1BCp F1BCy F1BC3和随后世代植物上形成的种子。 Progeny existing time (Instant) plants include F1, F2, F3, F4, F5, seed formation F6 and subsequent generation plants on, or BCp BC2, seeds formed BC3 and subsequent generation plants on, or F1BCp F1BCy F1BC3 and subsequent generations formed on the seed plants. 名称F1是指遗传上不同的两个亲本之间杂交的后代。 F1 refers to the name on the genetic offspring of a cross between two different parents. 名称F2、F3、F4、F5和F6是指F1植物自花传粉后代或近亲缘粉后代的随后世代。 Name F2, F3, F4, F5 and F6 refer to subsequent generations F1 progeny of self-pollinated plants or close relative margin of powder future generations.

[0062] “调节区”是指具有下述核苷酸序列的核酸,所述核苷酸序列影响转录或翻译起 [0062] "regulatory region" refers to a nucleic acid having a nucleotide sequence, said nucleotide sequence affects transcription or translation from

13始和速率,和转录或翻译产物的稳定性和/或迁移率。 13 the beginning and rate of transcription or translation product stability and / or mobility. 调节区包括但不限于启动子序列、 增强子序列、应答元件、蛋白质识别位点、可诱导元件、蛋白质结合序列、5'和3'非翻译区(UTR)、转录起始位点、终止序列、聚腺苷酸化序列、内含子及其组合。 Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 'and 3' untranslated regions (the UTR), transcriptional start sites, termination sequences , polyadenylation sequences, introns, and combinations thereof. 调节区通常包含至少一个核心(基础)启动子。 A regulatory region typically comprises at least a core (basal) promoter. 调节区也可包含至少一个控制元件,例如增强子序列、上游元件或下游激活区(UAR)。 Regulatory region may also comprise at least one control element, such as an enhancer sequence, upstream or downstream element activation region (UAR). 例如,合适的增强子是来自章鱼碱合酶(ocs)基因上游区的顺式调节元件(-212 到-154)。 For example, a suitable enhancer is a cis-regulatory element (-212 to -154) from the octopine synthase (OCS) gene upstream region. Fromm 等,The Plant Cell,1 :977_984 (1989)。 Fromm et al., The Plant Cell, 1: 977_984 (1989).

[0063] “上调”是指相对于基础或固有状态提高表达产物(mRNA、多肽或二者)水平的调节。 [0063] "up" refers to the natural state relative to the base or adjusted product (mRNA, polypeptide, or both) increased levels of expression.

[0064] “载体”是指可以向其中插入另一DNA区段从而导致插入的区段复制的复制子,例如质粒、噬菌体或黏粒。 [0064] "vector" refers to a DNA segment can be inserted into another causing wherein replicon replication of the inserted segment, such as a plasmid, phage or cosmid. 通常,载体与正确的控制元件结合时能够复制。 Typically, the carrier with the correct control element capable of replication when combined. 术语“载体”包括克隆和表达载体,以及病毒载体和整合载体。 The term "vector" includes cloning and expression vectors, as well as viral vectors and integrating vectors. “表达载体”是包含调节区的载体。 "Expression vector" is a vector comprising a regulatory region.

[0065] 氧化胁迫:植物物种耐受R0S/R0I/A0S的能力有所不同。 [0065] Oxidative stress: Plant species tolerant R0S / R0I / capacity A0S vary. “氧化胁迫”可以被定义为下述环境条件的集合,在所述环境条件下植物会开始遭受提高的R0S/R0I/A0S浓度的影响,例如酶活性的降低、DNA断裂、DNA-蛋白质交联、坏死和矮化的生长。 "Oxidative stress" can be defined by the following set of environmental conditions, a plant will begin to suffer increase in the ambient conditions R0S / R0I Effect / A0S concentration, such as reduced enzyme activity, the DNA breakage, DNA-protein crosslinks , necrosis and stunted growth. 因为这些原因,所以经历氧化胁迫的植物通常显示显著的生物量和/或产量降低。 For these reasons, the plants to oxidative stress typically exhibit a significant reduction in biomass and / or yield.

[0066] 提高的氧化胁迫可由天然的、地质学的过程和人类活动(例如污染)引起。 [0066] The oxidative stress may enhance the natural, geological processes and by human activities (e.g., contamination) caused. 因为植物物种耐受氧化胁迫的能力有所不同,所以不能概括引起胁迫的精确环境条件。 Because the ability of plant species resistant to oxidative stress is different, so you can not generalize exact cause environmental stress conditions. 然而,在氧化胁迫条件下,氧化胁迫耐性植物与非氧化胁迫耐性植物相比产生更高的生物量、产量和存活度。 However, under oxidative stress conditions, produce higher biomass, yield and viability compared to the non-oxidative stress tolerance of plants and plant oxidative stress tolerance. 可以定量生理外观、回收率和产量。 Quantifiable physiological appearance, recovery and production.

[0067] 光合作用效率:通过最大荧光信号Fm和可变荧光Fv之间的关系评估光合作用效率或通过光系统II的电子运输。 [0067] Photosynthetic efficiency: photosynthetic efficiency assessment by light or electron transport system II, the relationship between the maximum fluorescence signal and the variable fluorescence Fm Fv. 最适量子产量(Fv/Fm)的降低表明胁迫,并可用于监测盐或氧化胁迫条件下与非转基因植物相比转基因植物的性能。 Reducing optimum quantum yield (Fv / Fm) indicates stress, and for the performance of transgenic plants compared to non-transgenic plants under salt or oxidative stress conditions monitored.

[0068] 水杨酸生长指数(SAGI):光合作用效率χ幼苗面积。 [0068] Salicylic Acid Growth Index (SAGI): Photosynthetic efficiency χ seedling area.

[0069] 盐生长指数(SGI):光合作用效率χ幼苗面积(在盐胁迫条件下)。 Χ seedling photosynthetic efficiency area (under salt stress conditions): [0069] Salt growth index (SGI).

[0070] 盐度:植物物种耐受盐度的能力有所不同。 [0070] salinity: the ability to tolerate salinity species of plants vary. “盐度”可以被定义为下述环境条件的集合,所述环境条件下植物开始经受提高的盐浓度的影响,例如例子失衡、降低的气孔传导率、降低的光合作用、降低的生长速率、提高的细胞死亡、丧失膨压(萎蔫)或胚珠败育。 "Salinity" may be defined as a set of environmental conditions, the environmental condition at the plant starts to withstand the effects of elevated salt concentration, e.g. examples imbalance, decreased stomatal conductance, decreased photosynthesis, decreased growth rate, increased cell death, loss of turgor (wilting) or ovule abortion. 由于这些原因,所以经历盐度胁迫的植物通常显示生物量和/或产量的显著降低。 For these reasons, the salinity stress experienced Plants usually show a significant reduction of biomass and / or production.

[0071] 提高的盐度可由天然的、地理学过程和人类活动(例如污染)引起。 [0071] increase of salinity from natural, human activities and geographical processes (e.g., contamination) caused. 因为植物物种耐受盐度的能力有所不同,所以不能概括引起胁迫的精确环境条件。 Because the ability to tolerate salinity plant species are different, we can not generalize the exact cause environmental stress conditions. 然而,在盐条件下, 盐度耐性植物比非盐耐性植物产生更高的生物量、产量和存活率。 However, under salt conditions, salinity tolerant plants produce higher biomass, yield and survival than non-salt tolerant plants. 可以定量物理外观、回收率和产量的差异。 Quantitative differences in physical appearance may, recovery and yield.

[0072] 提高的盐度可由天然的、地理学过程和人类活动(例如灌溉)引起。 [0072] increase of salinity from natural, human activities and processes geography (e.g. irrigation) caused. 因为植物物种耐受水缺乏的能力有所不同,所以不能概括引起胁迫的精确环境盐条件。 Because of the lack of capacity of the water plant species tolerance is different, so you can not generalize precise environmental salt conditions caused by stress. 然而,在盐条件下,盐耐性植物比非盐耐性植物产生更高的生物量、产量和存活率。 However, under conditions of salt, salt tolerant plants produce higher biomass, yield and survival than non-salt tolerant plants. 可以定量并使用公知的测量和分析方法统计学分析物理外观、回收率和产量的差异。 And it can be quantified using known methods of measurement and analysis Statistical analysis The differences in physical appearance, recovery and yield.

[0073] II.多肽 [0073] II. Polypeptide

[0074] 本文所述多肽包括盐度耐性和/或氧化胁迫耐性调控多肽。 [0074] The polypeptides described herein include salinity tolerance and / or oxidative stress tolerance-modulating polypeptide. 盐度耐性和/或氧化胁迫耐性调控多肽在植物或植物细胞中表达时可以有效调控盐度耐性和/或氧化胁迫耐性水平。 Salinity tolerance and / or expression control can be effectively regulated polypeptide in a plant when the plant cell or oxidative stress tolerance salinity tolerance and / or oxidative stress tolerance levels. 这类多肽通常含有至少一个盐度耐性和/或氧化胁迫耐性调控多肽特征性(indicative)的结构域,如本文更详细描述的。 Such polypeptides typically contain at least a salinity tolerance and / or oxidative stress tolerance polypeptide characteristic regulation (Indicative) domain, as described in greater detail herein. 盐度耐性和/或氧化胁迫耐性调控多肽通常具有大于30的HMM 二进制值,如本文更详细描述的。 Salinity tolerance and / or oxidative stress tolerance-modulating polypeptides typically have HMM bit scores greater than 30, as described in greater detail herein. 在一些实施方案中,盐度耐性和/ 或氧化胁迫耐性调控多肽与SEQ ID NO :2、4、6、8、9、11、13、14、15、17、19、20、22、23、24、25、 27、29、30、31、33、35、36、37、38、39、41、42、43、44、45、47、49、50、52、54、56、58、60、62、63、 64、66、68、69、71、73、74、76、78、80、81、83、84、86、88、90、91、93、94、96、98、100、101、102、 104、106、107、109、110、112、114、116、118、119、121、122、123、125、126、127、128、129、130、 132、134、136、138、140、141、142、143、144、145、147、149、151、153、154、156、158、160、162、 163、165、166、167、168和SEQ ID NO :140的氨基酸等同物1到135具有大于85%的同一性,如本文更详细描述的。 In some embodiments, salinity tolerance and / or oxidative stress tolerance-modulating polypeptide and SEQ ID NO: 2,4,6,8,9,11,13,14,15,17,19,20,22,23, 24, 25, 27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60, 62, 63, 64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101, 102, 104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130, 132,134,136,138,140, 141,142,143,144,145,147,149,151,153,154,156,158,160,162, 163,165,166,167,168, and SEQ ID NO: 140 is the amino acid equivalents 1-135 identity greater than 85%, as described in greater detail herein.

[0075] A.請·禾口/贿碰迫屋膽碰白■赌 [0075] A. Please · Wo mouth / bribe touch forced to touch the white house bile ■ bet

[0076] 盐度耐性和/或氧化胁迫耐性调控多肽可含有IQ钙调蛋白结合基序结构域,所述结构域被预测是盐度耐性和/或氧化胁迫耐性调控多肽特征性的。 [0076] salinity tolerance and / or oxidative stress tolerance-modulating polypeptide can contain a IQ calmodulin-binding motif domain, the domain is predicted salinity tolerance and / or polypeptide characteristic of oxidative stress tolerance-modulating. 钙调蛋白(CaM)被认为是主要的钙传感器,并通过其与不同组的细胞蛋白质的相互作用管理(orchestrator)调节事件。 Calmodulin (of CaM) is recognized as a major calcium sensor, and adjusting the event management interaction with cellular proteins (Orchestrator) through which the different groups. 对许多已知的CaM结合蛋白而言存在三类识别基序;作为非Ca2+-依赖型结合共有序列的IQ基序,和用于Ca2+-依赖型结合的两种相关基序,其以保守的疏水残基PUBMED : 9141499的位置为基础称作18-14和1_5_10。 For many categories known in the presence of CaM binding proteins are concerned recognition motif; as a non-Ca2 + - dependent binding consensus sequence IQ motif, and a Ca2 + - dependent two related motifs bound, which is conserved hydrophobic residues PUBMED: 9141499 on the basis of the position 18-14 and referred 1_5_10.

[0077] 例如,扇贝肌球蛋白的调节区是三链蛋白质复合物,其应答Ca2+结合而在该马达上开关。 [0077] For example, the regulatory region of scallop myosin is a three-chain protein complex, which is bound Ca2 + response and the switch on the motor. 侧链相互作用将两条轻链与带有IQ-序列基序的重链的相邻区段串联连接。 The side-chain interactions two light chains and heavy chains of adjacent segments having IQ- sequence motif are connected in series. Ca2+_结合位点是主要轻链上新的EF-手基序,并且通过涉及重链和两条轻链的连接被稳定化,负责完整的肌球蛋白分子PUBMED =8127365中Ca2+结合和调节对所有三条链的要求。 _ Ca2 + binding site is a new major EF- hand motif of the light chain and is stabilized by connecting relates to heavy chains and two light chains, intact myosin molecule responsible PUBMED = 8127365 in Ca2 + binding and adjustment requirements for all three chains.

[0078]例如,SEQ ID NO :86 公开了本文中被鉴定为Ceres SEEDLINE IDno. ME08768 的拟南芥克隆的氨基酸序列,其被预测为编码含有来自残基116-136的IQ钙调蛋白结合基序结构域的多肽。 [0078] For example, SEQ ID NO: 86 is disclosed herein as Ceres SEEDLINE IDno identified amino acid sequence of an Arabidopsis clone ME08768, which is predicted to encode a IQ calmodulin-binding group derived from the residue 116-136. domain polypeptide sequence.

[0079] 在一些实施方案中,盐度耐性和/或氧化胁迫耐性调控多肽在天然存在的多肽的氨基端或羧基端被截短。 [0079] In some embodiments, salinity tolerance and / or oxidative stress tolerance-modulating polypeptide in a naturally occurring polypeptide of an amino terminal truncated or carboxy. 截短的多肽可保留天然存在的多肽的某些结构域,同时缺乏其他结构域。 Truncated polypeptide may retain certain domains of the naturally occurring polypeptide while lacking other domains. 因此,至多短或长5个氨基酸的长度变体通常显示截短多肽的盐度耐性和/或氧化胁迫耐性调控活性。 Thus, up to a length shorter or longer variants of 5 amino acids truncated polypeptides typically exhibit the salinity tolerance and / or oxidative stress tolerance-modulating activity. 在一些实施方案中,截短的多肽是显性负性多肽。 In some embodiments, a truncated polypeptide is a dominant negative polypeptide. SEQ ID N0:138公开了相对于天然存在的多肽而言在5'端被截短的盐度耐性和/或氧化胁迫耐性调控多肽的氨基酸序列。 SEQ ID N0: 138 discloses the truncated phase salinity tolerance and / or oxidative stress tolerance-modulating the amino acid sequence of the polypeptide is naturally occurring polypeptides at the 5 'end. 与不包含截短的对照植物和/或其组织中的相应水平相比,这类截短多肽在植物中的表达赋予植物和/或植物组织中盐度耐性和/或氧化胁迫耐性水平的差异。 Compared with / without respective horizontal tissues and control plants containing truncated thereof, such truncated polypeptide expression in a plant confers a plant and / or plant tissues salinity tolerance and / or oxidative stress tolerance level difference .

[0080] B.通过Reciprocal BLAST鉴定的功能性同源物 [0080] B. identified by Reciprocal BLAST functional homologues

[0081] 在一些实施方案中,由上文指出的一种或多种pfam描述定义的参考盐度耐性和/ 或氧化胁迫耐性调控多肽的一种或多种功能性同源物适合用作盐度耐性和/或氧化胁迫耐性调控多肽。 [0081] In some embodiments, the one or more indicated above described pfam defined reference salinity tolerance and / or oxidation of one or more functional homologues stress tolerance-modulating polypeptide suitable for use as salt of tolerance and / or oxidative stress tolerance-modulating polypeptide. 功能性同源物是与参照多肽具有序列相似性并且发挥参照多肽的一种或多种生物化学功能或生理功能的多肽。 Functional homolog is a polypeptide having sequence similarity to reference and to play the one or more polypeptides or physiologically functional biochemical functions of the reference polypeptide. 功能性同源物和参照多肽可以是天然存在的多肽,并且序列相似性可归因于趋同或趋异进化事件。 Functional homolog and the reference polypeptide may be a naturally occurring polypeptide, and attributable to sequence similarity or convergent divergent evolutionary events. 同样,功能性同源物有时在文献中被称作同源物、或直系同源物、或旁系同源物。 Similarly, functional homologs are sometimes referred to in the literature as homologs, or orthologs, or paralogs. 天然存在的功能性同源物的变体(例如野生型编码序列突变体所编码的多肽)自身可以是功能性同源物。 Variants of naturally occurring functional homolog (e.g. body of the encoded polypeptide coding sequence of wild-type mutant) itself can be a functional homolog. 功能性同源物也可以通过盐度耐性和 Functional homologues can also salinity tolerance and

15/或氧化胁迫耐性调控多肽编码序列的定点诱变产生,或通过将来自不同天然存在的盐度耐性和/或氧化胁迫耐性调控多肽的编码序列的结构域组合产生(“结构域交换”)。 15 / or oxidative stress tolerance-modulating polypeptide Site-directed mutagenesis of the coding sequence produced by or from the composite structures of different domains of naturally-occurring salinity tolerance and / or oxidative stress tolerance coding sequence regulated polypeptide produced ( "domain swapping") . 术语“功能性同源物”有时适用于编码功能同源性多肽的核酸。 The term "functional homologues" is sometimes applied to the nucleic acid encoding a functional homologous polypeptide.

[0082] 可以通过分析核苷酸和多肽序列比对来鉴定功能性同源物。 [0082] The alignment can be identified by analyzing the functional homologues nucleotide and polypeptide sequences. 例如,对核苷酸或多肽序列的数据库进行查询能够鉴定盐度耐性和/或氧化胁迫耐性调控多肽的同源物。 For example, a database of nucleotide or polypeptide sequences can identify query salinity tolerance and / or oxidative stress tolerance-modulating homolog polypeptide. 序列分析可涉及使用盐度耐性和/或氧化胁迫耐性调控多肽氨基酸序列作为参照序列,对非冗余的数据库进行BLAST、ReCipr0Cal BLAST或PSI-BLAST分析。 Sequence analysis can involve the use of a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide amino acid sequence as a reference sequence, the non-redundant database BLAST, ReCipr0Cal BLAST, or PSI-BLAST analysis. 在一些情况下,氨基酸序列从核苷酸序列演绎而来。 In some cases, the deduced amino acid sequence derived from the nucleotide sequence. 数据库中具有大于40%序列同一性的这些多肽是进一步评价作为盐度耐性和/或氧化胁迫耐性调控多肽的适应性的候选者。 These databases polypeptide having greater than 40% sequence identity are candidates for further evaluation of the adaptability of a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide. 氨基酸相似性允许保守的氨基酸替换,例如用一个疏水残基替换另一个,或用一个极性残基替换另一个。 Amino acid similarity allows for conservative amino acid substitutions, such as a hydrophobic residue with another, or substitutions of one polar residue with another. 需要时可以进行这类候选者的手检,从而缩小有待进一步评价的候选者的数量。 Such a candidate may be the subject's hand when needed, so that the number of candidates to be further evaluated reduction. 可以通过选择显示具有盐度耐性和/或氧化胁迫耐性调控多肽中存在的结构域(例如保守的功能结构域)来进行手检。 By selecting a display having a salinity tolerance and / or oxidative stress tolerance-modulating domain present in the polypeptide (e.g., conserved functional domains) to check the hand.

[0083] 可以通过在盐度耐性和/或氧化胁迫耐性调控多肽的一级氨基酸序列中定位下述区域来鉴定保守区域,所述区域是重复序列,形成一些二级结构(例如螺旋和β片层), 建立带正电或带负电的结构域,或代表蛋白质基序或结构域。 [0083] The following can be positioned in the region of a salinity tolerance and / or an amino acid sequence of oxidative stress tolerance-modulating polypeptide identified conserved regions, the region is a repetitive sequence, formation of some secondary structure (e.g. helices and β sheets layer), the establishment of positively or negatively charged domains, or represents a protein motif or domain. 见,例如万维网Sanger, ac. uk/Software/Pfam/和pfam. janeIia. org/上描述多种蛋白质基序和结构域的共有序列的Pfam网站。 See, for example, the World Wide Web Sanger, ac. Uk / Software / Pfam / and pfam. JaneIia. Site Pfam consensus sequence described in a variety of protein motifs and domains org / on. Pfam数据库中包含的信息的说明描述于Sonnhammer等,Nucl. AcidsRes., 26 :320-322(1998) ;Sonnhammer 等,Proteins, 28 :405-420 (1997);和Bateman 等,Nucl. Acids Res. ,27 =260-262(1999)中。 Description of the information contained in the Pfam database is described in Sonnhammer et, Nucl AcidsRes, 26: 320-322 (1998); Sonnhammer et, Proteins, 28:... 405-420 (1997); and the like Bateman, Nucl Acids Res. , 27 = 260-262 (1999). 还可以通过比对来自密切相关物种的相同或相关多肽的序列,来测定保守区。 By sequence alignment may also be the same or related polypeptides from closely related species, conserved regions determined. 密切相关物种优选来自相同的科。 Closely related species, preferably from the same department. 在一些实施方案中,来自两种不同物种的序列比对就足够。 In some embodiments, sequences from two different species is sufficient comparison.

[0084] 通常,显示至少约40%氨基酸序列同一性的多肽可用于鉴定保守区。 [0084] Generally, a display of at least about 40% amino acid sequence identity of polypeptides can be used to identify conserved regions. 相关多肽的保守区显示至少45%的氨基酸序列同一性(例如至少50%、至少60%、至少70%、至少80 %、或至少90 %的氨基酸序列同一性)。 Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). 在一些实施方案中,保守区显示至少92 %、94%、 In some embodiments, a conserved region exhibits at least 92%, 94%,

96 %、98 %或99 %的氨基酸序列同一性。 96%, 98%, or 99% amino acid sequence identity.

[0085] 图1和序列表中提供了SEQ ID NO :86中所示的多肽的功能性同源物的氨基酸序列。 [0085] FIGS. 1 and provided in the Sequence Listing SEQ ID NO: amino acid sequences of functional homologs of the polypeptide shown in FIG 86. 这类功能性同源物包括(SEQ ID NO :88、90、91、93、94、96、98、100、101、102、104、106 和107)。 Such functional homologs include (SEQ ID NO: 88,90,91,93,94,96,98,100,101,102,104,106 and 107). 在一些情况下,SEQ IDNO :86的功能性同源物具有下述氨基酸序列,所述氨基酸序列与SEQ IDNO :86中所示的氨基酸序列具有至少50%的序列同一性,例如50%、52%、56%、 59%,61%,65%,70%,75%,80%,85%,90%,95%,97%,98%^; 99%的序列同一性。 In some cases, SEQ IDNO: 86 functional homolog having the following amino acid sequence, the amino acid sequence of SEQ IDNO: 86 amino acid sequence shown in at least 50% sequence identity, e.g., 50%, 52 %, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% ^; 99% sequence identity.

[0086] 图2中提供了SEQ ID NO :41中所示的多肽的功能性同源物的氨基酸序列。 [0086] FIG. 2 is provided in SEQ ID NO: amino acid sequences of functional homologs of the polypeptide shown in 41. 这类功能性同源物包括(SEQ ID NO :42、43、44、45、47、49、50、52、54、56、58、60、62、63、64、66、 68、69、71、73、74、76、78、80、81、83 和84)。 Such functional homologs include (SEQ ID NO: 42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66, 68,69,71 , 73,74,76,78,80,81,83 and 84). 在一些情况下,SEQ ID NO :41 的功能性同源物具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :41中所示的氨基酸序列具有至少50% 的序列同一性,例如50%,52%,56%,59%,61%,65%,70%,75%,80%,85%,90%,95%, In some cases, SEQ ID NO: 41 functional homolog having the following amino acid sequence, the amino acid sequence SEQ ID NO: 41 amino acid sequence shown in at least 50% sequence identity, e.g., 50% , 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,

97 %、98 %或99 %的序列同一性。 97%, 98% or 99% sequence identity.

[0087] 图3中提供了SEQ ID NO :109中所示的多肽的功能性同源物的氨基酸序列。 [0087] FIG. 3 provides the SEQ ID NO: amino acid sequences of functional homologs of the polypeptide shown in 109. 这类功能性同源物包括(SEQ ID NO :110、112、114、116、118、119、121、122、123、125、126、127、128、129、130、132和134)。 Such functional homologs include (SEQ ID NO: 110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132 and 134). 在一些情况下,SEQ ID NO : 109的功能性同源物具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :109中所示的氨基酸序列具有至少50%的序列同一性,例如50%、52%、56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99%的序列同一性。 In some cases, SEQ ID NO: 109 is the functional homolog having the following amino acid sequence, the amino acid sequence SEQ ID NO: 109 amino acid sequence shown in at least 50% sequence identity, e.g., 50% , 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0088] 图4中提供了SEQ ID NO :140中所示的多肽的功能性同源物的氨基酸序列。 [0088] Figure 4 provides a SEQ ID NO: amino acid sequences of functional homologs of the polypeptide shown in 140. 这类功能性同源物包括(SEQ ID NO :136、138、140、141、142、143、144、145、147、149、151、153、 154、156、158、160、162、163、165、166、167 和168)。 Such functional homologs include (SEQ ID NO: 136,138,140,141,142,143,144,145,147,149,151,153, 154,156,158,160,162,163,165 166, 167 and 168). 在一些情况下,SEQ ID NO : 140 的功能性同源物具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :140中所示的氨基酸序列具有至少50% 的序列同一性,例如50%,52%, 56%,59%,61%,65%,70%,75%,80%,85%, 90%、95%、97%、98%或99% 的序列同一性。 In some cases, SEQ ID NO: 140 is the functional homologue having an amino acid sequence, the amino acid sequence SEQ ID NO: 140 amino acid sequence shown in at least 50% sequence identity, e.g., 50% , 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0089] 图5中提供了SEQ ID NO :2中所示的多肽的功能性同源物的氨基酸序列。 [0089] Figure 5 provides a SEQ ID NO: 2 amino acid sequences of functional homologs of the polypeptide shown in FIG. 这类功能性同源物包括(SEQ ID NO :2、4、6、8、9、11、13、14、15、17、19、20、22、23、24、25、27、29、30、 31和33)。 Such functional homologs include (SEQ ID NO: 2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30 , 31 and 33). 在一些情况下,SEQ ID NO :2的功能性同源物具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :2中所示的氨基酸序列具有至少50%的序列同一性,例如50%、52%、 56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99% 的序列同一性。 In some cases, SEQ ID NO: 2 is a functional homolog having the following amino acid sequence, the amino acid sequence SEQ ID NO: 2 amino acid sequence shown in at least 50% sequence identity, e.g., 50% , 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0090] 图6中提供了SEQ ID NO :35中所示的多肽的功能性同源物的氨基酸序列。 [0090] FIG. 6 is provided SEQ ID NO: amino acid sequences of functional homologs of the polypeptide shown in FIG 35. 这类功能性同源物包括(SEQ ID NO :35、36、37、38和39)。 Such functional homologs include (SEQ ID NO: 35,36,37,38, and 39). 在一些情况下,SEQ ID NO :35的功能性同源物具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :35中所示的氨基酸序列具有至少50% 的序列同一性,例如50%,52%,56%,59%,61%,65%,70%,75%,80%,85%, 90%、95%、97%、98%或99% 的序列同一性。 In some cases, SEQ ID NO: 35 functional homolog having the following amino acid sequence, the amino acid sequence SEQ ID NO: 35 amino acid sequence shown in at least 50% sequence identity, e.g., 50% , 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0091] 盐度耐性和/或氧化胁迫耐性调控多肽中保守区的鉴定有助于盐度耐性和/或氧化胁迫耐性调控多肽变体的产生。 Identification [0091] salinity tolerance and / or oxidative stress tolerance-modulating polypeptide conserved region conducive for production of salinity tolerance and / or oxidative stress tolerance-modulating polypeptide variants. 盐度耐性和/或氧化胁迫耐性调控多肽的变体通常在一级氨基酸序列中具有10个或更少的保守氨基酸替换,例如7个或更少的保守氨基酸替换, 5个或更少的保守氨基酸替换,或1个和5个之间的保守氨基酸替换。 Salinity tolerance and / or oxidative stress tolerance-modulating polypeptide variants typically have an amino acid sequence of 10 or fewer conservative amino acid substitutions, 7 or fewer conservative amino acid substitutions, for example, 5 or fewer conservative amino acid substitutions, conservative amino acid substitutions, or between 1 and 5. 可以基于图1到图6 中所示的比对之一构建有用的变体多肽。 Useful can be constructed based on one of the variant polypeptide compared to FIG. 1 to FIG. 6. 这类多肽包括保守区,所述保守区以图中所示从氨基端到羧基端的顺序排列。 Such polypeptides comprising the conserved region, the conserved regions shown in the drawings end to carboxy order from the amino terminal. 这类多肽也可在用虚线标记的位置中包含零个、一个或多于一个的氨基酸。 Such polypeptides may also be included in the zero position marked with dotted lines, one or more amino acids. 当虚线标记的位置上不存在氨基酸时,这类多肽的长度是所有保守区中氨基酸残基的总和。 When amino acids are present on the position marked by dashes, the length of such a polypeptide is the sum of all the conserved regions of amino acid residues. 用虚线标记的所有位置中均存在氨基酸时,这类多肽的长度是所有保守区和所有虚线中氨基酸残基的总和。 All positions are marked with dotted lines in the presence of amino acids, the length of such a polypeptide is the sum of broken line in all conserved regions and amino acid residues.

[0092] C.通过HMM鉴定的功能性同源物 [0092] C. identified by HMM functional homologues

[0093] 在一些实施方案中,有用的盐度和/或氧化胁迫耐性调控多肽包括适合以图1-6 任一中所示的多肽为基础的隐蔽马尔科夫模型的多肽。 [0093] In some embodiments, useful salinity and / or oxidative stress tolerance-modulating polypeptides include those suitable for Hidden Markov Models to a polypeptide shown in FIG claims 1-6 based polypeptide. 隐蔽马尔科夫模型(HMM)是一组功能性同源物的共有序列的统计学模型。 Hidden Markov Model (HMM) is a statistical model of a consensus sequence of set of functional homologs thereof. 参阅Durbin等,Biological Sequence Analysis : Probabilistic Models of Proteinsand Nucleic Acids, Cambridge University Press, Cambridge, UK(1998)。 See Durbin et al, Biological Sequence Analysis: Probabilistic Models of Proteinsand Nucleic Acids, Cambridge University Press, Cambridge, UK (1998). 使用功能性同源物组的序列作为输入,通过程序HMMER 2. 3. 2用默认程序参数产生HMM。 Using sequence functional homologues set as input, generates HMM 2.3.2 with default program parameters by the program HMMER. 使用一组默认参数通过1. 11版ProbCons (Do等,GenomeRes.,15(2): 330-40 (2005))产生多序列比对,所述参数为:—c,一一致性REPS为2 ;-ir,—迭代-细分REPS为100 ;-pre,—pre-培训REPS为0。 By using a set of default parameters Version 1. 11 ProbCons (Do et, GenomeRes, 15 (2):. 330-40 (2005)) to generate multiple sequence alignment of the parameters: -c, to a consistency REPS 2; -ir, - iteration - REPS subdivided into 100; -pre, -pre- training REPS zero. ProbCons是由斯坦福大学提供的公开结构域软 ProbCons is public domain software provided by Stanford University

17件程序。 17 program.

[0094] 构建HMM(hmmbuild)的默认参数如下:MAP体系结构使用的默认“体系结构优先级” (archpri)为0. 85,用于测定有效序列数的默认截止阈值(idlevel)为0. 62。 [0094] Construction of HMM (hmmbuild) default parameters as follows: MAP architecture using default "priority Architecture" (archpri) is 0.85, the effective number of sequences used for the determination of the default cutoff threshold (idlevel) is 0.62 . HMMER 2. 3. 2在2003年10月3日在GNU —般公开许可证中准许,并且可在万维网上的多个来源处获得。 HMMER 2. 3. 2 on October 3, 2003 in GNU - general public license grant, and is available in a number of sources on the World Wide Web at. Hmmbuild作为文本文件输出模型。 Hmmbuild as a text file output model.

[0095] 可以使用一组功能性同源物的HMM来测定候选盐度耐性和/或氧化胁迫耐性调控多肽序列比下述无效HMM更好地适合具体HMM的可能性,所述无效HMM使用非结构或功能相关的一组序列产生。 [0095] can be determined candidate salinity tolerance and / or oxidative stress tolerance-modulating polypeptide sequence comparison the following invalid HMM better fit particular HMM likelihood of the HMM using the non-use of a valid set of functional homologs HMM structure or function associated with a set of sequences generated. 受试多肽序列比无效HMM更好地适合HMM的可能性由HMM 二进制值表示,所述HMM 二进制值是使用HMMER hmmsearch程序将候选序列拟合至HMM图谱时产生的数字。 The possibility of the subject polypeptide sequence better suited than the HMM HMM invalid binary value is represented by the HMM, the HMM bit values ​​are generated using the HMMER hmmsearch program candidate sequence number generated when fitted to the HMM pattern. 运行hmmsearch时使用以下的默认参数:默认E-值截止(E)为10. 0,默认二进制值截止(T)为负无穷大,数据库中默认的序列数(Z)是数据库中的真实序列数,per-结构域分级的hit列表的默认E值截止(domE)是无穷大,per-结构域分级的hit列表的默认二进制值截止(domT)为负无穷大。 When running hmmsearch using the following default parameters: default E- value cutoff (E) is 10.0, the default binary value cutoff (T) is negative infinity, the default number of the database (Z) is the real number of sequences in the sequence database, the default value per- domain E grade cut-off of the hit list (domE) is infinite, the default binary value per- domain graded hit list off (domT) is negative infinity. 高HMM 二进制值表示受试序列进行用于产生HMM的多肽的一种或多种生物化学功能或生理学功能的可能性更大。 High HMM bit score indicates a greater likelihood for the test sequence to produce a polypeptide of the HMM more biochemical or physiological function or functions. 高HMM 二进制值至少为20,通吊更尚。 High HMM bit score of at least 20, more still hanging on.

[0096] 本领域技术人员应当明白,序列表中提供的HMM得分仅用于举例。 [0096] Those skilled in the art will appreciate, HMM scores provided in the sequence listing are merely exemplary. 因为多序列比对算法例如ProbCons仅能产生接近最佳的结果,所以由于下述因素可能发生模型的轻微变异,所述因素例如序列被加工用于比对的顺序。 Since multiple sequence alignment algorithms, for example, only produces the best results ProbCons close, so because of the following factors may occur slight variation model, the factors such as the order of alignment of sequences are used for processing. 然而,HMM得分变异性较小,因此序列表中的HMM得分代表了用各个序列制备的模型。 Nevertheless, HMM score variability is small, so the HMM scores in the sequence listing represent a model prepared by each sequence.

[0097] 下文讨论的盐度和/或氧化胁迫调控多肽以大于20 (例如大于20、30、40、50、60、 70、80、90、100、200、300、400或500)的HMM 二进制值适合所示HMM。 [0097] discussed below salinity and / or oxidative stress-modulating polypeptide of greater than 20 (e.g. greater than 20,30,40,50,60, 70,80,90,100,200,300,400, or 500) of the HMM bit value for HMM FIG. 在一些实施方案中,下文讨论的盐度和/或氧化胁迫调控多肽的HMM 二进制值约为序列表中提供的功能性同源物HMM 二进制值的50 %、60 %、70 %、80 %、90 %或95 %。 In some embodiments, discussed below salinity and / or oxidative stress-modulating polypeptide HMM bit value of approximately 50% sequence table provided in functional homologues HMM bit score of 60%, 70%, 80%, 90% or 95%. 在一些实施方案中,下文讨论的盐度和/或氧化胁迫调控多肽以大于20的HMM 二进制值适合所示HMM,并具有盐度和/或氧化胁迫调控多肽的特征性结构域。 In some embodiments, discussed below salinity and / or oxidative stress-modulating polypeptide HMM bit score greater than a value for HMM shown in FIG. 20, and having a salinity and / or oxidative stress-modulating polypeptide domain characteristic. 在一些实施方案中,下文讨论的盐度和/或氧化胁迫调控多肽以大于20的HMM 二进制值适合所示HMM,并且与图1到6任一中所示氨基酸序列或序列表中与图1到6中任一相关的氨基酸序列具有85%或更高的序列同一性(75%、80%、 85%、90%、95%或100%的序列同一性)。 In some embodiments, discussed below salinity and / or oxidative stress-modulating polypeptide HMM bit score greater than 20 is suitable HMM shown in FIG. 1 and any one of a 1-6 sequence or amino acid sequence listing of FIG. 6 the amino acid sequence of any associated with a 85% or more sequence identity (75%, 80%, 85%, 90%, 95%, or 100% sequence identity).

[0098] 序列表中提供了与图1中所示的氨基酸序列产生的HMM拟合时,具有大于400、 450、500、550、600、650、700、750、800、850、900、950、1000、1050 或1100 的HMM 二进制值的多肽。 When [0098] Providing the HMM generated by fitting the amino acid sequence shown in Figure 1 Sequence Listing, greater than 400, 450,500,550,600,650,700,750,800,850,900,950, HMM polypeptide binary value of 1000, 1050 or 1100. 这类多肽包括CeresSEEDLINE ID no. ME08768、Ceres 克隆ID no. 1943807、Ceres ANNOT IDno. 1471392,Public GI ID no. 6715635、Ceres 克隆ID no. 910109、Public GIID no. 115474509、Ceres 克隆ID no. 1780908、Ceres ANNOT ID no. 1520883、Ceres 克隆ID no. 148018、Public GI ID no. 18378797、Public GI IDno. 21553500、Ceres ANNOT ID no. 1444522、Ceres ANNOT ID no.146751 和Public GI ID no. 125559938(SEQ ID NO :86、 88、90、91、93、94、96、98、100、101、102、104、106 和107)。 Such polypeptides include CeresSEEDLINE ID no. ME08768, Ceres Clone ID no. 1943807, Ceres ANNOT IDno. 1471392, Public GI ID no. 6715635, Ceres Clone ID no. 910109, Public GIID no. 115474509, Ceres Clone ID no. 1780908, Ceres ANNOT ID no. 1520883, Ceres clone ID no. 148018, Public GI ID no. 18378797, Public GI IDno. 21553500, Ceres ANNOT ID no. 1444522, Ceres ANNOT ID no.146751, and Public GI ID no. 125559938 (SEQ ID NO: 86, 88,90,91,93,94,96,98,100,101,102,104,106 and 107).

[0099] 序列表中提供了与图2中所示的氨基酸序列产生的HMM拟合时具有大于30、50、 100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950或1000 Greater than 30, 50, 100,150,200,250,300,350,400,450,500,550,600 when [0099] Providing the HMM generated by fitting the amino acid sequence shown in FIG. 2 in the Sequence Listing , 650,700,750,800,850,900,950 or 1000

的HMM 二进制值的多肽。 The HMM bit score of the polypeptide. 这类多肽包括Ceres SEEDLINE ID no. ME06748,Ceres SEEDLINEIDno. ME20711、Ceres SEEDLINE ID no. ME18973、Ceres SEEDLINE IDno. ME08732、Ceres SEEDLINE ID no. ME19657, Ceres 克隆ID no. 835818、Ceres 克隆ID no. 1796745、Public GI ID no. 125543896、Ceres ANNOT IDno. 1483984、Ceres 克隆ID no. 1924654、Ceres ANNOT ID no. 1468861、Ceres 克隆ID no. 1641776、Ceres ANNOT ID no. 1438750、Ceres ANNOT IDno. 1447395、Public GI ID no. 79482785、Public GI ID no. 3292832、Ceres 克隆ID no. 1559074、Ceres 克隆ID no. 1726548、Public GI IDno. 115459996、Ceres 克隆ID no. 697034、Ceres 克隆ID no. 353438、PublicGI ID no. 125593074、Ceres 克隆ID no. 1920115、Ceres 克隆ID no. 21821、Ceres 克隆ID no. 560066、Public GI ID no. 115453071、Ceres 克隆IDno. 1968211 和Public GI ID no. 116310011_(SEQ ID NO :42、 43、44、45、47、49、50、52、54、56、58、60、62、63、64、66、68、69、71、73、74、76、78、80、81、83 和84)。 Such polypeptides include Ceres SEEDLINE ID no. ME06748, Ceres SEEDLINEIDno. ME20711, Ceres SEEDLINE ID no. ME18973, Ceres SEEDLINE IDno. ME08732, Ceres SEEDLINE ID no. ME19657, Ceres Clone ID no. 835818, Ceres Clone ID no. 1796745, public GI ID no. 125543896, Ceres ANNOT IDno. 1483984, Ceres clone ID no. 1924654, Ceres ANNOT ID no. 1468861, Ceres clone ID no. 1641776, Ceres ANNOT ID no. 1438750, Ceres ANNOT IDno. 1447395, public GI ID no. 79482785, Public GI ID no. 3292832, Ceres clone ID no. 1559074, Ceres clone ID no. 1726548, Public GI IDno. 115459996, Ceres clone ID no. 697034, Ceres clone ID no. 353438, PublicGI ID no. 125593074 , Ceres cLONE ID no 1920115, Ceres clone ID no 21821, Ceres clone ID no 560066, Public GI ID no 115453071, Ceres clone IDno 1968211 and Public GI ID no 116310011_ (SEQ ID NO:...... 42, 43, 44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83 and 84) .

[0100] 序列表中提供了与图3中所示的氨基酸序列产生的HMM拟合时具有大于120、150、 200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1050、 1100、1150 或1200 的HMM二进制值的多肽。 When [0100] the sequence listing provided HMM generated by fitting the amino acid sequence shown in Figure 3 has greater than 120,150, 200,250,300,350,400,450,500,550,600,650,700 , 750,800,850,900,950,1000,1050 polypeptide HMM bit values, 1100,1150, or 1200. 这类多肽包括Ceres SEEDLINE ID no. ME19173、 PublicGI ID no. 115435054、Ceres 克隆ID no. 1847857、Ceres ANNOT IDno. 1455219、 Ceres 克隆ID no. 352452、Ceres 克隆ID no. 787908、CeresLOCUS ID no. 0s01m00929_ AP002743、Ceres 克隆ID no. 246398、PublicGI ID no. 125527441、Public GI ID no. 125595056、Ceres 克隆ID no. 236071、Public GI ID no. 125524760、Public GI ID no. 125569365、Public GI IDno. 115439499、Public GI ID no. 15225258、Public GI ID no.115465173、Ceres ANNOT ID no.1477059 和Ceres ANNOT ID no.1530547 (SEQ IDNO : 110、112、114、116、118、119、121、122、123、125、126、127、128、129、130、132 和134)。 Such polypeptides include Ceres SEEDLINE ID no. ME19173, PublicGI ID no. 115435054, Ceres Clone ID no. 1847857, Ceres ANNOT IDno. 1455219, Ceres Clone ID no. 352452, Ceres Clone ID no. 787908, CeresLOCUS ID no. 0s01m00929_ AP002743 , Ceres cLONE ID no. 246398, PublicGI ID no. 125527441, Public GI ID no. 125595056, Ceres clone ID no. 236071, Public GI ID no. 125524760, Public GI ID no. 125569365, Public GI IDno. 115439499, Public GI . ID no 15225258, Public GI ID no.115465173, Ceres ANNOT ID no.1477059, and Ceres ANNOT ID no.1530547 (SEQ IDNO: 110,112,114,116,118,119,121,122,123,125,126 , 127,128,129,130,132 and 134).

[0101] 序列表中提供了与图4中所示的氨基酸序列产生的HMM拟合时具有大于150、200、 250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1050、1100、 1150、1200、1250、1300或1350的HMM 二进制值的多肽。 When [0101] the sequence listing provided HMM generated by fitting the amino acid sequence shown in Figure 4 of greater than 150,200, 250,300,350,400,450,500,550,600,650,700,750 , 800,850,900,950,1000,1050,1100, polypeptide or 1150,1200,1250,1300 HMM bit score of 1350. 这类多肽包括Ceres SEEDLINE IDno. ME24091、Ceres 克隆ID no. 375578、Ceres 克隆ID no. 375578、CeresSEEDLINE ID no. ME10681、Ceres SEEDLINE ID no. ME03140, CeresSEEDLINE ID no. ME24076、Ceres SEEDLINE ID no. ME24217, Public GIID no. 115440873、Ceres 克隆ID no. 826796、Ceres ANNOT ID no. 1465047、Ceres 克隆ID no. 1919901、Ceres 克隆ID no. 520008、Public GI IDno. 7413581、Ceres 克隆ID no. 228069、Ceres 克隆ID no. 467508、Ceres 克隆ID no. 1829581、Ceres 克隆ID no. 229668、Public GI ID no. 125550655、Ceres 克隆ID no. 106263、Public GI ID no. 15231175、Public GI IDno. 145357576 和Public GI ID no. 125528277(SEQ ID NO : 136、138、140、141、142、143、144、145、147、149、151、153、154、 156、158、160、162、163、165、166、167 和168)。 Such polypeptides include Ceres SEEDLINE IDno. ME24091, Ceres Clone ID no. 375578, Ceres Clone ID no. 375578, CeresSEEDLINE ID no. ME10681, Ceres SEEDLINE ID no. ME03140, CeresSEEDLINE ID no. ME24076, Ceres SEEDLINE ID no. ME24217, public GIID no. 115440873, Ceres clone ID no. 826796, Ceres ANNOT ID no. 1465047, Ceres clone ID no. 1919901, Ceres clone ID no. 520008, public GI IDno. 7413581, Ceres clone ID no. 228069, Ceres cLONE ID no. 467508, Ceres clone ID no. 1829581, Ceres clone ID no. 229668, Public GI ID no. 125550655, Ceres clone ID no. 106263, Public GI ID no. 15231175, Public GI IDno. 145357576 and Public GI ID no. 125528277 (SEQ ID NO: 136,138,140,141,142,143,144,145,147,149,151,153,154, 156,158,160,162,163,165,166,167 and 168) .

[0102] 序列表中提供了与图5中所示的氨基酸序列产生的HMM拟合时具有大于425、 450、500、550、600、650、700、750、800、850、900、950、1000、1050、1100、1150、1200、1250、 1300、1350、1400、1450、1500或1550的HMM 二进制值的多肽。 When [0102] Providing the HMM generated by fitting the amino acid sequence shown in FIG. 5 in the Sequence Listing greater than 425, 450,500,550,600,650,700,750,800,850,900,950,1000 , 1050,1100,1150,1200,1250, 1300,1350,1400,1450,1500 polypeptide or HMM bit score of 1550. 这类多肽包括Ceres克隆IDno. 1792354、Ceres 克隆ID no. 1925477、Ceres ANNOT ID no. 1521592、Ceres 克隆ID no. 463594、Public GI ID no. 22330633、Ceres 克隆IDno. 345954、Ceres LOCUS ID no. 0s01m05025 AP003288, GI ID no. 56784330、Public GI ID no. 125527495、PublicGI ID no. 125553119、Ceres 克隆ID no. 236431、Ceres 克隆ID no. 908518、Public GI ID no. 115465121、Ceres 克隆ID no. 1791910、Public GI ID no. 125595019、Public GI IDno. 42568886、Public GI ID no. 2947062、Ceres ANNOT ID no. 1468228、Ceres 克隆ID no. 1942388、Public GI ID no. 12324824、Public GI IDno. 5882749 和Ceres 克隆ID no. 325403(SEQ ID NO :2、4、6、8、9、11、13、14、15、17、19、20、22、23、24、25、27、29、30、31 和33)。 Such polypeptides include Ceres CLONE IDno. 1792354, Ceres Clone ID no. 1925477, Ceres ANNOT ID no. 1521592, Ceres Clone ID no. 463594, Public GI ID no. 22330633, Ceres clone IDno. 345954, Ceres LOCUS ID no. 0s01m05025 AP003288, GI ID no. 56784330, Public GI ID no. 125527495, PublicGI ID no. 125553119, Ceres clone ID no. 236431, Ceres clone ID no. 908518, Public GI ID no. 115465121, Ceres clone ID no. 1791910, Public GI ID no. 125595019, Public GI IDno. 42568886, Public GI ID no. 2947062, Ceres ANNOT ID no. 1468228, Ceres clone ID no. 1942388, Public GI ID no. 12324824, Public GI IDno. 5882749 and Ceres cLONE ID no . 325403 (SEQ ID NO: 2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31 and 33).

[0103] 序列表中提供了与图6中所示的氨基酸序列产生的HMM拟合时具有大于550、600、 650或700的HMM 二进制值的多肽。 [0103] Sequence Listing provides the polypeptide having greater than 550,600, HMM binary value 650 or 700 when fitted to an HMM generated with the amino acid sequence shown in FIG. 这类多肽包括CeresGI ID no. 56784328、Public GI ID no. 56784330、Public GI IDno. 125528718、Public GI ID no.125572975 和Public GI ID no. 125528716(SEQ ID NO :35、36、37、38 和39)。 Such polypeptides include CeresGI ID no 56784328, Public GI ID no 56784330, Public GI IDno 125528718, Public GI ID no.125572975, and Public GI ID no 125528716 (SEQ ID NO:.... 35,36,37,38, and 39 ).

[0104] P.同一件百分比 [0104] P. with a percentage

[0105] 在一些实施方案中,盐度和/或氧化胁迫耐性调控多肽具有与SEQ IDNO :2、4、6、 8、9、11、13、14、15、17、19、20、22、23、24、25、27、29、30、31、33、35、36、37、38、39、41、42、43、 44、45、47、49、50、52、54、56、58、60、62、63、64、66、68、69、71、73、74、76、78、80、81、83、84、 86、88、90、91、93、94、96、98、100、101、102、104、106、107、109、110、112、114、116、118、119、 121、122、123、125、126、127、128、129、130、132、134、136、138、140、141、142、143、144、145、 147、149、151、153、154、156、158、160、162、163、165、166、167、168 和SEQ ID NO :140 的氨基酸等同物1到135中所示的氨基酸序列具有至少50%的序列同一性的氨基酸序列,例如50%、52%、56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99% 的序列同一性的氨基酸序列。 [0105] In some embodiments, salinity and / or oxidative stress tolerance-modulating polypeptide has SEQ IDNO: 2,4,6, 8,9,11,13,14,15,17,19,20,22, 23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43, 44,45,47,49,50,52,54,56,58, 60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84, 86,88,90,91,93,94,96,98,100, 101,102,104,106,107,109,110,112,114,116,118,119, 121,122,123,125,126,127,128,129,130,132,134,136,138, 140,141,142,143,144,145, 147,149,151,153,154,156,158,160,162,163,165,166,167,168 and SEQ ID NO: 1 equivalents of amino acid 140 the 135 amino acid sequence identity to an amino acid sequence having at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 97%, 98%, or an amino acid sequence 99% sequence identity. 具有这类序列同一性百分比的多肽通常具有盐度和/或氧化胁迫调控多肽的特征性结构域和/或具有大于20的HMM 二进制值,如上文所述。 Polypeptide having a percentage identity of such sequences usually have a salinity and / or oxidative stress-modulating polypeptide domain characteristic and / or HMM bit score greater than 20, as described above. 图1-6中提供了与SEQ ID NO :2、4、6、8、9、11、13、14、15、17、19、20、22、23、24、25、27、29、30、31、33、35、 36、37、38、39、41、42、43、44、45、47、49、50、52、54、56、58、60、62、63、64、66、68、69、71、73、 74、76、78、80、81、83、84、86、88、90、91、93、94、96、98、100、101、102、104、106、107、109、110、 112、114、116、118、119、121、122、123、125、126、127、128、129、130、132、134、136、138、140、 141、142、143、144、145、147、149、151、153、154、156、158、160、162、163、165、166、167、168 和SEQ ID NO :140的氨基酸等同物1到135中所示的氨基酸序列之一具有至少85%序列同一性的盐度和/或氧化胁迫耐性调控多肽的氨基酸序列的实例。 FIG 1-6 is provided with SEQ ID NO: 2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30, 31, 33, 36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68, 69,71,73, 74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109, 110, 112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140, ​​141,142,143,144,145, 147,149,151,153,154,156,158,160,162,163,165,166,167,168 and SEQ ID NO: 1 and illustrated in one of the 135 amino acid sequence having at least 140 equivalents example 85% sequence identity to salinity and / or amino acid sequence of oxidative stress tolerance-modulating polypeptide.

[0106] “序列同一性百分比”是指任何给定的参照序列和候选的盐度和/或氧化胁迫调控序列之间的序列同一性程度,所述参照序列例如为SEQ ID NO :2、4、6、8、9、11、13、14、15、 17、19、20、22、23、24、25、27、29、30、31、33、35、36、37、38、39、41、42、43、44、45、47、49、50、 52、54、56、58、60、62、63、64、66、68、69、71、73、74、76、78、80、81、83、84、86、88、90、91、93、 94、96、98、100、101、102、104、106、107、109、110、112、114、116、118、119、121、122、123、125、 126、127、128、129、130、132、134、136、138、140、141、142、143、144、145、147、149、151、153、 154、156、158、160、162、163、165、166、167、168 和SEQ ID NO :140 的氨基酸等同物1 到135。 [0106] "Percentage of sequence identity" refers to any given reference sequence and a candidate salinity and / or oxidative stress-modulating the degree of sequence identity between the sequences, the reference sequence, for example SEQ ID NO: 2,4 , 6,8,9,11,13,14,15, 17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41 , 42,43,44,45,47,49,50, 52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81 , 83,84,86,88,90,91,93, 94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122 , 123, 125, 126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153, 154,156, 158,160 , 162,163,165,166,167,168, and SEQ ID NO: 140 amino acids 1 to 135 equivalents. 候选序列通常具有参照序列长度80%到200%的长度,例如参照序列长度的82%、85%、 87%,89%,90%,93%,95%,97%,99% ,100% ,105% ,110% ,115% ,120% ,130% ,140%, 150 % ,160% ,170%, 180 %、190 %、或200 %。 Candidate sequence typically has a length of the reference sequence length of 80% to 200%, for example, reference to 82% of the sequence length, 85%, 87%, 89%, 90%, 93%, 95%, 97%, 99%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. 可如下测定任何候选核酸或多肽相对于参照核酸或多肽的同一性百分比。 It may be determined as follows any candidate nucleic acid or polypeptide relative to the percent identity of the reference nucleic acid or polypeptide. 使用计算机程序Clustaiwa. 83版,默认参数)将参照序 Computer program Clustaiwa. 83 Edition, default parameters) with reference to the sequence

20列(例如核酸序列或氨基酸序列)与一个或多个候选序列比对,所述计算机程序ClustalW 允许在核酸或多肽序列的整个长度上进行比对(全局比对)。 20 (e.g., a nucleic acid or amino acid sequence) with one or more candidate sequence alignments allow the computer program ClustalW alignment (global alignment) over the entire length of the nucleic acid or polypeptide sequences. Cherma等、Nucleic Acids Res. ,31(13) :3497_500 (2003)。 Cherma etc., Nucleic Acids Res, 31 (13):. 3497_500 (2003).

[0107] Clustalff计算参照序列和一个或多个候选序列之间的最佳匹配,并对它们进行比对,从而能够测定同一性、相似性和差异。 [0107] Clustalff calculates the best match between the reference sequence and one or more candidate sequences and comparing them, it is possible to determine identity, similarities and differences. 可以向参照序列、候选序列或该二者中插入一个或多个残基的缺口,以最大化序列比对。 It may be insertion of one or more residues of the reference sequence, a candidate sequence, or both of the notch, to maximize sequence alignments. 对核酸序列的快速配对比对而言,使用以下的默认参数:字段长度(wordsize) :2 ;窗口尺寸:4 ;记分法:百分比;顶部对角线数(number of topdiagonal) :4;和缺口罚分:5。 Fast pairwise alignment of nucleic acid sequences, the following default parameters: Field Length (wordsize): 2; window size: 4; scoring method: percentage; top number (number of topdiagonal) diagonals: 4; and a notch penalty: 5. 对于核酸序列的多重比对而言,使用以下的参数:缺口开放罚分:10.0 ;缺口延伸罚分:5.0 ;权重转换:是。 For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; weight conversion: Yes. 对于蛋白质序列的快速配对比对而言,使用以下的参数:字段长度:1 ;窗口尺寸:5 ;记分法:百分比;顶部对角线数:5 ;和缺口罚分:3。 For fast pairwise alignment of protein sequences, the following parameters: Field Length: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; and a gap penalty: 3. 对于蛋白质序列的多重比对而言,使用以下的参数:权重矩阵(weight matrix): blosum ;缺口开放罚分:10. 0 ;缺口延伸罚分:0. 05 ;亲水缺口:开;亲水残基Gly、Pro、Ser、 Asn、Asp、Gln、Glu、Arg和Lys ;残基特异的缺口罚分:开。 For multiple alignment of protein sequences, the following parameters: weight matrix (weight matrix): blosum; gap opening penalty: 100; gap extension penalty: 0.05; hydrophilic gaps: on; Hydrophilic residues Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. ClustalW输出的是反映序列之间相互关系的序列比对。 ClustalW output is a reflection of the relationship between the sequences of sequence. ClustalW可以在万维网上的例如Baylor College of Medicine Search Launcher 网页(searchlauncher. bcm. tmc. edu/multi-align/multi-align. html) 上禾口EuropeanBioinformatics Institute 网页(ebi.ac.uk/clustalw)上运ίΐ。 ClustalW can be run, for example, Baylor College on the World Wide Web page of Medicine Search Launcher (searchlauncher. Bcm. Tmc. Edu / multi-align / multi-align. Html) transported on Wo port EuropeanBioinformatics Institute website (ebi.ac.uk/clustalw) ίΐ.

[0108] 为了测定候选核酸或氨基酸序列与参照序列的同一性百分比,使用ClustalW比对序列,用比对中相同匹配的数量除以参照序列的长度,并将结果乘以100。 [0108] To determine percent identity of a candidate nucleic acid or amino acid sequence to a reference sequence, sequence alignment using ClustalW with the number of identical matches in the alignment is divided by the length of the reference sequence, and multiplying the result by 100. 注意可以将同一性百分比数值估值为最接近的十分之一。 Note that the percentage of identity as one can estimate the value nearest tenth. 例如,可以将78. 11,78. 12,78. 13和78. 14估值为78. 1,将78. 15,78. 16,78. 17,78. 18 和78. 19 估值为78. 2。 For example, 78. 11,78. 12,78. 13 and 78.14 to 78.1 valuation may be the 78. 15,78. 16,78. 17,78. 18 and 78 is valued 78.19 . 2.

[0109] 在一些情况下,盐度和/或氧化胁迫耐性调控多肽具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :86中所示的一种或多种氨基酸序列具有至少50%的序列同一性, 例如50%、52%、56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99%的序列同一性。 [0109] In some cases, salinity and / or oxidative stress tolerance-modulating polypeptide has an amino acid sequence, the amino acid sequence SEQ ID NO: of one or more amino acid sequence shown in at least 50% of the 86 sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity. 序列表中提供了与SEQ ID NO :86中所示的多肽具有高度序列同一性的多肽的氨基酸序列。 It provides the sequence listing SEQ ID NO: 86 polypeptide shown in amino acid sequence of the polypeptide having a high degree of sequence identity. 这类多肽包括SEQ ID NO :88、90、91、93、94、96、98、100、101、102、104、 106 和107。 Such polypeptides include SEQ ID NO: 88,90,91,93,94,96,98,100,101,102,104, 106 and 107.

[0110] 在一些情况下,盐度和/或氧化胁迫耐性调控多肽具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :41中所示的氨基酸序列具有至少50%的序列同一性,例如50%、 52%、56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99% 的序列同一性。 [0110] In some cases, salinity and / or oxidative stress tolerance-modulating polypeptide has an amino acid sequence, the amino acid sequence SEQ ID NO: 41 amino acid sequence shown in at least 50% sequence identity, e.g. 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity. 序列表中提供了与SEQ ID Ν0:41中所示的多肽具有高度序列同一性的多肽的氨基酸序列。 It provides the sequence listing SEQ ID Ν0: 41 polypeptide shown in amino acid sequence identity to the polypeptide sequence of height. 这类多肽包括SEQ ID NO :42、43、44、45、47、49、50、52、54、56、58、60、62、63、64、 66、68、69、71、73、74、76、78、80、81、83 和84。 Such polypeptides include SEQ ID NO: 42,43,44,45,47,49,50,52,54,56,58,60,62,63,64, 66,68,69,71,73,74, 76,78,80,81,83 and 84.

[0111] 在一些情况下,盐度和/或氧化胁迫调控多肽具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :109中所示的氨基酸序列具有至少50%的序列同一性,例如50%、52%、 56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99% 的序列同一性。 [0111] In some cases, salinity and / or oxidative stress-modulating polypeptide has an amino acid sequence, the amino acid sequence SEQ ID NO: 109 amino acid sequence shown in at least 50% sequence identity, e.g., 50 %, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity. 序列表中提供了与SEQ ID NO :109中所示的多肽具有高度序列同一性的多肽的氨基酸序列。 It provides the sequence listing SEQ ID NO: 109 polypeptide shown in amino acid sequence of the polypeptide having a high degree of sequence identity. 这类多肽包括SEQ ID NO :110、112、114、116、118、119、121、122、123、125、126、127、 128、129、130、132 和134。 Such polypeptides include SEQ ID NO: 110,112,114,116,118,119,121,122,123,125,126,127, 128,129,130,132, and 134.

[0112] 在一些情况下,盐度和/或氧化胁迫调控多肽具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :140中所示的氨基酸序列具有至少50%的序列同一性,例如50%、52%、 56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99% 的序列同一性。 [0112] In some cases, salinity and / or oxidative stress-modulating polypeptide has an amino acid sequence, the amino acid sequence SEQ ID NO: 140 amino acid sequence shown in at least 50% sequence identity, e.g., 50 %, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity. 序列表中提供了与SEQ ID NO :140中所示的多肽具有高度序列同一性的多肽的氨基酸序列。 It provides the sequence listing SEQ ID NO: 140 polypeptide shown in amino acid sequence of the polypeptide having a high degree of sequence identity. 这类多肽包括SEQ ID NO :136、138、141、142、143、144、145、147、149、151、153、154、 156、158、160、162、163、165、166、167 和168。 Such polypeptides include SEQ ID NO: 136,138,141,142,143,144,145,147,149,151,153,154, 156,158,160,162,163,165,166,167 and 168.

[0113] 在一些情况下,盐度和/或氧化胁迫调控多肽具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :2中所示的氨基酸序列具有至少50%的序列同一性,例如50%、52%、 56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99% 的序列同一性。 [0113] In some cases, salinity and / or oxidative stress-modulating polypeptide has an amino acid sequence, the amino acid sequence SEQ ID NO: 2 amino acid sequence shown in at least 50% sequence identity, e.g., 50 %, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity. 序列表中提供了与SEQ ID NO :2中所示的多肽具有高度序列同一性的多肽的氨基酸序列。 It provides the sequence listing SEQ ID NO: 2 polypeptide shown in amino acid sequence of the polypeptide having a high degree of sequence identity. 这类多肽包括SEQ ID NO :4、6、8、9、11、13、14、15、17、19、20、22、23、24、25、27、29、30、 31 禾口33。 Such polypeptides include SEQ ID NO: 4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30, 31 Wo port 33.

[0114] 在一些情况下,盐度和/或氧化胁迫调控多肽具有下述氨基酸序列,所述氨基酸序列与SEQ ID NO :35中所示的氨基酸序列具有至少50%的序列同一性,例如50%、52%、 56%、59%、61%、65%、70%、75%、80%、85%、90%、95%、97%、98% 或99% 的序列同一性。 [0114] In some cases, salinity and / or oxidative stress-modulating polypeptide has an amino acid sequence, the amino acid sequence SEQ ID NO: 35 amino acid sequence shown in at least 50% sequence identity, e.g., 50 %, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity. 序列表中提供了与SEQ ID NO :35中所示的多肽具有高度序列同一性的多肽的氨基酸序列。 It provides the sequence listing SEQ ID NO: 35 shown in polypeptide having the amino acid sequence of the polypeptide of the high degree of sequence identity. 这类多肽包括SEQ ID NO :36、37、38和39。 Such polypeptides include SEQ ID NO: 36,37,38 and 39.

[0115] E.其他序列 [0115] E. Other sequences

[0116] 应当明白,盐度和/或氧化胁迫耐性调控多肽可包括不涉及盐度和/或氧化胁迫耐性调控的其他氨基酸,因此这类多肽可能比其他情况下更长。 [0116] It should be appreciated, salinity and / or oxidative stress tolerance-modulating polypeptide may comprise other amino acids not involved in salinity and / or oxidative stress tolerance-modulating, and thus such a polypeptide can be longer than would otherwise be the case. 例如,盐度和/或氧化胁迫耐性调控多肽可包含添加在氨基端或羧基端的纯化标签,叶绿体转运肽、造粉体转运肽、线粒体转运肽或前导序列。 For example, salinity and / or oxidative stress tolerance-modulating polypeptide can contain added amino or carboxy terminus of a purification tag, a chloroplast transit peptide, amyloplast transit peptide, a mitochondrial transit peptide or leader sequence. 在一些实施方案中,盐度和/或氧化胁迫耐性调控多肽包含发挥报道基因功能的氨基酸序列,例如绿色荧光蛋白或黄色荧光蛋白。 In some embodiments, a salinity and / or oxidative stress tolerance-modulating polypeptide comprising the amino acid sequence of a reporter gene play function, such as green fluorescent protein or yellow fluorescent protein.

[0117] III.核酸 [0117] III. Nucleic acid

[0118] 本文所述的核酸包括在植物或植物细胞中转录时有效调控盐度和/或氧化胁迫耐性的核酸。 [0118] The nucleic acids described herein includes effectively regulate salinity and / or oxidative stress tolerance when transcribed in a plant or plant cell a nucleic acid. 这类核酸包括但不限于编码盐度和/或氧化胁迫耐性调控多肽的核酸,和能够用于通过基于核酸的方法抑制盐度耐性和/或氧化胁迫耐性调控多肽表达的核酸。 Such nucleic acids include, but are not limited to coding salinity and / or oxidative stress tolerance-modulating nucleic acid polypeptides, nucleic acids, and for salinity tolerance and / or oxidative stress tolerance-modulating polypeptide by inhibiting the expression of the nucleic acid based methods.

[0119] A. ^^^jtat^^n / ^MAmmmim.^mmm [0119] A. ^^^ jtat ^^ n / ^ MAmmmim. ^ Mmm

[0120] 本文描述了编码盐度耐性和/或氧化胁迫耐性调控多肽的核酸。 [0120] Described herein are nucleic acids encoding salinity tolerance and / or oxidative stress tolerance-modulating polypeptide. 这类核酸包括SEQ ID NO :1、3、5、7、10、12、16、18、21、26、28、32、34、40、46、48、51、53、55、57、59、61、65、 67、70、72、75、77、79、82、85、87、89、92、95、97、99、103、105、108、111、113、115、117、120、 124、131、133、135、137、139、146、148、150、152、155、157、159、161 和164,如下文更详细描述的。 Such nucleic acids include SEQ ID NO: 1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59, 61, 65, 67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120, 124, 131,133,135,137,139,146,148,150,152,155,157,159,161, and 164, as described in more detail below.

[0121] 盐度耐性和/或氧化胁迫耐性调控核酸可包含SEQ ID NO :85中所示的核苷酸序列。 [0121] salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can comprise SEQ ID NO: 85 in the nucleotide sequence shown. 或者,盐度耐性和/或氧化胁迫耐性调控核酸可以是具有SEQ ID N0:85中所示的核苷酸序列的核酸变体。 Alternatively, the salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have SEQ ID N0: nucleic acid nucleotide sequence as shown in 85 variants. 例如,盐度耐性和/或氧化胁迫耐性调控核酸可以具有下述核苷酸序列,所述核苷酸序列与SEQ IDN0 :85、87、89、92、95、97、99、103和105中所示的核苷酸序列具有至少80%的序列同一性,例如81%、85%、90%、95%、97%、98%或99%的序列同一性。 For example, salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence, said nucleotide sequence SEQ IDN0: 85,87,89,92,95,97,99,103 and 105 nucleotide sequence having at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0122] 盐度耐性和/或氧化胁迫耐性调控核酸可包含SEQ ID NO :40中所示的核苷酸序 [0122] salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can comprise SEQ ID NO: 40 is the nucleotide sequence shown in

22列。 22. 或者,盐度耐性和/或氧化胁迫耐性调控核酸可以是具有SEQ ID N0:40中所示的核苷酸序列的核酸变体。 Alternatively, the salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have SEQ ID N0: nucleotide sequence of the nucleic acid in the variant 40 as shown. 例如,盐度耐性和/或氧化胁迫耐性调控核酸可以具有下述核苷酸序列,所述核苷酸序列与SEQ IDN0 :40、46、48、51、53、55、57、59、61、65、67、70、72、75、77、79 和82中所示的核苷酸序列具有至少80%的序列同一性,例如81%、85%、90%、95%、97%、 98%或99%的序列同一性。 For example, salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence, said nucleotide sequence SEQ IDN0: 40,46,48,51,53,55,57,59,61, the nucleotide sequence shown 65,67,70,72,75,77,79 and 82 have at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0123] 盐度耐性和/或氧化胁迫耐性调控核酸可包含SEQ ID NO :108中所示的核苷酸序列。 [0123] salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can comprise SEQ ID NO: 108 in the nucleotide sequence shown. 或者,盐度耐性和/或氧化胁迫耐性调控核酸可以是具有SEQ ID N0:108中所示的核苷酸序列的核酸变体。 Alternatively, the salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have SEQ ID N0: nucleic acid nucleotide sequence as shown in 108 variants. 例如,盐度耐性和/或氧化胁迫耐性调控核酸可以具有下述核苷酸序列,所述核苷酸序列与SEQID NO : 108、111、113、115、117、120、124、131和133中所示的核苷酸序列具有至少80%的序列同一性,例如81%、85%、90%、95%、97%、98%或99%的序列同一性。 For example, salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence, said nucleotide sequence SEQID NO: 108,111,113,115,117,120,124,131 and 133 nucleotide sequence having at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0124] 盐度耐性和/或氧化胁迫耐性调控核酸可包含SEQ ID NO :139中所示的核苷酸序列。 [0124] salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can comprise SEQ ID NO: 139 in the nucleotide sequence shown. 或者,盐度耐性和/或氧化胁迫耐性调控核酸可以是具有SEQ ID N0:139中所示的核苷酸序列的核酸变体。 Alternatively, the salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have SEQ ID N0: nucleic acid nucleotide sequence as shown in 139 variants. 例如,盐度耐性和/或氧化胁迫耐性调控核酸可以具有下述核苷酸序列,所述核苷酸序列与SEQID NO :135、137、139、146、148、150、152、155、157、159、161 和164 中所示的核苷酸序列具有至少80%的序列同一性,例如81%、85%、90%、95%、97%、98% 或99%序列同一性。 For example, salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence, said nucleotide sequence SEQID NO: 135,137,139,146,148,150,152,155,157, 159, 161 and 164 nucleotide sequence having at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0125] 盐度耐性和/或氧化胁迫耐性调控核酸可包含SEQ ID NO :1中所示的核苷酸序列。 [0125] salinity tolerance and / or regulatory nucleic acid may comprise oxidative stress tolerance SEQ ID NO: 1 nucleotide sequence shown in FIG. 或者,盐度耐性和/或氧化胁迫耐性调控核酸可以是具有SEQID N0:1中所示的核苷酸序列的核酸变体。 Alternatively, the salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have SEQID N0: nucleic acid nucleotide sequence as shown in a variant. 例如,盐度耐性和/或氧化胁迫耐性调控核酸可以具有下述核苷酸序列,所述核苷酸序列与SEQ ID NO :1、3、5、7、10、12、16、18、21、26、28和32中所示的核苷酸序列具有至少80%的序列同一性,例如81%、85%、90%、95%、97%、98%或99%的序列同一性。 For example, salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence, said nucleotide sequence SEQ ID NO: 1,3,5,7,10,12,16,18,21 , 26, 28 and 32 in the nucleotide sequence having at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0126] 盐度耐性和/或氧化胁迫耐性调控核酸可包含SEQ ID NO :34中所示的核苷酸序列。 [0126] salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can comprise SEQ ID NO: 34 in the nucleotide sequence shown. 或者,盐度耐性和/或氧化胁迫耐性调控核酸可以是具有SEQ ID N0:34中所示的核苷酸序列的核酸变体。 Alternatively, the salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have SEQ ID N0: nucleic acid variants of the nucleotide sequence shown in FIG 34. 例如,盐度耐性和/或氧化胁迫耐性调控核酸可以具有下述核苷酸序列,所述核苷酸序列与SEQ IDN0 :34中所示的核苷酸序列具有至少80%的序列同一性,例如81%、85%、90%、95%、97%、98%或99% 的序列同一性。 For example, salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence, said nucleotide sequence SEQ IDN0: 34 nucleotide sequence having at least 80% sequence identity, For example, 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

[0127] 可以使用标准技术产生分离的核酸分子。 [0127] The isolated nucleic acid molecule may be produced using standard techniques. 例如,可以使用聚合酶链式反应(PCR) 技术获得含有本文所述核苷酸序列的分离的核酸。 For example, using polymerase chain reaction (PCR) techniques to obtain an isolated nucleic acid comprising the nucleotide sequence described herein. 可以使用PCR从DNA以及RNA(包括来自总基因组DNA或总细胞RNA的序列)中扩增特定的序列。 PCR can be used to amplify specific sequences from DNA as well as RNA (including sequences from total genomic DNA or total cellular RNA). 多种PCR方法描述于例如PCR Primer :A LaboratoryManual, Dieffenbach and Dveksler 编,Cold Spring Harbor LaboratoryPress, 1995中。 Various PCR methods described, for example PCR Primer: A LaboratoryManual, Dieffenbach and Dveksler eds, Cold Spring Harbor LaboratoryPress, 1995 in the. 通常,使用来自目的区域末端或之外的序列信息设计寡核苷酸引物,所述引物在序列上与待扩增的模板的相对链相同或相似。 Typically, oligonucleotides are designed using sequence information from the region of interest or beyond the end of oligonucleotide primers, said primers identical or similar in sequence to opposite strands of the template to be amplified. 还可以获得多种PCR策略, 通过所述策略可以将位点特异性核苷酸序列修饰引入模板核酸中。 Multiple PCR strategies may also be obtained, site-specific nucleotide sequence modifications can be introduced into a template nucleic acid by the policy. 还可以化学合成分离的核酸,例如作为单一核酸分子合成(例如使用亚磷酰胺技术,在3'到5'方向上使用自动化DNA合成)或作为一系列寡核苷酸合成。 It can also be synthesized chemically isolated nucleic acids, e.g., as a single molecule of nucleic acid synthesis (e.g. phosphoramidite technique, using an automated DNA synthesis in the 3 'to 5' direction), or as a series of oligonucleotide synthesis. 例如,可以合成含有预期序列的一对或多对长寡核苷酸(例如> 100个核苷酸),每个对含有短的互补性区段(例如约15个核苷酸),使得寡核苷酸对退火时形成双链体。 For example, one or more pairs may be synthesized long oligonucleotides (e.g.,> 100 nucleotides) containing the expected sequence for each segment of complementarity (e.g., about 15 nucleotides) containing a short, such that oligonucleotide nucleotide duplexes formed during annealing. 使用DNA聚合酶延伸寡核苷酸,每个寡核苷酸对得到单一的、双链的核酸分子,所述核酸分子随后可与载体连接。 Using a DNA polymerase extension oligonucleotides, each oligonucleotide to obtain a single, double-stranded nucleic acid molecule, the nucleic acid molecule may then be linked to the carrier. 也可以通过诱变例如天然存在的DNA来获得本发明的分离的核酸。 It may be obtained according to the invention the isolated nucleic acid, for example, by mutagenesis of a naturally-occurring DNA.

[0128] B.核酸调控多肽表汰的用涂 [0128] B. regulatory nucleic acid coated with a polypeptide table jig

[0129] i.舰讀禾口/贿碰i白讀膽碰白腫汰 [0129] i. I read port ship / i White bribe touch touch reading bladder elimination swollen white

[0130] 多肽 [0130] polypeptide

[0131] 通常通过用下述核酸转化植物细胞,用编码本文所述盐度耐性和/或氧化胁迫耐性调控多肽的核酸在目的植物物种中表达多肽,所述核酸具有以有义方向与一个或多个调节区有效连接的编码多肽的序列。 [0131] typically by transforming a plant cell with a nucleic acid, as described herein encoding a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide nucleic acid is expressed in the plant species of the polypeptide, the nucleic acid having a sense orientation or with one or sequence encoding the polypeptide operably linked to regulatory regions of the plurality. 应当明白,由于遗传密码子的简并性,大量核酸可编码一个特定的盐度耐性和/或氧化胁迫耐性调控多肽,即对许多氨基酸而言,存在多于一种发挥该氨基酸密码子作用的核苷酸三联体。 It should be appreciated that due to the degeneracy of the genetic code, a large number of nucleic acid can encode a particular salinity tolerance and / or oxidative stress tolerance-modulating polypeptide, i.e., for many amino acids, there is more than one amino acid codon play the role of nucleotide triplet. 因此,可以使用针对具体植物物种的合适密码子偏好表,修饰给定盐度耐性和/或氧化胁迫耐性调控多肽的编码序列中的密码子,从而获得所述植物物种中的最优表达。 Thus, using appropriate codon bias tables for specific plant species, to modify a given salinity tolerance and / or oxidative stress tolerance-modulating sequence encoding a polypeptide codon, so as to obtain an optimal expression of the plant species.

[0132] 在一些情况下,盐度耐性和/或氧化胁迫耐性调控多肽的表达抑制内源多肽的一种或多种功能。 [0132] In some cases, or expression of a salinity tolerance and / oxidative stress tolerance-modulating polypeptide inhibits one or more functions of an endogenous polypeptide. 例如,可以使用编码显性负相多肽的核酸抑制蛋白质功能。 For example, a nucleic acid encoding a dominant negative polypeptide inhibit protein function. 显性负相多肽通常相对于内源野生型多肽而言是突变或截短的,并且其在细胞中的存在抑制细胞中野生型多肽的一种或多种功能,即显性负相多肽是遗传显性的,并且赋予功能的丧失。 Dominant negative polypeptide typically relative to an endogenous wild-type polypeptides is a mutant or truncated, and its presence in a cell of wild-type polypeptide inhibits one or more functions of a cell, i.e. a dominant negative polypeptide is genetically dominant, and the loss given function. 显性负相多肽赋予这种表型的机制可以变化,但是常常涉及蛋白质-蛋白质相互作用或蛋白质-DNA相互作用。 Such dominant negative polypeptide imparting mechanism phenotype can vary but often involves a protein - protein interaction or protein -DNA interaction. 例如,显性负相多肽可以是相对于固有野生型酶被截短的酶,从而截短的多肽保留涉及与第一蛋白质结合的结构域但是缺乏涉及与第二蛋白质结合的结构域。 For example, a dominant negative polypeptide can be an enzyme relative to wild type enzyme inherent in a truncated, so that the truncated polypeptide retains binding domains relates to the first protein but lacks domains and the second relates to protein binding. 因此,截短的多肽不能正确地调控第二蛋白质的活性。 Thus, a truncated polypeptide is not properly regulated activity of the second protein. 见例如US 2007/0056058。 See for example, US 2007/0056058. 作为另一个例子,催化结构域中导致非保守氨基酸替代的点突变能够导致显性负相多肽。 As another example, the catalytic domain resulting in non conservative amino acid substitution point mutation can result in a dominant negative polypeptide. 见例如US 2005/032221。 See, for example US 2005/032221. 作为另一个例子,显性负相多肽可以是相对于固有的野生型转录因子被截短的转录因子,从而截短的多肽保留一个或多个DNA结合结构域但是缺乏一个或多个活化结构域。 As another example, a dominant negative polypeptide can be relative to the wild-type transcription factor inherent truncated transcription factors, such truncated polypeptides retain one or more DNA binding domains but lack the activation domain with one or more . 这类截短的多肽能够抑制野生型转录因子与DNA结合,从而抑制转录活化。 Such a truncated polypeptide capable of inhibiting wild-type transcription factor binding to DNA, thereby inhibiting transcription activation.

[0133] ii.抑制盐度耐性和/或氧化胁迫耐性调控多肽的表达 [0133] ii. Inhibiting expression of a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide

[0134] 本文所述的多核苷酸和重组构建体可用于抑制目的植物物种中盐度耐性和/或氧化胁迫耐性调控多肽的表达。 [0134] As used herein and recombinant polynucleotide constructs can be used for the purpose of inhibiting the expression of plant species salinity tolerance and / or oxidative stress tolerance-modulating polypeptide. 见例如nature, com/reviews/focus/mai上的Matzke和Birchler, Nature ReviewsGenetics 6 :24~35 (2005) ;Akashi 等,Nature Reviews Mol. Cell BioloRy6 :413~422(2005) ;Mittal, Nature Reviews Genetics 5:355-365(2004); Dorsett 禾口Tuschl, Nature Reviews PruR Discovery 3 :318_329(2004);禾口Nature Reviews RNA interference collection, 2005年10月。 See, e.g., Matzke and Birchler on the nature, com / reviews / focus / mai, Nature ReviewsGenetics 6: 24 ~ 35 (2005); Akashi et, Nature Reviews Mol Cell BioloRy6:. 413 ~ 422 (2005); Mittal, Nature Reviews Genetics 5: 355-365 (2004); Dorsett Wo mouth Tuschl, Nature Reviews PruR Discovery 3: 318_329 (2004); Wo mouth Nature Reviews RNA interference collection, in October 2005. 已知大量基于核酸抑制植物中的基因表达,所述方法包括反义RNA、核酶定向RNA切割、转录后基因沉默(PTGS)的方法,例如RNA干扰(RNAi)和转录基因沉默(TGS)。 Based on a large number of nucleic acids are known to inhibit gene expression in plants, the method comprising antisense RNA, ribozyme directed RNA cleavage, post-transcriptional gene silencing (PTGS) method, such as RNA interference (RNAi), and transcriptional gene silencing (TGS). 反义技术是一种公知的方法。 Antisense technology is one well-known method. 在这种方法中,将来自要阻抑的基因的核酸区段克隆并与调节区和转录终止序列有效连接,使得RNA的反义链被转录。 In this method, from the gene to be repressed is cloned and operably linked to a nucleic acid segment to a regulatory region and a transcription termination sequence, so that the antisense strand of RNA is transcribed. 然后将重组构建体转化进植物中,如本文所述,并产生RNA的反义链。 The recombinant construct is then transformed into plants, as described herein, and the antisense strand of RNA. 核酸区段不需要是要阻抑的基因的整个序列,但是通常应与要阻抑的基因有义链的至少一部分基本互补。 Nucleic acid segment need not be the entire sequence of the gene to be repressed, but typically should have the gene to be repressed substantially complementary to at least a portion of the sense strand. 通常,可以使用较高的同源性来补偿较短序列的使用。 Generally, higher homology can be used to compensate for the use of a shorter sequence. 通常使用至少30个核苷酸的序列,例如至少40、50、80、100、200、500个核苷酸或更多。 Usually at least 30 nucleotides of the sequence, e.g. at least 40,50,80,100,200,500 nucleotides or more.

[0135] 在另一种方法中,可以将核酸转录为影响mRNA表达的核酶或催化性RNA。 [0135] In another approach, a nucleic acid can be transcribed into a ribozyme, or catalytic RNA affect the expression of mRNA. 见美国专利No. 6,423,885。 See US Patent No. 6,423,885. 核酶可以被设计为实际上与任何靶RNA特异性配对,并在特异性位置上切割磷酸二酯主链,从而使靶RNA功能性失活。 Ribozymes can be designed to specifically pair with practically any target RNA, and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. 异源核酸能够编码被设计为切割具体mRNA转录物的核酶,从而预防多肽的表达。 Heterologous nucleic acid can encode ribozymes designed to be specific cleavage of mRNA transcripts, thus preventing expression of a polypeptide. 锤头状核酶可用于破坏具体的mRNA,尽管可以使用在位点特异性识别序列上切割mRNA的多种核酶。 Hammerhead ribozymes may be used to destroy specific mRNA, although using site-specific recognition sequences on various ribozymes that cleave mRNA. 锤头状核酶在下述侧翼区指定的位置切割mRNA,所述侧翼区与靶mRNA形成互补的碱基对。 Hammerhead ribozyme flanking region specified in the following position of the cutting mRNA, complementary to flanking regions of the base is formed with the target mRNA pair. 唯一的要求是靶RNA含有5'_UG_3' 核苷酸序列。 The only requirement is that the target RNA containing the 'nucleotide sequence 5'_UG_3. 锤头状核酶的构建和产生是本领域已知的。 Construction and production of hammerhead ribozymes is known in the art. 见例如美国专利No. 5,254,678 和TO 02/46449及其中引用的参考文献。 See, for example, references cited 02/46449 and in U.S. Pat. No. 5,254,678 and TO. 锤头状核酶序列可以被植入稳定的RNA例如转运RNA(tRNA)中,以提高体内切割效率。 Hammerhead ribozyme sequences can be implanted, for example, it stabilized RNA transport RNA (tRNA) in order to increase cleavage efficiency in vivo. Perriman 等,Proc.Natl.Acad.Sci.USA,92(13): 6175-6179(1995) ;de Feyter 和Gaudron,Methods inMolecular Biology,第74 卷,第43 章,"Expressing Ribozymes in Plants,,,Turner,PC编著,Humana Press Inc. ,Totowa, NJ.已描述的RNA内切核糖核酸酶例如嗜热四膜虫(Tetrahymena thermophila)中天然存在的RNA内切核糖核酸酶可以是有用的。见例如美国专利No. 4,987,071和6,423,885。 Perriman, etc., Proc.Natl.Acad.Sci.USA, 92 (13): 6175-6179 (1995); de Feyter and Gaudron, Methods inMolecular Biology, Vol. 74, Chapter 43, "Expressing Ribozymes in Plants ,,, Turner, PC, ed., Humana Press Inc., Totowa, NJ. endoribonuclease RNA have been described e.g. endoribonuclease Tetrahymena thermophila (Tetrahymena thermophila) naturally occurring RNA may be useful. see e.g. US Patent No. 4,987,071 and 6,423,885.

[0136] PTGS例如RNAi也可以用于抑制基因的表达。 [0136] PTGS, for example, RNAi may be used to inhibit gene expression. 例如,可以制备包含以下序列的构建体,所述序列被转录为能够自身退火的RNA,例如具有茎-环结构的双链RNA。 For example, constructs may be prepared comprising the following sequence, it is transcribed into an RNA capable of self-annealing of the sequences, e.g. having a stem - loop structure of the double-stranded RNA. 在一些实施方案中,双链RNA茎部分的一条链包含下述序列,所述序列与盐度耐性和/或氧化胁迫耐性调控多肽的有义编码序列相似或相同并且长度为约10个核苷酸到约2,500个核苷酸。 In some embodiments, one strand of a double stranded RNA stem portion comprising the sequence that salinity tolerance and / or oxidative stress tolerance-modulating polypeptide has similar or identical to the sense coding sequence and a length of about 10 nucleotides acid to about 2,500 nucleotides. 与有义编码序列相似或相同的序列的长度可以从10个核苷酸到500个核苷酸,从15个核苷酸到300个核苷酸,从20个核苷酸到100个核苷酸,或从25个核苷酸到100个核苷酸。 With similar or identical to the sense coding sequence of the sequence length from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides acid, or from 25 nucleotides to 100 nucleotides. 双链RNA茎部分的另一条链包含下述序列,所述序列与盐度耐性和/或氧化胁迫耐性调控多肽编码链的反义链相似或相同并且可以具有比有义序列相应长度更短、相同或更长的长度。 Other strand of the stem portion of a double stranded RNA comprising the sequence, and may have a similar or identical shorter than the corresponding length of the sequence and the sense sequence of a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide coding strand antisense strand, the same length or longer. 在一些情况下,双链RNA茎部分的一条链包含与编码盐度耐性和/或氧化胁迫耐性调控多肽的mRNA的3'或5'非翻译区相似或相同的序列,并且双链RNA茎部分的另一条链包含分别与编码盐度耐性和/或氧化胁迫耐性调控多肽的mRNA 3'或5'非翻译区的互补序列相似或相同的序列。 In some cases, one strand of a double stranded RNA stem portion of the mRNA encoding the polypeptide comprising a salinity tolerance and / or oxidative stress tolerance-modulating the 3 'or 5' untranslated region of similar or identical sequences, and the double stranded RNA stem portion each of the other chain comprises encoding salinity tolerance and / or oxidative stress tolerance-modulating polypeptide mRNA 3 'or 5' untranslated region of the complementary sequences similar or identical sequences. 在其他实施方案中,双链RNA茎部分的一条链包含与编码盐度耐性和/或氧化胁迫耐性调控多肽的前-mRNA中内含子序列相似或相同的序列,并且茎部分的另一条链包含与前-mRNA中内含子序列的互补序列相似或相同的序列。 In other embodiments, the front -mRNA intron sequence one strand of a double stranded RNA stem portion comprises encoding a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide sequence is similar or identical, and the other strand of the stem portion comprising a sequence similar or identical to the front -mRNA complementary intron sequence. 双链RNA的环部分可以是3个核苷酸到5,000个核苷酸,例如3个核苷酸到25个核苷酸、15个核苷酸到1,000个核苷酸、20个核苷酸到500个核苷酸或25个核苷酸到200个核苷酸。 The loop portion of a double stranded RNA can be from 3 nucleotides to 5,000 nucleotides, e.g. 3 nucleotides to 25 nucleotides, 15 nucleotides to 1,000 nucleotides, 20 nucleotides to 500 nucleotides, or 25 nucleotides to 200 nucleotides. RNA的环部分可包含内含子。 RNA loop portion may comprise an intron. 双链RNA可具有0、1、2、3、4、5、6、7、8、9、10或更多个茎-环结构。 Or may have a double-stranded RNA 0,1,2,3,4,5,6,7,8,9,10 more stem - loop structure. 将包含下述序列的构建体转化进本文所述的植物中,所述序列与调节区和转录终止序列有效连接并且被转录为能够形成双链RNA的RNA。 The sequences comprising the construct transformed into plants as described herein, the sequence and a regulatory region operably linked to a transcription termination sequence is transcribed into RNA and is capable of forming a double-stranded RNA. 使用RNAi抑制基因表达的方法是本领域技术人员已知的。 Use of RNAi to suppress gene expression are known to the skilled person. 见例如美国专利5,034,323 ;6,326,527 ;6,452,067 ;6,573,099 ;6,753,139 ; 和6,777,588。 See, e.g., U.S. Patent No. 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. 还见W0 97/01952 ;W0 98/53083 ;W0 99/32619 ;W0 98/36083 ;和美国专利公开20030175965, 20030175783,20040214330 和20030180945。 See also W0 97/01952; W0 98/53083; W0 99/32619; W0 98/36083; and U.S. Patent Publication 20030175965, 20030175783,20040214330, and 20030180945.

[0137] 也可以使用下述构建体抑制基因的表达,所述构建体含有以有义方向与核酸分子有效连接的调节区。 [0137] may also be used to construct expression inhibition of the gene, the construct contains a regulatory region in the sense orientation operably linked to a nucleic acid molecule described below. 转录产物可以与盐度耐性和/或氧化胁迫耐性调控多肽的有义编码序列相似或相同。 Polypeptide transcription product can be salinity tolerance and / or oxidative stress tolerance-modulating sense coding sequence similar or identical. 转录产物也可以是未聚腺苷酸化的、缺乏5'帽结构或含有不可剪接的内含子。 Transcription product may be non-polyadenylated, they lack a 5 'cap structure, or contain non-splicing intron. 使用全长cDNA以及部分cDNA序列抑制基因表达的方法是本领域已知的。 Full-length cDNA and the partial cDNA sequence to inhibit gene expression are known in the art. 见例如美国专利No. 5,231,020。 See, for example, US Patent No. 5,231,020.

25[0138] 在一些实施方案中,用含有下述核酸的构建体抑制基因的表达,所述核酸有至少一条链是彼此互补的有义和反义序列的模板。 25 [0138] In some embodiments, a construct containing a nucleic acid inhibiting the expression of a gene, said nucleic acid having at least one strand are complementary to each other template sense and antisense sequences. 有义和反义序列可以是更大核酸分子的部分,或者可以是具有不互补序列的单独核酸分子的部分。 The sense and antisense sequences can be part of a larger nucleic acid molecule, or may be part of a single nucleic acid molecule having a sequence not complementary. 有义或反义序列可以是与编码盐度耐性和/或氧化胁迫耐性调控多肽的mRNA序列、mRNA的3'或5'非翻译区或前-mRNA中的内含子相同或互补的序列。 Sense or antisense sequence may be mRNA sequence encoding a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide, and the same 3 'or 5' untranslated region or intron before -mRNA or sequence complementary to the mRNA. 在一些实施方案中,有义或反义序列与下述调节区的序列相同或互补,所述调节区驱动编码盐度耐性和/或氧化胁迫耐性调控多肽的基因的转录。 In some embodiments, the sense or antisense sequence of the region following the regulatory sequence identical or complementary to the regulatory region driving encoding a salinity tolerance and / or transcription of a gene or oxidative stress tolerance-modulating polypeptide. 在每种情况下,有义序列是与反义序列互补的序列。 In each case, the sense and antisense sequence is a sequence complementary to the sequence.

[0139] 有义和反义序列可以是大于约12个核苷酸(例如13、14、15、16、17、18、19、20、21、 22、23、24、25、26、27、28、29、30或更多个核苷酸)的任何长度。 [0139] The sense and antisense sequences may be greater than about 12 nucleotides (e.g. 13,14,15,16,17,18,19,20,21, 22,23,24,25,26,27, 28, 29, or more nucleotides) of any length. 例如,反义序列长度可以是21或22个核苷酸。 For example, an antisense sequence length may be 21 or 22 nucleotides. 通常,有义和反义序列的长度范围为从约15个核苷酸到约30个核苷酸,例如从约18个核苷酸到约28个核苷酸,或从约21个核苷酸到约25个核苷酸。 Typically, there is a length of the sense and antisense sequences range of from about 15 nucleotides to about 30 nucleotides, e.g., from about 18 nucleotides to about 28 nucleotides, or from about 21 nucleotides acid to about 25 nucleotides.

[0140] 在一些实施方案中,反义序列是与编码本文所述盐度耐性和/或氧化胁迫耐性调控多肽的mRNA序列互补的序列。 [0140] In some embodiments, the antisense sequence is an mRNA sequence encoding the herein salinity tolerance and / or oxidative stress tolerance-modulating polypeptide sequence complementary. 与反义序列互补的有义序列可以是存在于盐度耐性和/ 或氧化胁迫耐性调控多肽的mRNA中的序列。 Antisense sequence complementary to the sense sequence may be present in salinity tolerance / or oxidative stress tolerance-modulating polypeptide mRNA in sequence. 通常,有义和反义序列被设计为对应于靶mRNA 的15-30个核苷酸的序列,使得靶mRNA的水平降低。 Typically, the sense and antisense sequences are designed to be 15-30 nucleotides corresponding to sequences of the target mRNA, such that the level of the target mRNA is reduced.

[0141] 在一些实施方案中,可以使用含有下述核酸的构建体抑制基因的表达,所述核酸有至少一条链是多于一条有义序列(例如2、3、4、5、6、7、8、9、10或更多有义序列)的模板。 Gene expression [0141] In some embodiments, the nucleic acid may be used which contains a construct to suppress, at least one strand of said nucleic acid is more than one sense sequence (e.g. 2,3,4,5,6,7 , 9, 10 or more sense sequences) template. 类似地,可以使用含有下述核酸的构建体抑制基因的表达,所述核酸有至少一条链是多于一条反义序列(例如2、3、4、5、6、7、8、9、10或更多反义序列)的模板。 Similarly, a nucleic acid containing the gene construct to suppress expression of the nucleic acid is at least one strand more than one antisense sequence (e.g., 9, 10 or more antisense sequences) template. 例如,构建体可含有下述核酸,所述核酸有至少一条链是两条有义序列和两条反义序列的模板。 For example, the construct may contain the following nucleic acid is at least one strand template two sense sequences and two antisense sequences. 多条有义序列可以相同或不同,并且多条反义序列可以相同或不同。 A plurality of sense sequences can be identical or different, and a plurality of antisense sequences can be identical or different. 例如,构建体可具有下述核酸,所述核酸有一条链是两条相同有义序列和与所述两条相同有义序列互补的两条相同反义序列的模板。 For example, the construct may have the following nucleic acid has a template strand are two identical two identical antisense sequences of the sense sequences and two identical sense sequences complementary. 或者,分离的核酸可具有一条链是以下的模板:(1)长度为20个核苷酸的两条相同的有义序列,(2)长度为20个核苷酸的、与两条相同有义序列互补的一条反义序列,(3) 长度为30个核苷酸的有义序列,和(4)长度为30个核苷酸的、与有义序列互补的三条相同的反义序列。 Alternatively, an isolated nucleic acid can have one strand is the following template: (1) the same length as the two 20-nucleotide sense sequence, (2) 20 nucleotides in length, have the same two an antisense sequence complementary to the sense sequence, (3) a length of 30 nucleotides sense sequence, and (4) 30 nucleotides in length, the same antisense sequence complementary to the sense sequence three. 本文提供的构建体可以被设计为具有有义和反义序列的任何排列。 The constructs provided herein can be designed to have any arrangement of sense and antisense sequences. 例如,两条相同的有义序列可以紧跟两条相同的反义序列,或者可以被置于两条相同的反义序列之间。 For example, two identical sense sequences can be followed by two identical antisense sequences or can be placed between two identical antisense sequences.

[0142] 具有至少一条链是一条或多条有义和/或反义序列模板的核酸可以与调节区有效连接,以驱动含有一条或多条有义和/或反义序列的RNA分子的转录。 [0142] at least one strand of the nucleic acid is one or more sense and / or antisense sequence of the template can be linked to a regulatory region to drive transcription of containing one or more sense and / or antisense RNA molecule having the sequence . 另外,这样的核酸可与转录终止子序列例如胭脂碱合酶(nos)基因的终止子有效连接。 Further, such a nucleic acid can be operably linked to a transcription terminator sequence, for example, the nopaline synthase terminator (NOS) gene. 在一些情况下,两个调节区能够指导两个转录物的转录:一个来自于顶部链,一个来自于底部链。 In some cases, two regulatory regions can direct transcription of two transcripts: one from the top strand, and one from the bottom strand. 见例如Yan 等,Plant Physiol.,141 :1508-1518 (2006)。 See for example, Yan et al., Plant Physiol, 141:. 1508-1518 (2006). 两个调节区可以相同或不同。 Two regulatory regions can be the same or different. 两个转录物可形成诱导靶RNA降解的双链RNA分子。 Two transcripts can form double-stranded RNA degradation induce target RNA molecule. 在一些情况下,核酸可以置于T-DNA或来自植物的转化DNA (P-DNA)内,使得P-DNA的左侧和右侧T-DNA边界序列或左侧和右侧边界样序列在核酸的两侧或任一侧。 In some cases, the nucleic acid may be placed within the T-DNA or plant-derived transfer DNA (P-DNA), such that the left and right T-DNA border sequences, or left and right border-like sequences of the P-DNA in both sides or either side of the nucleic acid. 见US2006/0265788。 See US2006 / 0265788. 两个调节区之间的核酸序列长度可从约15个到约30个核苷酸。 The length of the nucleic acid sequence between the two regulatory regions can be from about 15 to about 30 nucleotides. 在一些实施方案中,两个调节区之间的核酸序列长度从约15到约200 个核苷酸,长度从约15到约100个核苷酸,长度从约15到约50个核苷酸,长度从约18到约50个核苷酸,长度从约18到约40个核苷酸,长度从约18到约30个核苷酸,或长度从约 In some embodiments, the length of the nucleic acid sequence between the two regulatory regions is from about 15 to about 200 nucleotides in length from about 15 to about 100 nucleotides in length from about 15 to about 50 nucleotides in length from about 18 to about 50 nucleotides in length from about 18 to about 40 nucleotides in length from about 18 to about 30 nucleotides in length, or from about

2618到约25个核苷酸。 2618 to about 25 nucleotides.

[0143] 在用于抑制植物中基因表达的一些基于核酸的方法中,合适的核酸可以是核酸类似物。 [0143] In some nucleic-acid based methods for inhibition of gene expression in plants, a suitable nucleic acid can be a nucleic acid analog. 核酸类似物可以在碱基部分、糖部分或磷酸主链上被修饰,以提高核酸的例如稳定性、杂交或溶解度。 Nucleic acid analogs can be modified at the base moiety, sugar moiety or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. 碱基部分上的修饰包括脱氧尿苷替换脱氧胸苷,和5-甲基_2' -脱氧胞苷和5-溴-2' -脱氧胞苷替换脱氧胞苷。 Modifications at the base moiety include deoxyuridine for deoxythymidine Alternatively, and 5-methyl _2 '- deoxycytidine and 5-bromo-2' - deoxycytidine Alternatively deoxycytidine. 糖部分的修饰包括修饰核糖的2'羟基形成2'-0_甲基或2'-0_烯丙基糖。 Modified sugar moiety include modification of the ribose 2 'hydroxyl form 2'-methyl or 2'-0_ 0_-allyl sugars. 可以修饰脱氧核糖磷酸主链产生吗啉代核酸,其中每个碱基部分与六元吗啉代环连接,或产生肽核酸,其中用假肽主链替代脱氧磷酸主链并保留四个喊基。 Deoxyribose phosphate backbone can be modified to produce morpholino nucleic acid strand, wherein each base portion and six yuan morpholino ring connection, or generate peptide nucleic acids in which a pseudopeptide backbone Alternatively deoxyphosphate backbone and retain four call group . 见例如Summerton禾PWeller,1997,Antisense Nucleic AcidDrug Dev. ,7 : 187-195 ; Hyrup等,Bioorgan. Med. Chem.,4 =5-23(1996)。 See e.g. Summerton Wo PWeller, 1997, Antisense Nucleic AcidDrug Dev, 7: 187-195; Hyrup et, Bioorgan Med Chem, 4 = 5-23 (1996)..... 另外,可以将脱氧磷酸主链替换为例如硫代磷酸酯或二硫代磷酸酯主链,磷酰胺酯(phosphoroamidite)或烷基磷酸三酯主链。 In addition, the deoxyphosphate backbone can be replaced, for example, a phosphorothioate or phosphorodithioate backbone, phosphoramidate (phosphoroamidite) or an alkyl phosphotriester backbone.

[0144] C.构津体/载体 [0144] C. Jin configuration / carriers

[0145] 可以使用本文提供的重组构建体转化植物或植物细胞,从而调控盐度耐性和/或氧化胁迫耐性水平。 [0145] provided herein using recombinant construct was transformed plant or plant cell, thereby regulating salinity tolerance and / or oxidative stress tolerance levels. 重组的核酸构建体可包含编码本文所述盐度耐性和/或氧化胁迫耐性调控多肽的核酸,其与适合在植物或细胞中表达盐度耐性和/或氧化胁迫耐性调控多肽的调节区有效连接。 The recombinant nucleic acid construct described herein may comprise encoding salinity tolerance and / or oxidative stress tolerance-modulating nucleic acid polypeptide, operably linked to suitable expression in a plant cell or a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide regulatory region of . 因此,核酸可包含编码SEQ ID NO :1、3、5、7、10、12、16、18、21、26、28、32、 34、40、46、48、51、53、55、57、59、61、65、67、70、72、75、77、79、82、85、87、89、92、95、97、99、 103、105、108、111、113、115、117、120、124、131、133、135、137、139、146、148、150、152、155、 157、159、161和164中所示的任何盐度耐性和/或氧化胁迫耐性调控多肽的编码序列。 Thus, the nucleic acid may comprise coding SEQ ID NO: 1,3,5,7,10,12,16,18,21,26,28,32, 34,40,46,48,51,53,55,57, 59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99, 103,105,108,111,113,115,117,120, 124,131,133,135,137,139,146,148,150,152,155, 157,159,161, and any salinity tolerance and / or oxidative stress tolerance-modulating the coding sequence of the polypeptide shown in 164. 编码盐度耐性和/或氧化胁迫耐性调控多肽的核酸的例子示于SEQID N0 : 2、4、6、8、9、11、13、 14、15、17、19、20、22、23、24、25、27、29、30、31、33、35、36、37、38、39、41、42、43、44、45、47、 49、50、52、54、56、58、60、62、63、64、66、68、69、71、73、74、76、78、80、81、83、84、86、88、90、 91、93、94、96、98、100、101、102、104、106、107、109、110、112、114、116、118、119、121、122、 123、125、126、127、128、129、130、132、134、136、138、140、141、142、143、144、145、147、149、 151、153、154、156、158、160、162、163、165、166、167、168 和SEQ ID NO :140 的氨基酸等同物1到135中。 Examples of salinity tolerance or encoding and / oxidative stress tolerance-modulating polypeptide is a nucleic acid shown in SEQID N0: 2,4,6,8,9,11,13, 14,15,17,19,20,22,23,24 , 25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47, 49,50,52,54,56,58,60,62 , 63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90, 91,93,94,96,98,100,101,102 , 104,106,107,109,110,112,114,116,118,119,121,122, 123,125,126,127,128,129,130,132,134,136,138,140,141 , 142,143,144,145,147,149, 151,153,154,156,158,160,162,163,165,166,167,168 and SEQ ID NO: 140 amino acids 1 to 135 equivalents . 重组核酸编码的盐度耐性和/或氧化胁迫耐性调控多肽可以是固有的盐度耐性和/或氧化胁迫耐性调控多肽,或者可以对细胞是异源的。 Recombinant nucleic acid encoding a salinity tolerance and / or oxidative stress tolerance-modulating polypeptide may be inherent salinity tolerance and / or oxidative stress tolerance-modulating polypeptide, or can be heterologous to the cell. 在一些情况下,重组构建体含有抑制盐度耐性和/或氧化胁迫耐性调控多肽表达的核酸,其与调节区有效连接。 In some cases, the recombinant construct comprising an inhibitory salinity tolerance and / or nucleic acid expression of oxidative stress tolerance-modulating polypeptide, operably linked to a regulatory region. 合适的调节区的例子描述于标题为“调节区”的部分中。 Examples of suitable regulatory regions are described in the section entitled "regulatory region" of.

[0146] 还提供了含有重组核酸构建体(如本文所述重组核酸构建体)的载体。 [0146] Also provided are vectors comprising the recombinant nucleic acid construct (as described herein recombinant nucleic acid construct). 合适的载体主链包括例如本领域常规使用的载体主链,例如质粒、病毒、人造染色体、BAC、YAC或PAC。 Suitable vector backbones include, for example vector backbone conventionally used in the art, e.g. plasmids, viruses, artificial chromosomes, BAC, YAC, or PAC. 合适的表达载体包括但不限于,来自例如噬菌体、杆状病毒和反转录病毒的质粒和病毒载体。 Suitable expression vectors include but are not limited to, for example, bacteriophage, baculoviruses and retroviruses and viral vectors derived from plasmids. 大量载体和表达系统可商业得自例如Novagen (Madison,WI)、Clontech (Palo Alto, CA), Stratagene(La Jolla, CA)禾口Invitrogen/Life Technologies(Carlsbad, CA) 的公司。 Numerous vectors and expression systems such as commercially available from Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA) Wo mouth Invitrogen / Life Technologies (Carlsbad, CA) of the company.

[0147] 本文提供的载体可包含例如复制起点、支架附着区(SAR)和/或标记物。 [0147] The vectors provided herein may comprise, for example, origins of replication, scaffold attachment regions (SAR) and / or marker. 标记物基因能够赋予植物细胞可选择的表型。 Marker gene capable of conferring a selectable phenotype on plant cells. 例如,标记物能够赋予抗生剂抗性,例如对抗生素(例如卡那霉素、G418、博来霉素或潮霉素)的抗性,或对除草剂(例如草甘膦、氯磺隆(chlorsulfuron)或草胺膦(phosphinothricin))的抗性。 For example, marker agents capable of imparting antibiotic resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin or hygromycin) or herbicides (e.g., glyphosate, chlorsulfuron ( Chlorsulfuron) or phosphinothricin (, phosphinothricin)) resistance. 另外,表达载体可包含被设计为便于操作或检测(例如纯化或定位)所表达的多肽的标签序列。 Further, the expression vector may comprise a polypeptide to be designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed sequence tag. 标签序列例如萤光素 Tag sequences such as luciferase

27酶、葡糖醛酸糖苷酶(GUS)、绿色荧光蛋白(GFP)、谷胱甘肽S-转移酶(GST)、聚组氨酸、 c-myc、血凝素或Flag™标签(Kodak,New Haven, CT)序列通常被表达为与所编码的多肽的融合物。 27 enzymes, enzyme glucuronidase (the GUS), green fluorescent protein (GFP), glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag ™ tag (Kodak , New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. 这类标签可以被插入多肽中任何地方,包括羧基或氨基任一端。 Such tags can be inserted anywhere in a polypeptide, including an amino group or a carboxyl group at either end.

[0148] D.调节区 [0148] D. regulatory region

[0149] 待包含在重组构建体中的调节区的选择取决于若干因素,包括但不限于效率、选择性、可诱导性、期望的表达水平和细胞或组织优先表达。 [0149] The choice of regulatory regions to be included in the construct depends on several factors in the body in a recombinant, including but not limited to, efficiency, selectability, inducibility, desired expression preferential expression level, and cell- or tissue. 通过适当地选择和放置与编码序列相关的调节区来调节编码序列的表达对本领域技术人员而言是常规问题。 To modulate the expression of the coding sequence by appropriately selecting and placing the coding sequence of the regulatory region associated to those skilled in the art that conventional problems. 可以通过类似的方式调控核酸的转录。 You can regulate transcription of a nucleic acid in a similar manner.

[0150] 一些合适的启动子仅在或主要在某些细胞类型中起始转录。 [0150] Some suitable promoters only, or predominantly, in certain cell types to initiate transcription. 待包含在重组构建体中的调节区的选择取决于若干因素,包括但不限于效率、选择性、可诱导性、期望的表达水平和细胞或组织优先表达。 The choice of regulatory regions to be included in a construct in recombinant depend on several factors, including but not limited to, efficiency, selectability, inducibility, desired expression preferential expression level, and cell- or tissue. 通过适当地选择和放置与编码序列相关的调节区来调节编码序列的表达对本领域技术人员而言是常规问题。 To modulate the expression of the coding sequence by appropriately selecting and placing the coding sequence of the regulatory region associated to those skilled in the art that conventional problems. 可以通过类似的方式调控核酸的转录。 You can regulate transcription of a nucleic acid in a similar manner.

[0151] 一些合适的调节区仅在或主要在某些细胞类型中起始转录。 [0151] Some suitable regulatory regions only, or predominantly, in certain cell types to initiate transcription. 用于在植物基因组DNA中鉴定和表征调节区的方法是已知的,包括例如以下参考文献中描述的方法Jordano 等,Plant Cell, 1 :855_866 (1989) ;Bustos 等,Plant Cell, 1 :839_854 (1989) ;Green 等, EMBO J. ,7 :4035-4044(1988) ;Meier 等,Plant Cell, 3 :309-316 (1991);和Zhang等,Plant Physiology, 110 : 1069-1079(1996)。 For plant genomic DNA methods to identify and characterize regulatory regions are known, including for example, a method described in references like Jordano, Plant Cell, 1: 855_866 (1989); Bustos et, Plant Cell, 1: 839_854 (1989); Green et, EMBO J., 7: 4035-4044 (1988); Meier et, Plant Cell, 3: 309-316 (1991); and Zhang et al., Plant Physiology, 110: 1069-1079 (1996) .

[0152] 多种调节区种类的实例在下文描述。 [0152] Various examples of adjusting the type regions described below. 下文指出的一些调节区以及其他调节区在美国专利申请序列号60/505,689 ;60/518,075 ;60/544,771 ;60/558,869 ;60/583,691 60/619,181 ;60/637,140 ;60/757,544 ;60/776,307 ;10/957,569 ;11/058,689 11/172,703 ; 11/208,308 ; 11/274,890 ;60/583,609 ;60/612, 891 ;11/097,589 11/233,726 ;11/408,791 ;11/414,142 ;10/950,321 ;11/360,017 ;PCT/US05/011105 PCT/US05/23639 ;PCT/US05/034308 ;PCT/US05/034343 ;禾口PCT/US06/038236 ;PCT/ US06/040572 ;和PCT/US07/62762 中更详细地描述。 Some regulatory regions indicated below as well as other regulatory regions in U.S. Patent Application Serial No. 60 / 505,689; 60 / 518,075; 60 / 544,771; 60 / 558,869; 60 / 583,691 60 / 619,181 ; 60 / 637,140; 60 / 757,544; 60 / 776,307; 10 / 957,569; 11 / 058,689 11 / 172,703; 11/208, 308; 11 / 274,890; 60 / 583,609; 60/612, 891; 11 / 097,589 11 / 233,726; 11 / 408,791; 11 / 414,142; 10 / 950,321; 11 / 360,017; PCT / US05 / 011105 PCT / US05 / 23639; PCT / US05 / 034308; PCT / US05 / 034343; Wo port PCT / US06 / 038236; PCT / US06 / 040572; and PCT / US07 / 62762 described in more detail.

[0153]例如,调节区 p326、YP0144、YP0190、pl3879、YP0050、p32449、21876、YP0158、 YP0214、YP0380、PT0848、PT0633、YP0128、YP0275、PT0660、PT0683、PT0758、PT0613、PT0672、 PT0688、PT0837、YP0092、PT0676、PT0708、YP0396、YP0007、YP0111、YP0103、YP0028、YP0121、 YP0008、YP0039、YP0115、YP0119、YP0120、YP0374、YP0101、YP0102、YP0110、YP0117、YP0137、 YP0285、YP0212、YP0097、YP0107、YP0088、YP0143、YP0156、PT0650、PT0695、PT0723、PT0838、 PT0879、PT0740、PT0535、PT0668、PT0886、PT0585、YP0381、YP0337、PT0710、YP0356、YP0385、 YP0384、YP0286、YP0377、PD1367、PT0863、PT0829、PT0665、PT0678、YP0086、YP0188、YP0263、 PT0743和YP0096的序列示于PCT/US06/040572的序列表中;调节区PT0625的序列示于PCT/US05/034343 的序列表中;调节区PT0623、YP0388、YP0087、YP0093、YP0108、YP0022 和YP0080的序列公开于美国专利申请序列号11/172,703的序列表中;调节区PR0924的序列公开于 [0153] For example, regulatory regions p326, YP0144, YP0190, pl3879, YP0050, p32449,21876, YP0158, YP0214, YP0380, PT0848, PT0633, YP0128, YP0275, PT0660, PT0683, PT0758, PT0613, PT0672, PT0688, PT0837, YP0092 , PT0676, PT0708, YP0396, YP0007, YP0111, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115, YP0119, YP0120, YP0374, YP0101, YP0102, YP0110, YP0117, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143 , YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886, PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286, YP0377, PD1367, PT0863, PT0829, PT0665, PT0678, YP0086 , YP0188, YP0263, PT0743 sequence and YP0096 are shown in the sequence Listing PCT / US06 / 040572 in; sequence of the regulatory region PT0625 is shown in the sequence Listing PCT / US05 / 034343 in; regulatory regions PT0623, YP0388, YP0087, YP0093, YP0108 , YP0022, and YP0080 sequences are disclosed in U.S. Patent application serial No. 11 sequence Listing / 172,703 in; sequence of the regulatory region PR0924 is disclosed in PCT/US07/62762 的序列表中;调节区p530cl0、p0sFIE2_2、pOsMEA、p0sYpl02 和p0sYp285的序列示于PCT/US06/038236的序列表中。 SEQUENCE LISTING PCT / US07 / 62762 in; regulatory region p530cl0, sequence p0sFIE2_2, pOsMEA, p0sYpl02 p0sYp285 and are shown in the Sequence Listing PCT / US06 / 038236 in.

[0154] 应当明白调节区可以基于它在一种植物物种中的活性满足一种分类标准,但是基于它在另一种植物物种中的活性满足不同的分类标准。 [0154] It should be understood that it may be based on a regulatory region active in a plant species a classification criterion is satisfied, but based on its activity in another plant species to satisfy the different classification criteria.

[0155] i.广泛表达的启动子[0156] 当启动子在许多(但不必须是全部)植物组织中促进转录时,其可以被称作是“广泛表达的”。 Widely expressed [0155] i promoter [0156] Where the promoter in many (but not necessarily all) plant tissues promote transcription, it may be referred to as "widely expressed." 例如,广泛表达的启动子可以在一个或多个枝条、枝端(顶端)和叶中促进有效连接的序列转录,但是在如根或茎的组织中弱促进或完全不促进转录。 For example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more branches of branches (the top), and leaves, but the root or stem tissue, such as weakly or not promote transcription promotion. 另一个实例是,广泛表达的启动子能够在一个或多个茎、枝条、枝端(顶端)和叶中促进有效连接的序列转录,但是在如花和产生种子的生殖组织中能够弱促进或完全不促进转录。 As another example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more stems, shoots, branches end (top), and leaves, but can promote or completely in weak reproductive tissues of the flower and produce seed does not promote transcription. 可以包含在本文提供的核酸构建体中的广泛表达启动子的非限制性实例包括P326、YP0144、YP0190、 pl3879、YP0050、p32449、21876、YP0158、YP0214、YP0380、PT0848 和PT0633 启动子。 Construct may comprise non-limiting examples of broadly expressing promoters include body P326, YP0144, YP0190, pl3879, YP0050, p32449,21876, YP0158, YP0214, YP0380, PT0848, and PT0633 promoters in the nucleic acid provided herein. 其他的实例包括花椰菜花叶病毒(CaMV)35S启动子、甘露碱合酶(MAS)启动子、来自根瘤农杆菌(Agrobacteriumtumefaciens)T-DNA 的1,或2,启动子、玄参(figmort)花叶病毒34S 启动子、诸如稻肌动蛋白启动子的肌动蛋白启动子,以及诸如玉米泛素-1启动子的泛素启动子。 Other examples include the cauliflower mosaic virus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter from Agrobacterium tumefaciens (Agrobacteriumtumefaciens) T-DNA of 1 or 2, a promoter, figwort (figmort) Flowers mosaic virus 34S promoter, such as the rice actin promoter, actin promoter, as well as the maize ubiquitin-1 promoter is ubiquitin promoter. 在一些情况下,CaMV 35S启动子从广泛表达的启动子范畴中被排除。 In some cases, CaMV 35S promoter is excluded from the category of broadly expressing promoters in.

[0157] ii.根启动子 [0157] ii. Root promoter

[0158] 根活性启动子在根组织(例如根内皮层、根表皮或根维管组织)中赋予转录。 [0158] Root-active promoters confer transcription in root tissue (e.g., root endodermis, root epidermis, or root vascular tissues). 在一些实施方案中,根活性启动子是根优先的启动子,即仅在或主要在根组织中赋予转录。 In some embodiments, root-active promoters are root-preferential promoters, i.e., confer transcription only or predominantly in root tissue. 根优先的启动子包括YP0128、YP0275、PT0625、PT0660、PT0683和PT0758启动子。 Root preferential promoters include YP0128, YP0275, PT0625, PT0660, PT0683, and PT0758 promoters. 其他根优先的启动子包括PT0613)、PT0672、PT0688和PT0837启动子,其主要在根组织中驱动转录, 在胚珠和/或种子中较低程度地驱动转录。 Other root-preferential promoters include the PT0613), PT0672, PT0688, and PT0837 promoters, which drive transcription primarily in root tissue, drive transcription in ovules and / or seed a lesser extent. 根优先的启动子的其他实例包括CaMV 35S启动子的根特异的亚结构域(Lam等人,Proc. Natl. Acad. Sci.美国86 =7890-7894 (1989)), Conkling等人,Plant Physiol. 93 :1203_1211 (1990)报导的根细胞特异启动子和烟草RD2 启动子。 Other examples of root-preferential promoters include the root specific CaMV 35S promoter subdomains (Lam et al., Proc. Natl. Acad. Sci. USA 86 = 7890-7894 (1989)), Conkling et al., Plant Physiol 93: 1203_1211 (1990) reported root cell specific promoters and the tobacco RD2 promoter.

[0159] iii.成熟胚乳启动子 [0159] iii. Maturing endosperm promoters

[0160] 在一些实施方案中,在成熟中的胚乳中驱动转录的启动子可以是有用的。 [0160] In some embodiments, the maturing endosperm promoters drive transcription may be useful. 来自成熟胚乳启动子的转录通常在受精之后开始,并主要发生于种子发育期间的胚乳组织中,并且通常在细胞化期(cellularization phase)最高。 Transcription from mature endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development, and generally the highest in the cells of the period (cellularization phase). 最合适的是主要在成熟中的胚乳中有活性的启动子,尽管有时能够使用在其他组织中也有活性的启动子。 The most suitable is the main promoter active in maturing endosperm, though sometimes can be used in other organizations are also active promoters. 可以包含在本文提供的核酸构建体中的成熟中胚乳启动子的非限制性实例包括:油菜籽蛋白启动子、Arcelin-5 启动子、菜豆蛋白启动子(Bustos等人Plant Cell 1(9) :839_853 (1989))、大豆胰蛋白酶抑制剂启动子(Riggs 等人Plant Cell 1(6) :609_621 (1989))、ACP 启动子(Baerson 等人Plant Mol Biol, 22 (2) :255_267 (1993))、硬脂酰-ACP 去饱和酶启动子(Slocombe 等人Plant Physiol 104(4) : 167-176 (1994) )、3 -伴大豆球蛋白(conglycinin)的大豆a '亚基启动子(Chen等人Proc. Natl. Acad. Sci.美国83 :8560_8564(1986))、油质蛋白启动子(Hong等人Plant MolBiol 34(3) :549_555 (1997))和玉米醇溶蛋白启动子(如15kD玉米醇溶蛋白启动子、16kD玉米醇溶蛋白启动子、19kD玉米醇溶蛋白启动子、22kD玉米醇溶蛋白启动子和27kD玉米醇溶蛋白启动子)。 Non-limiting examples provided herein may comprise a nucleic acid construct maturation in the endosperm promoters include: the napin promoter, Arcelin-5 promoter, the phaseolin promoter (Bustos et al., Plant Cell 1 (9): 839_853 (1989)), soybean trypsin inhibitor promoter (Riggs et al., Plant Cell 1 (6): 609_621 (1989)), ACP promoter (Baerson et et al., Plant Mol Biol, 22 (2): 255_267 (1993) ), -ACP stearoyl desaturase promoter (Slocombe et al, Plant Physiol 104 (4): 167-176 (1994)), 3 - conglycinin (conglycinin,) soybean a 'subunit promoter (Chen et al., Proc Natl Acad Sci USA 83:.... 8560_8564 (1986)), oleosin promoter (Hong et al., Plant MolBiol 34 (3): 549_555 (1997)), and zein promoter (e.g. 15kD promoter, zein, 16kD zein promoter, 19kD zein promoter, 22kD zein promoter and 27kD zein promoter). 也合适的是来自稻谷蛋白-1基因的Osgt-1启动子(Zheng等人Mol. Cell Biol. 13 =5829-5842 (1993)), 3 -淀粉酶基因启动子和大麦醇溶蛋白基因启动子。 Also suitable are the Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al., Mol Cell Biol 13 = 5829-5842 (1993)..), 3 - amylase gene promoter, and the barley hordein gene promoter . 其他成熟中胚乳启动子包括YP0092、PT0676和PT0708启动子。 Other maturing endosperm promoters include the YP0092, PT0676, and PT0708 promoters.

[0161] iv.子房组织启动子 [0161] iv. Ovary tissue promoter

[0162] 在子房组织如胚珠壁和中果皮中有活性的启动子也可以是有用的,例如聚半乳糖醛酸酐酶(polygalacturonidase)启动子、香蕉TRX启动子、瓜肌动蛋白启动子、YP0396和 [0162] There ovary tissues such as the ovule wall and mesocarp active promoters can also be useful, for example, polygalacturonase anhydrase (a polygalacturonidase) promoter, the banana TRX promoter, the melon actin promoter, and YP0396

29PT0623。 29PT0623. 主要在胚珠中有活性的启动子的实例为YP0007、YP0111、YP0092、YP0103、YP0028、 YP0121、YP0008、YP0039、YP0115、YP0119、YP0120 和YP0374。 A promoter active primarily in ovules Examples YP0007, YP0111, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115, YP0119, YP0120, and YP0374.

[0163] v.胚囊/早期胚乳启动子 [0163] v. Embryo Sac / Early Endosperm Promoters

[0164] 为了在胚囊/早期胚乳中实现表达,可以使用下述调节区,所述调节区在极核(polar nuclei)和/或中央细胞中,或在极核前体中有活性,但不在卵细胞或卵细胞前体中有活性。 [0164] To achieve expression in embryo sac / early endosperm, regulatory regions can be used below, the polar nuclei in the regulatory region (polar nuclei) and / or the central cell, active in polar nuclei or precursors but egg or egg is not active precursor. 最合适的是仅在或主要在极核或其前体,和/或中央细胞中驱动表达的启动子。 Most suitable are the only or predominantly in polar nuclei precursor, and / or the central cell, or a promoter to drive expression. 使用胚囊/早期胚乳优先的启动子也可以发现从极核扩展进入早期胚乳发育的转录模式, 尽管在细胞化期期间和之后转录在晚期胚乳发育中通常显著降低。 Embryo sac / early endosperm-preferential promoters can be found in early endosperm development into the dubbing mode from the extension pole core, although the cells of transcription during the period after and generally significantly decreases in later stage of endosperm development. 合子或正在发育的胚中的表达通常不与胚囊/早期胚乳启动子一起存在。 Zygote or embryo expressed the developing embryo sac is usually not present with / early endosperm promoters.

[0165] 可适用的启动子包括来自以下基因的启动子:拟南芥viviparous-1 (见GenBank 号U93215);拟南芥atmycl(见Urao (1996)Plant Mol. Biol. ,32 :571_57 ; Conceicao (1994) Plant, 5 :493_505);拟南芥FIE (GenBank 号AF129516);拟南芥MEA ;拟南芥FIS2 (GenBank号AF096096)和FIE 1. 1 (美国专利6,906,244)。 [0165] applicable promoters include the promoter from the following genes: Arabidopsis viviparous-1 (see GenBank No. U93215); Arabidopsis atmycl (see Urao (1996) Plant Mol Biol, 32:.. 571_57; Conceicao (1994) Plant, 5: 493_505); Arabidopsis FIE (GenBank No. AF129516); Arabidopsis the MEA; Arabidopsis FIS2 (GenBank No. AF096096) and FIE 1. 1 (U.S. Patent No. 6,906,244). 可适用的其他启动子可包括来自以下基因的启动子:玉米MAC1 (见Sheridan (1996)Genetics,142 :1009-1020); 玉米Cat3(见GenBank 号L05934 ;Abler (1993) Plant Mol. Biol. ,22 :10131-1038)。 Applicable Other promoters may include promoters from the following genes: maize MACl (see Sheridan (1996) Genetics, 142: 1009-1020); maize Cat3 (see GenBank No. L05934; Abler (1993) Plant Mol Biol,.. 22: 10131-1038). 其他启动子包括以下的拟南芥启动子:YP0039、YP0101、YP0102、YP0110、YP0117、YP0119、YP0137、 DME、YP0285和YP0212。 Other promoters include the following Arabidopsis promoters: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119, YP0137, DME, YP0285, and YP0212. 可适用的其他启动子包括以下的稻启动子:p530cl0、p0sFIE2_2、 pOsMEA、p0sYpl02 和p0sYp285。 Applicable Other promoters include the following rice promoters: p530cl0, p0sFIE2_2, pOsMEA, p0sYpl02 and p0sYp285.

[0166] vi.胚启动子 [0166] vi. Embryo promoter

[0167] 优先在受精后的合子细胞中驱动转录的调节区能够提供胚优先的表达。 [0167] preferentially drive transcription in zygotic cells regulatory region capable of providing the fertilized embryo preferential expression. 最合适的是在心形阶段之前优先在早期胚中驱动转录的启动子,但是晚期和成熟胚中的表达也是合适的。 Most suitable are promoters that preferentially drive transcription in early stage embryos prior to the heart-shaped stage, but the expression of advanced and mature embryos is also suitable. 胚优先的启动子包括大麦脂质转移蛋白(Ltpl)启动子(Plant Cell Rep (2001) 20 : 647-654),YP0097、YP0107、YP0088、YP0143、YP0156、PT0650、PT0695、PT0723、PT0838、 PT0879 和PT0740。 Embryo-preferential promoters include the barley lipid transfer protein (LTPL) promoter (Plant Cell Rep (2001) 20: 647-654), YP0097, YP0107, YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, and PT0740.

[0168] vii.光合组织启动子 [0168] vii. Photosynthetic tissues promoter

[0169] 在光合组织中有活性的启动子在绿色组织(如叶和茎)中赋予转录。 [0169] with a promoter active in photosynthetic tissue confer transcription in green tissues (such as leaves and stems) of. 最合适的是仅在或主要在这类组织中驱动表达的启动子。 The most suitable is the only major promoter that drives expression in such organizations or in. 这类启动子的实例包括核酮糖-1,5-二磷酸羧化酶(RbcS)启动子,如来自美洲落叶松(Larixlaricina)的RbcS启动子、松树cab6启动子(Yamamoto 等人Plant CellPhysiol. 35 :773_778 (1994))、来自小麦的Cab_l 基因启动子(Fejes 等人Plant Mol. Biol. 15 :921_932 (1990))、来自菠菜的CAB-1 启动子(Lubberstedt 等人Plant Physiol. 104 :997_1006 (1994))、来自稻的cablR启动子(Luan等人Plant Cell 4 :971-981(1992))、来自玉米的丙酮酸正磷酸二激酶(PPDK)启动子(Matsuoka等人Proc Natl Acad. Sci 美国90 :9586_9590 (1993))、烟草Lhcbl*2 启动子(Cerdan 等人Plant Mol. Biol. 33 :245-255(1997))、拟南芥SUC2蔗糖-H+同向转运体启动子(Truernit等人Planta 196 :564-570(1995))和来自菠菜的类囊体膜蛋白质启动子(psaD、psaF、psaE、PC、FNR、 atpC、atpD、cab、rbcS)。 Examples of such promoters include the ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS from eastern larch (Larixlaricina) promoter, promoters cab6 pine (Yamamoto et al Plant CellPhysiol. 35: 773_778 (1994)), Cab_l gene promoter from wheat (. Fejes et al, Plant Mol Biol 15:. 921_932 (1990)), CAB-1 promoter from spinach (Lubberstedt et al Plant Physiol 104:. 997_1006 (1994)), cablR promoter from rice (Luan et al, Plant Cell 4: 971-981 (1992)), from maize pyruvate, orthophosphate dikinase (the PPDK) promoter (Matsuoka et al., Proc Natl Acad Sci. USA 90: 9586_9590 (1993)), the tobacco Lhcbl * 2 promoter (Cerdan et al, Plant Mol Biol 33:.. 245-255 (1997)), Arabidopsis thaliana SUC2 sucrose-started with -H + symporter promoter (Truernit et people Planta 196: 564-570 (1995)) and thylakoid membrane protein promoter (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS) from spinach. 其他光合组织启动子为PT0535、PT0668、PT0886、PR0924、YP0144、 YP0380 和PT0585。 Other photosynthetic tissues promoters PT0535, PT0668, PT0886, PR0924, YP0144, YP0380, and PT0585.

[0170] viii.维管组织启动子 [0170] viii. Vascular tissue promoter

[0171] 在维管束中具有高活性或优先活性的启动子的实例包括YP0087、YP0093、YP0108、 Examples [0171] having a high priority activity or a promoter activity in vascular bundles include YP0087, YP0093, YP0108,

30YP0022和YP0080。 30YP0022 and YP0080. 其他维管组织优先的启动子包括富含甘氨酸的细胞壁蛋白质GRP 1.8 启动子(Keller 和Baumgartner, PlantCell, 3 (10) : 1051-1061 (1991))、鸭跖草属(Commelina)黄斑病毒(CoYMV)启动子(Medberry 等,Plant Cell,4(2) : 185-192 (1992)) 和稻东格鲁杆状病毒(tungro bacilliform virus, RTBV))启动子(Dai 等,Proc. Natl. Acad. Sci. USA,101(2) :687_692(2004))。 Other vascular tissue-preferential promoters include glycine-rich cell wall protein GRP 1.8 promoter (Keller and Baumgartner, PlantCell, 3 (10): 1051-1061 (1991)), Commelina (Commelina) macular virus (CoYMV ) promoter (like, Medberry, Plant Cell, 4 (2): 185-192 (1992)) and rice tungro bacilliform virus (tungro bacilliform virus, RTBV)) promoter (Dai et, Proc Natl Acad... . Sci USA, 101 (2): 687_692 (2004)).

[0172] ix.诱导型启动子 [0172] ix. Inducible promoter

[0173] 诱导型启动子响应外界刺激(如化学物质或环境刺激)而赋予转录。 [0173] an inducible promoter responsive to external stimuli (e.g., chemical or environmental stimuli) confer transcription. 例如,诱导型启动子可响应激素(如赤霉素或乙烯)或响应光照或干旱而赋予转录。 For example, inducible promoters may be responsive to hormones (e.g., ethylene or gibberellic acid) or in response to light or drought confer transcription. 干旱诱导型启动子包括YP0380、PT0848、YP0381、YP0337、YP0337、PT0633、YP0374、PT0710、YP0356、 YP0385、YP0396、YP0388、YP0384、PT0688、YP0286、YP0377、PD1367 和PD0901。 Drought inducible promoters include YP0380, PT0848, YP0381, YP0337, YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384, PT0688, YP0286, YP0377, PD1367 and PD0901. 氮诱导型启动子的实例包括PT0863、PT0829、PT0665和PT0886。 Examples of nitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886. 黑暗诱导型启动子的实例包括PR0924 和PT0678。 Examples of dark-inducible promoters include PR0924 and PT0678. 盐诱导的启动子的一个实例是rd29A(Kasuga等(1999)Nature Biotech 17: 287-291)。 One example of a promoter induced by salt is rd29A (Kasuga et (1999) Nature Biotech 17: 287-291).

[0174] x.基础启动子 [0174] x. Basal promoter

[0175] 基础启动子是转录起始所需的转录复合物装配必需的最小序列。 [0175] A basal promoter is the minimal sequence transcription of a transcription complex required for assembly of necessary starting. 基础启动子常常包含可位于转录起始位点上游约15个到约35个核苷酸之间的“TATA盒”元件。 Basal promoters frequently comprises upstream of the transcription start site to about 15, "the TATA box" element of between about 35 nucleotides. 基础启动子还可包含“CCAAT盒”元件(一般为序列CCAAT)和/或GGGCG序列,其可位于转录起点上游约40和约200个核苷酸之间,一般为上游约60到约120个核苷酸之间。 Basal promoters also contain "a CCAAT box" element (typically a CCAAT sequence) and / or a GGGCG sequence, which can be located upstream of the transcription start between about 40 and about 200 nucleotides, typically from about 60 to about 120 upstream of nuclei glycosides between acid.

[0176] xi.其他启动子 [0176] xi. Other promoters

[0177] 其他种类的启动子包括但不仅限于枝条优先的、愈伤组织优先的、藻丝细胞优先的、保卫细胞优先的如PT0678、块茎优先的、薄壁组织细胞优先的和衰老优先的启动子。 [0177] Other classes of promoters include, but are not limited to branches of priority, the priority of callus, trichome cells preferentially to defend the priority of cells such as PT0678, tubers priority, the priority of parenchyma cells and senescence-preferential promoters child. 如上述参考专利申请中所述,被命名为YP0086、YP0188、YP0263、PT0758、PT0743、PT0829、 YP0119和YP0096的启动子也可是有用的。 As described in the above-referenced patent application, it is named YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, and YP0096 promoters also can be useful.

[0178] xii.其他调节区 [0178] xii. Other regulatory region

[0179] 5'非翻译区(UTR)可以包含在本文所述的核酸构建体中。 [0179] 5 'untranslated region (UTR) may comprise a nucleic acid construct described herein. 5' UTR被转录,但是不被翻译,并位于转录物的起点和转录起始密码子之间,并可包括+1核苷酸。 5 'UTR is transcribed, but not translated, and lies between the starting point and a transcription start codon of the transcript, and may include the +1 nucleotide. 3' UTR可位于转录终止密码子和转录物末端之间。 3 'UTR may be located between the transcription termination codon and the end of the transcript. UTR可具有如提高mRNA稳定性或减弱翻译的具体功能。 UTR may have specific functions such as increasing mRNA stability or attenuating translation. 3' UTR的实例包括但不限于聚腺苷酸化信号和转录终止序列,例如胭脂氨酸合酶终止序列。 'Example 3 UTR include, but are not limited to polyadenylation signals and transcription termination sequence, e.g. nopaline synthase termination sequence.

[0180] 应当理解多于一种调节区可存在于重组多核苷酸中,例如内含子、增强子、上游激活区、转录终止子和诱导型元件。 [0180] It should be understood that more than one regulatory region may be present in a recombinant polynucleotide, such as introns, enhancers, upstream activation regions, transcription terminators, and inducible elements. 因此,例如多于一种调节区可以与编码盐和/或氧化胁迫耐性调控多肽的多核苷酸序列有效连接。 Thus, for example, more than one regulatory region can be operably linked / or salt thereof and a polynucleotide sequence encoding oxidative stress tolerance-modulating polypeptide.

[0181] 调节区(例如内源基因的启动子)可以通过化学合成获得,或通过从包含此类调节区的基因组DNA中亚克隆获得。 [0181] A regulatory region (e.g., promoter of the endogenous gene) can be obtained by chemical synthesis, or obtained by subcloning from a genomic DNA comprising such a regulatory region. 包含这类调节区的核酸也可以包含含有限制性酶切位点的侧翼序列,所述限制性酶切位点有助于随后的操作。 A nucleic acid comprising such a regulatory region can also include flanking sequences containing restriction enzyme sites, the restriction sites facilitate subsequent operation.

[0182] 或者,可以使用两组分系统完成异常表达,其中第一组分由转基因植物组成,所述转基因植物包含与启动子有效连接的转录激活因子,第二组分由下述转基因植物组成,所述转基因植物包含与转录激活因子的靶结合序列/区有效连接的本发明的核酸分子。 [0182] Alternatively, a two component system is completed abnormally expressed, wherein the first component consists of the composition of a transgenic plant, the transgenic plant comprising a transcriptional activator operatively linked to a promoter, a second component consists of a transgenic plant the following composition , a nucleic acid molecule of the invention comprises the transgenic plant transcriptional activator of the target binding sequence / region operably linked. 将两种转基因植物杂交,并且本发明的核酸分子在所述植物后代中表达。 The two hybrid gene transferred plant and a nucleic acid molecule of the invention is expressed in the progeny plant. 在本发明的另一备选的实施方案中,可以通过将两组分系统的序列转化进同一转基因植物品系中完成异常表达。 In another alternative embodiment of the present invention, can be transformed into a sequence of two-component systems the same transgenic plant lines is completed abnormal expression.

[0183] IV.转基因植物和植物细胞 [0183] IV. The transgenic plants and plant cells

[0184] A.转化 [0184] A. Transformation

[0185] 本发明还涉及包含至少一种本文所述重组核酸构建体的转基因植物细胞和植物。 [0185] The present invention further relates to compositions comprising at least one recombinant nucleic acid construct described herein, plant cells and transgenic plants body. 植物或植物细胞可以通过将构建体整合进其基因组中来转化,即能够被稳定转化。 Plant or plant cell constructs can be integrated into its genome by transformation, i.e., can be stably transformed. 稳定转化的细胞通常在每次细胞分裂时保留引入的核酸。 Stably transformed cells typically retain the introduced nucleic acid with each cell division. 植物或植物细胞也可以被瞬时转化,使得构建体不整合进其基因组中。 Plant or plant cell can also be transiently transformed such that the construct is not integrated into its genome. 瞬时转化的细胞通常在每次细胞分裂时丢失所有或部分引入的核酸构建体,使得引入的核酸在足量的细胞分裂后不能在子细胞中检出。 Transiently transformed cells typically lose all or part of a nucleic acid is introduced in the construct with each cell division, so that the nucleic acid is introduced after a sufficient amount of cell division can not be detected in daughter cells. 瞬时转化和稳定转化的转基因植物和植物细胞却可用于本文所述的方法中。 Transiently transformed and stably transformed transgenic plants and plant cells but may be used in the methods described herein.

[0186] 本文所述方法中使用的转基因植物可组成整株植物的部分或全部。 [0186] The transgenic plants used in the methods described herein may constitute part or all of a whole plant. 这类植物可以在培养箱中、温室中或者大田中,以适合所述物种的方式生长。 Such plants can be in the incubator, in a greenhouse or in the field to suit the way of the growth species. 转基因植物可以根据期望育种来实现具体的目的,例如将重组核酸引入其他品系中,将重组核酸转移至其他物种,或进一步选择其他想要的性状。 The transgenic plant can be achieved as desired breeding specific object, for example a recombinant nucleic acid into other lines, the recombinant nucleic acid is transferred to other species or for further selection of other desired traits. 或者,对适合这类技术的物种而言,可以营养繁殖转基因植物。 Or, in terms of species suitable for this type of technology can be vegetative propagation of transgenic plants. 在本文中使用时,转基因植物也表示具有转基因的初始转基因植物后代。 As used herein, transgenic plants having said initial transgene progeny plant genes. 可以培养转基因植物产生的种子,然后将其自交(或异型杂交和自交),获得对核酸构建体而言纯合的种子。 It may be cultured in the seed of the transgenic plants produced, and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid member constructed.

[0187] 可以在悬浮培养物或组织或器官培养物中培养转基因植物。 [0187] The transgenic plant can be grown in suspension culture or tissue or organ culture. 就本发明的目的而言,可以使用固体和/或液体组织培养技术。 For purposes of the present invention, it can be solid and / or liquid tissue culture techniques. 使用固体培养基时,可以将转基因植物细胞直接置于培养基上,或置于滤器上,然后将该滤器与培养基接触放置。 When using solid medium, transgenic plant cells can be placed directly on a medium, or placed on a filter, the filter is then placed in contact with the medium. 使用液体培养基时,可以将转基因植物细胞置于漂浮设备(例如接触液体培养基的多孔膜)上。 When using liquid medium, transgenic plant cells can be placed floatation device (e.g., a porous membrane in contact with the liquid medium) on. 固体培养基可以是例如Murashige and Skoog(MS)培养基,其含有琼脂和合适浓度的生长素例如2,4_ 二氯苯氧乙酸(2,4-D),和合适浓度的细胞分裂素(cytokinin)例如细胞分裂素(kinetin)。 The solid medium may be, for example, Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, for example, cytokinin (2,4-D), and a suitable concentration 2,4_-dichlorophenoxyacetic acid (AND GRAINS ), for example, cytokinins (kinetin).

[0188] 使用瞬时转化的植物细胞时,转化步骤中可包含编码具有报道基因活性的报道基因多肽的报道基因序列,并可在转化后合适的时间进行报道基因活性或表达测定。 [0188] When transiently transformed plant cells are used, the conversion step may comprise a reporter gene encoding a reporter gene polypeptide sequence of reporter gene activity, reporter gene activity can be measured or expressed in a suitable time after transformation. 进行测定的合适时间通常为转化后约1-21天,例如约1-14天、约1-7天或约1-3天。 Suitable measurement time is typically about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days. 瞬时测定的使用对不同物种中的快速分析而言尤其便利,或者用于证实具体受体细胞中先前未证实其表达的异源盐度耐性和/或氧化胁迫耐性调控多肽的表达。 Use of transient assays is particularly convenient for rapid analysis in different species, or to confirm a particular receptor and cells not previously confirmed the expression or the expression of a heterologous salinity tolerance / oxidative stress tolerance-modulating polypeptide.

[0189] 将核酸引入单子叶植物和双子叶植物中的技术是本领域已知的,并且包括但不限于农杆菌介导的转化、病毒载体介导的转化、电穿孔和粒子枪转化,例如美国专利5,538,880 ;5, 204,253 ;6, 329,571和6,013,863。 [0189] introducing nucleic acids into monocotyledonous and dicotyledonous plants techniques are known in the art, and include, but are not limited to Agrobacterium-mediated transformation, and viral vector-mediated transformation, electroporation and particle gun transformation, e.g. U.S. Patent No. 5,538,880; 5, 204,253; 6, 329,571 and 6,013,863. 如果使用细胞或培养的组织作为转化的受体组织,则需要时可以通过本领域技术人员已知的技术从转化的培养物中再生植物。 If the plants can be regenerated using tissue culture of cells or tissue transformed as a recipient, the need is known to those skilled in the art from the culture transformed.

[0190] B.筛选/选择 [0190] B. Screening / selection

[0191] 可以在转基因植物种群中筛选和/或选择下述种群成员,其具有转基因的表达所赋予的性状或表型。 [0191] can be screened for the transgenic plant population and / or selection of the following members of the population, with expression of the transgene conferred trait or phenotype. 例如,可以从单次转化事件的后代种群中筛选下述植物,所述植物具有期望的盐度耐性和/或氧化胁迫耐性调控多肽或核酸的表达水平。 For example, the following can be screened from the progeny plant population in a single transformation event, the plant having a desired salinity tolerance and / or oxidative stress tolerance-modulating polypeptide or nucleic acid expression levels. 可以使用物理和生物化学方法鉴定表达水平。 The expression levels may be identified using physical and biochemical methods. 这些方法包括用于检测多核苷酸的Southern分析或PCR扩增;用于检测RNA转录物的Northern印迹、SI RNase保护、引物延伸或RT-PCR扩增;用于检测多肽和多核苷酸的酶或核酶活性的酶测定;和用于检测多肽的蛋白质凝胶电泳、Western印迹、 These include methods for detecting polynucleotide Southern analysis or PCR amplification; Northern blots for detection of RNA transcripts, SI RNase protection, primer-extension, or RT-PCR amplification; polypeptides and polynucleotides for the detection of enzyme enzyme or ribozyme activity assay; and protein gel electrophoresis for detecting the polypeptide, Western blots,

32免疫沉淀和酶联免疫测定。 32 immunoprecipitation and enzyme-linked immunosorbent assay. 也可以使用其他技术例如原位杂交、酶染色和免疫染色来检测多肽和/或多核苷酸的存在或表达。 Other techniques may also be used such as in situ hybridization, enzyme staining, and immunostaining to detect the presence or expression of polypeptides and / or polynucleotides. 进行所有引用的技术的方法是已知的。 All technical methods cited are known. 作为一种备选方案,可以在包含独立转化事件的植物种群中筛选具有期望性状的植物,例如受调节的盐度耐性和/或氧化胁迫耐性水平。 As an alternative, it can be screened for plants having a desired trait in a plant population comprising independent transformation events, e.g. modulated salinity tolerance and / or oxidative stress tolerance levels. 可以在一代或多代中、和/或多于一个地理位置中进行选择和/或筛选。 It may be in one or more generations, and / or more than one location in the selection and / or screening. 在一些情况下,可以在下述条件下生长和选择转基因植物,所述条件诱导期望表型或者以其他在转基因植物中产生期望表型所必需的方式。 In some cases, it can be grown and selection of transgenic plants under the following conditions which induce a desired phenotype or other transgenic plants to produce a desired phenotype in the necessary manner. 另外,可以在具体的发育阶段期间应用选择和/或筛选,所述发育阶段中期望由植物表现所述表型。 In addition, application selection and / or screening during particular stages of development, the stage of development of the desired plant exhibits the phenotype. 可以进行选择和/或筛选来选择相对于缺乏转基因的对照植物而言在盐度耐性和/或氧化胁迫耐性水平中具有统计学显著差异的转基因植物。 You may be selected and / or screening to select lacking the transgene with respect to control plants transgenic plant having statistically significant difference in a salinity tolerance and / or oxidative stress tolerance levels. 选择或筛选的转基因植物与相应的对照植物相比具有改变的表型,如本文“转基因植物表型”部分中所述。 Selecting or screening transgenic plants and the corresponding control plant having an altered phenotype, as described in the "transgenic plant phenotype" section herein.

[0192] 可以在转基因植物种群中筛选和/或选择下述种群成员,其具有转基因的表达所赋予的性状或表型。 [0192] can be screened for the transgenic plant population and / or selection of the following members of the population, with expression of the transgene conferred trait or phenotype. 例如,可以从单次转化事件的后代种群中筛选下述植物,所述植物具有期望的盐和/或氧化胁迫耐性调控多肽和/或核酸的表达水平。 For example, the following can be screened from the progeny plant population in a single transformation event, the plant having a desired salts and / or oxidative stress tolerance-modulating polypeptide and / or nucleic acid expression levels. 可以使用物理和生物化学方法鉴定表达水平。 The expression levels may be identified using physical and biochemical methods. 这些方法包括用于检测多核苷酸的Southern分析或PCR扩增;用于检测RNA转录物的Northern印迹、SI RNase保护、引物延伸或RT-PCR扩增;用于检测多肽和多核苷酸的酶或核酶活性的酶测定;和用于检测多肽的蛋白质凝胶电泳、Western印迹、免疫沉淀和酶联免疫测定。 These include methods for detecting polynucleotide Southern analysis or PCR amplification; Northern blots for detection of RNA transcripts, SI RNase protection, primer-extension, or RT-PCR amplification; polypeptides and polynucleotides for the detection of enzyme enzyme assays or ribozyme activity; and protein gel electrophoresis for detecting the polypeptide, Western blots, immunoprecipitation, and enzyme-linked immunosorbent assay. 也可以使用其他技术例如原位杂交、酶染色和免疫染色来检测多肽和/或多核苷酸的存在或表达。 Other techniques may also be used such as in situ hybridization, enzyme staining, and immunostaining to detect the presence or expression of polypeptides and / or polynucleotides. 进行所有引用的技术的方法是已知的。 All technical methods cited are known. 作为一种备选方案,可以在包含独立转化事件的植物种群中筛选具有期望性状的植物,例如受调节的盐度耐性和/或氧化胁迫耐性水平。 As an alternative, it can be screened for plants having a desired trait in a plant population comprising independent transformation events, e.g. modulated salinity tolerance and / or oxidative stress tolerance levels. 可以在一代或多代中、和/或多于一个地理位置中进行选择和/或筛选。 It may be in one or more generations, and / or more than one location in the selection and / or screening. 在一些情况下,可以在下述条件下生长和选择转基因植物,所述条件诱导期望表型或者以其他方式是转基因植物中产生期望表型所必需的。 In some cases, it can be grown and selection of transgenic plants under the following conditions which induce a desired phenotype or are otherwise produced in a transgenic plant necessary for the desired phenotype. 另外,可以在具体的发育阶段期间应用选择和/或筛选,所述发育阶段中期望由植物表现所述表型。 In addition, application selection and / or screening during particular stages of development, the stage of development of the desired plant exhibits the phenotype. 可以进行选择和/或筛选来选择相对于缺乏转基因的对照植物而言,在盐度耐性和/或氧化胁迫耐性水平中具有统计学显著差异的转基因植物。 You may be selected and / or screening to select lacking the transgene with respect to control plants, transgenic plants having a statistically significant difference in a salinity tolerance and / or oxidative stress tolerance levels. 选择或筛选的转基因植物与相应的对照植物相比具有改变的表型,如本文“转基因植物表型”部分中所述。 Selecting or screening transgenic plants and the corresponding control plant having an altered phenotype, as described in the "transgenic plant phenotype" section herein.

[0193] C.植物物种 [0193] C. Plant Species

[0194] 可以使用本文所述多核苷酸和载体转化大量单子叶植物和双子叶植物和植物细胞体系,包括来自以下科之一的物种:爵床科(Acanthaceae)、葱科(Alliaceae)、六出花禾斗(Alstroemeriaceae)、石蒜禾斗(Amaryllidaceae)、夹竹桃禾斗(Apocynaceae)、掠榈禾斗(Arecaceae)、菊禾斗(Asteraceae)、小檗禾斗(Berberidaceae)、红木禾斗(Bixaceae)、 十字花禾斗(Brassicaceae)、凤梨禾斗(Bromeliaceae)、大麻禾斗(Cannabaceae)、石竹禾4" (Caryophy 1 laceae). ~ 4c ^ (Cephalotaxaceae)、胃禾4" (Chenopodiaceae) > ^C 7jC 仙禾斗(Colchicaceae)、葫声禾斗(Cucurbitaceae)、薯裁禾斗(Dioscoreaceae)、麻黄禾斗(Ephedraceae)、古柯禾斗(Erythroxylaceae)、大戟禾斗(Euphorbiaceae)、ii禾斗(Fabaceae)、 唇形禾斗(Lamiaceae)、亚麻禾斗(Linaceae)、石松禾斗(Lycopodiaceae)、锦葵禾斗(Malvaceae) > 黑药花科(Melanthiaceae)、芭蕉科(Musaceae)、桃金娘科(Myrtaceae [0194] The polynucleotides described herein may be used and a large number of vectors were transformed monocotyledonous and dicotyledonous plants and plant cell systems, including species from one of the following families: Acanthaceae (Acanthaceae), Alliaceae (Alliaceae), six He took a bucket (Alstroemeriaceae), Lycoris Wo bucket (Amaryllidaceae), oleander Wo bucket (Apocynaceae), swept palm Wo bucket (Arecaceae), Wo Ju bucket (Asteraceae), Berberis Wo bucket (Berberidaceae), mahogany Wo bucket (Bixaceae), cruciferous Wo bucket (Brassicaceae), pineapple Wo bucket (Bromeliaceae), cannabis Wo bucket (Cannabaceae), Dianthus Wo 4 "(Caryophy 1 laceae). ~ 4c ^ (Cephalotaxaceae), stomach Wo 4" (Chenopodiaceae )> ^ C 7jC cents Wo bucket (Colchicaceae), gourd sound Wo bucket (Cucurbitaceae), potato cut Wo bucket (Dioscoreaceae), ephedra Wo bucket (Ephedraceae), coca Wo bucket (Erythroxylaceae), Euphorbia Wo bucket (Euphorbiaceae) , ii Wo bucket (Fabaceae), Wo bucket lip (Lamiaceae), flax Wo bucket (Linaceae), Lycopodium Wo bucket (Lycopodiaceae), mallow Wo bucket (Malvaceae)> melanthiaceae (melanthiaceae), Musa (Musaceae ), the myrtle family (Myrtaceae )、珙桐科(Nyssaceae)、S粟禾斗(Papaveraceae)、松禾斗(Pinaceae)、车前禾斗(Plantaginaceae)、禾本科(Poaceae)、蔷薇科(Rosaceae)、茜草科(Rubiaceae)、杨柳科(Salicaceae)、无患子科(Sapindaceae)、爺禾斗(Solanaceae)、红ii杉禾斗(Taxaceae)、山茶禾斗(Theaceae)或葡萄禾斗(Vitaceae)。 ), Dove Branch (Nyssaceae), S Su Wo bucket (Papaveraceae), sonhwa bucket (Pinaceae), the vehicle in front Wo bucket (Plantaginaceae), Gramineae (Poaceae), rose family (Rosaceae), Rubiaceae (Rubiaceae), Willow Branch ( Salicaceae), Sapindaceae (Sapindaceae), Ye Wo bucket (Solanaceae), red cedar ii Wo bucket (Taxaceae), camellia Wo bucket (Theaceae) or grape Wo bucket (Vitaceae).

[0195] 合适的物种可包括以下属的成员:秋葵属(Abelmoschus)、冷杉属(Abies)、 槭属(Acer)、剪股颖属(Agrostis)、葱属(Allium)、六出花属(Alstroemeria)、风梨属(Ananas)、穿心莲属(Andrographis)、须芒草属(Andropogon)、蒿属(Artemisia)、声竹属(Arundo)、颠茄属(Atropa)、小檗属(Berberis)、甜菜属(Beta)、红木属(Bixa)、芸苔属(Brassica)、金盖花属(Calendula)、山茶属(Camellia)、喜树属(Camptotheca)、大麻属(Cannabis) > M® M (Capsicum) > il^lt^M (Carthamus) > (Catharanthus) > 三尖杉属(Cephalotaxus)、商嵩属(Chrysanthemum)、金鸡纳属(Cinchona)、西瓜属(Citrullus)、咖啡属(Cof f ea)、秋水仙属(Colchicum)、锦紫苏属(Coleus)、香瓜属(Cucumis)、南瓜属(Cucurbita)、狗牙根属(Cynodon)、曼陀罗属(Datura)、石竹属(Dianthus)、毛地黄属(Digitalis)、薯蓣属(Dioscorea)、油棕属(Elaeis)、麻黄属(Ephedra)、! [0195] Suitable species may include subordinate members: okra genus (Abelmoschus), Abies (Abies), genus Acer (Acer), Agrostis (Agrostis), allium (Allium), the genus Alstroemeria (alstroemeria), wind pear (Ananas), genus Andrographis (Andrographis), the genus Andropogon (Andropogon), Artemisia (Artemisia), the sound of bamboo genera (Arundo), atropa (Atropa), barberry (Berberis) , sugar beet belongs to (Beta), species of the genus (Bixa), Brassica (Brassica), gold cap is a flower (Calendula), Camellia (Camellia), genus acuminata (Camptotheca), cannabis (cannabis)> M® M (Capsicum)> il ^ lt ^ M (Carthamus)> (Catharanthus)> Cephalotaxus (Cephalotaxus), commercially Song genus (Chrysanthemum), cinchona genus (cinchona), watermelon genus (Citrullus), Coffea (Cof f ea), colchicine is a (Colchicum), the genus Coleus (Coleus), cantaloupe genus (Cucumis), squash (Cucurbita), Cynodon (Cynodon), Datura (Datura), carnation (Dianthus) , Foxglove (digitalis), Dioscorea (Dioscorea), oil palm genus (Elaeis), ephedra (ephedra) ,! ^茅属(Erianthus)、古柯属(Erythroxylum)、按属(Eucalyptus)、羊茅属(Festuca)、草莓属(Fragaria)、雪花莲属(Galanthus)、大豆属(Glycine)、草棉属(Gossypium)、向日葵属(Helianthus)、橡胶树属(Hevea)、大麦属(Hordeum)、天仙子属(Hyoscyamus)、麻风树属(Jatropha)、莴苣属(Lactuca)、亚麻属(Linum)、黑麦草属(Lolium)、羽扇豆属(Lupinus)、番爺属(Lycopersicon)、石松属(Lycopodium)、 木薯属(Manihot)、苜蓿属(Medicago)、薄荷属(Mentha)、芒属(Miscanthus)、芭蕉属(Musa)、烟草属(Nicotiana)、稻属(Oryza)、黍属(Panicum)、罂粟属(Papaver)、银胶菊属(Parthenium)、狼尾草属(Pennisetum)、矮牵牛属(Petunia)、草芦属(Phalaris)、梯牧草属(Phleum)、松属(Pinus)、早熟禾属(Poa)、大戟属(Poinsettia)、杨属(Populus)、萝芙木属(Rauwolfia)、蓖麻属(Ricinus)、蔷薇属(Rosa)、甘蔗属(Saccharum)、杨柳属(Salix)、 血 ^ Festuca (Erianthus), the genus coca (Erythroxylum), according to the case (Eucalyptus), fescue (Festuca), Strawberries (Fragaria), the genus snowdrop (Galanthus), genus soybean (Glycine), cotton genus ( Gossypium), Helianthus (Helianthus), Hevea (Hevea), barley (Hordeum), Hyoscyamus (Hyoscyamus), jatropha (jatropha), the genus Lactuca (Lactuca), the genus flax (Linum), Lolium (Lolium), lupine (Lupinus), Fan Ye genus (Lycopersicon), lycopodium (Lycopodium), Manihot (Manihot), alfalfa (Medicago), mint (Mentha), miscanthus (Miscanthus), Musa (Musa), Nicotiana (Nicotiana), Oryza (Oryza), Panicum (Panicum), poppy (Papaver), parthenium (Parthenium), Pennisetum (Pennisetum), petunia (petunia ), reed canary grass genus (Phalaris), Phleum (Phleum), Pinus (Pinus), bluegrass (Poa), Euphorbia (Poinsettia), Populus (Populus), Rauwolfia (Rauwolfia), Ricinus (Ricinus), Rosa (Rosa), Saccharum (Saccharum), the genus willow (Salix), blood 草属(Sanguinaria)、赛直菪属(Scopolia)、黑麦属(Secale)、爺属(Solanum)、高梁(sorghum)、大米草属(Spartina)、菠菜属(Spinacea)、菊蒿属(Tanacetum)、红豆杉属(Taxus)、可可属(Theobroma)、小黑麦属(Triticosecale)、小麦属(Triticum)、Uniola、藜芦属(Veratrum)、蔓长春花属(Vinca)、葡萄属(Vitis)和玉蜀黍属(Zea)。 Genus (Sanguinaria), henbane is a straight race (Scopolia), rye (Secale), Lord genus (Solanum), sorghum (sorghum), rice genus (Spartina), spinacia (Spinacea), Tanacetum (Tanacetum ), Taxus (Taxus), Theobroma (Theobroma), small rye (Triticosecale), triticale (Triticum), Uniola, Veratrum (Veratrum), vinca (vinca), Vitis (Vitis ) and Zea (Zea).

[0196] 合适的物种包括黍属物种、高粱属物种、芒属物种、甘蔗属物种、蔗茅属物种、 杨属物禾中、大须芒草(Andropogon gerardii)、象草(Pennisetumpurpureum)、丝带草(Phalaris arundinacea)(草声)、狗牙根(Cynodondactylon)(铁线草)、高羊茅(Festuca arundinacea) (Spartinapectinata) (Medicago sativa) (Arundo donax)、黑麦(Secalecereale)、杨柳属物种(柳树)、桉属物种(尤加利树)、小黑麦属(小麦属-小麦X黑麦)和竹子。 [0196] Suitable species include Panicum spp., Sorghum spp., Miscanthus species, sugarcane spp., Erianthus spp., Populus was Wo, the big bluestem (Andropogon gerardii), elephant grass (Pennisetumpurpureum), ribbon grass (Phalaris arundinacea) (grass voice), bermudagrass (Cynodondactylon) (wire grass), tall fescue (Festuca arundinacea) (Spartinapectinata) (Medicago sativa) (Arundo donax), rye (Secalecereale), willow species (willow ), Eucalyptus species (Eucalyptus), triticale genus (Triticum - wheat X rye) and bamboo.

[0197] 白勺•禾中日,(Helianthus annuus) ^X^E (Carthamustinctorius) > 麻风树(Jatropha curcas)、蓖麻(Ricinus communis)、油掠榈(Elaeis guineensis)、亚麻(Linum usitatissimum)禾口#胃(Brassicajuncea)。 [0197] • Wo white spoon and Japan, (Helianthus annuus) ^ X ^ E (Carthamustinctorius)> Jatropha (Jatropha curcas), castor (Ricinus communis), swept the oil palm (Elaeis guineensis), flax (Linum usitatissimum) Wo port # stomach (Brassicajuncea).

[0198] 合适的物种还包括甜菜(Beta vulgaris)和木薯(Manihot esculenta)。 [0198] Suitable species also include sugar beet (Beta vulgaris) and cassava (Manihot esculenta).

[0199] 合适的物种还包括番爺(Lycopersicon esculentum)、莴苣(Lactucasativa)、 ^; ^ (Musa paradisiacal) ( # ^ ), S, # W (Solanum tuberosum) ( ± M ) > M [0199] Suitable species also include Fan Ye (Lycopersicon esculentum), lettuce (Lactucasativa), ^; ^ (Musa paradisiacal) (# ^), S, # W (Solanum tuberosum) (± M)> M

(Brassica oleracea)(花茎甘蓝、花椰菜、抱子甘蓝)、茶树(Camellia sinensis)(茶)、草 (Brassica oleracea) (broccoli, cauliflower, Brussels sprouts), tea (Camellia sinensis) (tea), grass

34莓(Fragaria ananassa)、可可(Theobromacacao)、小果咖啡(Coffea arabica)(咖啡)、 葡萄(Vitis vinifera)、凤梨(Ananas comosus)(菠萝)、辣椒(Capsicum annum)(辣椒禾口舌甘椒)、洋葱(Allium cepa)、舌甘瓜(Cucumis melo)(瓜)、黄瓜(Cucumis sativus)、舆瓜(Cucurbita maxima)(南瓜)、南瓜(Cucurbita moschata)(南瓜)、菠菜(Spinacea oleracea)、西瓜(Citrullus lanatus)、咖啡黄葵(Abelmoschusesculentus)(羊角豆)禾口^p (Solanum melongena)。 34 berries (Fragaria ananassa), cocoa (Theobromacacao), Coffea arabica (Coffea arabica) (Coffee), grapes (Vitis vinifera), pineapple (Ananas comosus) (pineapple), pepper (Capsicum annum) (chili Wo tongue pimento) , onion (Allium cepa), the tongue Gan melon (Cucumis melo) (melon), cucumber (Cucumis sativus), Yu melon (Cucurbita maxima) (pumpkin), pumpkin (Cucurbita moschata) (pumpkin), spinach (Spinacea oleracea), watermelon (Citrullus lanatus), coffee Huang Kui (Abelmoschusesculentus) (okra) Hekou ^ p (Solanum melongena).

[0200] 合适的物种还包括罂粟(Papaver somniferum)、东方罂粟(Papaverorientale)、 欧洲红豆杉(Taxus baccata)、短叶红豆杉(Taxus brevifolia)、青蒿(Artemisia annua) >(Cannabis sativa) (Camptothecaacuminate) (Catharanthus roseus)、长春花(Vinca rosea)、金鸡纳树(Cinchona officinalis) > 秋7jC 仙(Colchicum autumnale)、Veratrumcalifornica.、狭叶毛地黄(Digitalis lanata)、毛地黄(Digitalis purpurea)、暮! [0200] Suitable species also include poppy (Papaver somniferum), Oriental poppy (Papaverorientale), the European yew (Taxus baccata), short leaves of yew (Taxus brevifolia), sweet wormwood (Artemisia annua)> (Cannabis sativa) (Camptothecaacuminate ) (Catharanthus roseus), periwinkle (vinca rosea), the cinchona tree (cinchona officinalis)> autumn 7jC cents (Colchicum autumnale), Veratrumcalifornica., narrow leaf foxglove (digitalis lanata), foxglove (digitalis purpurea), twilight! i 属物禾中、穿莲(Andrographis paniculata)、真页(Atropabelladonna)、Datura stomonium、小檗属物种、三尖杉属物种、草麻黄(Ephedra sinica)、j^HM•禾中、(Erythroxylum coca) > Galanthuswornorii> ^^HM^fK 千层塔(Lycopodium serratum = Huperziaserrata)、石松属物禾中、蛇根木(Rauwolfia serpentina)、萝芙木属物禾中、血卞艮草(Sanguinaria canadensis)、天il]子属物禾中、金 I was in the case of i wear Lin (Andrographis paniculata), real page (Atropabelladonna), Datura stomonium, Berberis species, three sharp cedar species, Ephedra (Ephedra sinica), j ^ in HM • Wo, (Erythroxylum coca )> Galanthuswornorii> ^^ HM ^ fK Melaleuca tower (Lycopodium serratum = Huperziaserrata), Lycopodium is a matter Wo, the snake root wood (Rauwolfia serpentina), Rauwolfia was Wo, the blood Bian Gen grass (Sanguinaria canadensis), day IL] was Wo genus, gold

^ (Calendulaoff icinalis) > ^ (Chrysanthemum parthenium)、^;喉 # ^ (Calendulaoff icinalis)> ^ (Chrysanthemum parthenium), ^; throat #

(Coleusforskohlii)禾口zj、白菊(Tanacetum parthenium)。 (Coleusforskohlii) Wo mouth zj, feverfew (Tanacetum parthenium).

[0201] 合适的物种还包括银胶菊(Parthenium argentatum)、橡胶树属物种(橡胶)、 留兰香(Mentha spicata)(薄荷)、欧薄荷(Mentha piperita)(薄荷)、胭脂树(Bixa orellana)和六出花属物种。 [0201] Suitable species also include guayule (Parthenium argentatum), Hevea spp. (Rubber), spearmint (Mentha spicata) (mint), peppermint (Mentha piperita) (mint), annatto (Bixa orellana) and six flower species.

[0202] 合适的物种还包括蔷薇属物种(玫瑰)、麝香石竹(Dianthuscaryophyllus)(康乃馨)、矮牵牛属物种(矮牵牛)和猩猩草(Poinsettiapulcherrima)。 [0202] Suitable species also include Rosa spp (Rose), carnation (Dianthuscaryophyllus) (carnation), Petunia spp. (Petunia) and orangutan grass (Poinsettiapulcherrima).

[0203] 合适的物种还包括烟草(Nicotiana tabacum)、白羽扇豆(Lupinusalbus)(羽扇豆)、Uniola paniculata (燕麦)、bentgrass (剪股颖属物禾中)、Populus tremuloides (白杨)、松属物种(松树)、冷杉属物种(冷杉)、槭属物种(槭树)、大麦(Hordeum vulgare)、 草地早熟禾(Poa pratensis)(蓝草)、黑麦草属物种(黑麦草)和猫尾草(Phleum pratense)(梯牧草)。 [0203] Suitable species also include tobacco (Nicotiana tabacum), White Lupine (Lupinusalbus) (lupine), Uniola paniculata (oats), bentgrass (Agrostis was Wo in), Populus tremuloides (aspen), Pinus species (pine), species Abies (fir), maple species (maple), barley (Hordeum vulgare), Kentucky bluegrass (Poa pratensis) (blue grass), Lolium species (ryegrass) and Timothy (Phleum pratense) (timothy).

[0204] 因此,可以在宽泛的植物物种范围中使用所述方法和组合物,包括来自双子叶属芸苔属、红蓝花属、大豆属、草棉属、向日葵属、麻风树属、银胶菊属、杨属和蓖麻属;和单子叶属油棕属、羊茅属、大麦属、黑麦草属、稻属、黍属、狼尾草属、梯牧草属、早熟禾属、甘蔗属、黑麦属、茄属、小黑麦属、小麦属和玉蜀黍属的物种。 [0204] Thus, using the methods and compositions in a wide range of plant species, including dicotyledonous genera Brassica, red and blue flowers genus, genus soybean, cotton, Helianthus, Jatropha, silver from Parthenium genus Populus and Ricinus; and monocots belong to oil palm, Festuca, Hordeum, Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, sugar cane , Secale, Solanum, triticale, Triticum and Zea species. 在一些实施方案中,植物是柳枝稷、 两色蜀黍(高粱,双色高粱)、巨芒草(芒属)、甘蔗属物种(能源蔗)、香脂白杨(白杨)、玉蜀黍(玉米)、大豆(黄豆)、欧洲油菜(卡诺拉油菜)、普通小麦(小麦)、陆地棉(棉花)、 稻(水稻)、向日葵、紫苜蓿(苜蓿)、甜菜或御谷(珍珠粟)物种的成员。 In some embodiments, the plant is a Panicum virgatum, Sorghum bicolor (sorghum, Sorghum bicolor), giant Miscanthus (miscanthus), Saccharum species (energy cane), balsam poplar (poplar), Zea mays (maize), soybeans (soy) , oilseed rape (canola), common wheat (wheat), upland cotton (cotton), rice (rice), sunflower, alfalfa (Medicago sativa), beets or members of bajra (pearl millet) species.

[0205] 在某些实施方案中,可以使用本文所述多核苷酸和载体转化大量单子叶和双子叶植物和植物细胞体系,其中这类植物是不同物种或物种变种(例如甘蔗属物种X芒属物种) 的杂种。 [0205] In certain embodiments, the polynucleotides described herein may be used to transform a number of monocotyledonous and vectors and dicotyledonous plants and plant cell systems, wherein such plants are of different species or varieties (such as sugarcane species X Mountain hybrid species) of.

[0206] D.转基因植物表型[0207] 在一些实施方案中,其中盐度和/或氧化胁迫调控多肽的表达得到调控的植物可具有提高的盐度和/或氧化胁迫耐性水平。 [0206] D. Transgenic Plant Phenotypes [0207] In some embodiments, wherein the salinity and / or oxidative stress-modulating polypeptide expression in plants can be regulated obtained having increased salinity and / or oxidative stress tolerance levels. 例如,可以在转基因植物中表达本文所述的盐度和/或氧化胁迫调控多肽,得到提高的盐度和/或氧化胁迫耐性水平。 For example, it can be expressed herein salinity and / or oxidative stress in transgenic plants modulating polypeptide, improved salinity and / or oxidative stress tolerance levels. 与不表达转基因的相应对照植物中的水平相比,盐度和/或氧化胁迫耐性水平可以提高至少2%,例如2%,3%,4%,5%,6%,7%,8%,9% ,10% ,11% >12% ,13% ,14% ,15% ,16% ,17% ,18%, 19%、20%、25%、30%、35%、40%、45%、50%、55%、60%或大于60%。 Compared to a corresponding control plant that does not express the transgene in the level of salinity and / or oxidative stress tolerance levels can be increased by at least 2%, e.g. 2%, 3%, 4%, 5%, 6%, 7%, 8% , 9%, 10%, 11%> 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 55%, 60%, or greater than 60%.

[0208] 本发明的核酸分子和多肽是感兴趣的,因为当核酸分子被异常表达(即在非天然位点表达,或相对于野生型以被提高或降低的量表达)时,它们产生下述植物,所述植物与野生型植物相比,显示提高的盐耐性和/或氧化胁迫耐性,如下文公开的多个实验的结果所部分证实。 Under [0208] the nucleic acid molecules and polypeptides of the present invention are of interest because when the nucleic acid molecule is abnormally expressed (i.e., non-native site in the expression, relative to wild type or amount is increased or decreased expression), they produce said plant, said plant compared to wild type plants, exhibit improved salt tolerance and / or oxidative stress tolerance, the results of multiple experiments disclosed in the section below confirmed. 具体地,用本发明的核酸分子和多肽转化的植物可具有大量与野生型植物相比经修饰的特征。 In particular, with nucleic acid molecules and polypeptides of the invention may have a large number of transformed plants compared to wild type plants modified features. 经修饰的特征的实例包括可以通过植物高度、叶或莲座面积或干物质测量的光合效率、幼苗面积和生物量。 Examples of features include modified by plant height, leaf or rosette area photosynthetic efficiency or dry matter measured, and the area on the biomass. 可以在不同的植物发育阶段例如种子、幼苗、抽苔、衰老等等中观察和测量经修饰的特征。 May, for example seed, seedling, bolting, aging and the like was observed in the measurement of the modified features of different stages of development of plants. 通常,盐或氧化耐性可以被表述为测量的比例或组合,例如盐生长指数值或水杨酸生长指数值。 Typically, salt or oxidative tolerance can be expressed as the ratio of measured or in combination, such as salt growth index values, or salicylic acid growth index values. 例如,用本发明序列转化的植物可显示至少5%、至少10%、至少25%、至少50%、至少75%、至少100%、至少200%、至少300%、至少400% 或甚至至少500%的SGI、幼苗面积和/或SAGI值提高。 For example, plant sequences transformed according to the present invention can display at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, or even at least 500 % of the SGI, area and / or SAGI values ​​increase seedling. 这些性状可以被用于开发或最大化植物产品。 These traits can be used to develop or maximize plant products. 例如,本发明的核酸分子和多肽被用于提高基因的表达,这引起植物在盐和/ 或氧化条件下与相同条件下的野生型植物相比具有提高的生物量、生长速率和/或幼苗活力。 For example, nucleic acid molecules and polypeptides of the invention are used to increase the expression of a gene, which causes plants under salt and / or oxidative conditions compared to a wild type plant under the same conditions with increased biomass, growth rate and / or seedling vitality.

[0209] 因为公开的序列和方法提高盐和/或氧化胁迫条件下的营养生长和生长速率,所以公开的方法能够用于增加在盐和/或氧化条件下生长的植物中的植物生长。 [0209] Because the disclosed sequences and methods increase vegetative growth and growth rate in a salt and / or oxidative stress conditions, the disclosed methods can be used for increased plant under salt and / or growth of the oxidation conditions of plant growth. 例如,本发明的植物在盐和/或氧化条件下与下述植物相比显示提高的光合成效率和提高的幼苗面积,所述植物是相同物种但是未经遗传修饰为主要是营养生长。 For example, plants of the present invention is described below in comparison with plants under salt and / or oxidative conditions exhibits an enhanced area of ​​the light synthesis efficiency and increased seedling, the plant is of the same species but not genetically modified to be primarily vegetative growth. 生物量产生提高的实例包括与相同条件下相同物种的野生型植物的生物量产生相比,至少5%、至少20%、或甚至至少50%的提高。 Examples include an increased biomass production as compared to wild-type plant biomass produces the same species under the same conditions, at least 5%, at least 20%, or even at least 50% increase.

[0210] 通常,使用适当参数或非参数统计学(例如x方检验、学生t_检验、 Marm-Whitney检验或F-检验)时,转基因植物或细胞相对于对照植物或细胞而言对盐度和/或氧化胁迫耐性量的差异在p < 0. 05时被认为是统计学显著的。 When [0210] Generally, using the appropriate parametric or nonparametric statistics (e.g., x-square test, Student test t_, Marm-Whitney test or test F-), a transgenic plant or cell relative to a control plant or cell of salinity differences and / or oxidative stress tolerance amount is considered when p 05 <0.01 was statistically significant. 在一些实施方案中,盐度和/或氧化胁迫耐性量的差异在P < 0. 01、p < 0. 005或? In some embodiments, differences in salinity and / or oxidative stress tolerance in the amount of P <0. 01, p <0. 005 or? < 0. 001时是统计学显著的。 <0.001 when is statistically significant.

[0211] 相对于对照植物评价转基因植物的表型。 [0211] Transgenic plant phenotypic evaluation of the transgenic plants. 当植物显示的多肽量或编码多肽的mRNA量少于目的植物显示量的10%,例如少于9%、8%、7%、6%、5%、4%、3%、2%、1%、 0.5%、0.1%、0.01%或0.001%时,植物被称作“不表达”所述多肽。 When the amount of the polypeptide or mRNA encoding the polypeptide plants display object is less than 10% of the plants showed an amount of, for example, less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1%, 0.01%, or 0.001%, the plant is referred to as "not expressed" the polypeptide. 可以使用下述方法评价表达,所述方法包括例如RT-PCR、Northern印迹、SI RNase保护、引物延伸、Western印迹、 蛋白质凝胶电泳、免疫沉淀、酶联免疫测定、芯片测定和质谱法。 The method may be used to evaluate the following expression, the method comprising, for example, RT-PCR, Northern blot, SI RNase protection, primer extension, Western blots, protein gel electrophoresis, immunoprecipitation, enzyme-linked immunoassays, chip assays and mass spectrometry. 应当注意,如果多肽在组织优先或广泛表达启动子的控制下表达,则可以在整株植物或选择的组织中评价表达。 It should be noted that, if the tissue-preferential or broadly expressing polypeptide under the control of a promoter the expression can be evaluated in a whole plant or in a selected tissue expression. 类似地,如果多肽在特定时间(例如发育或诱导后的特定时间)表达,则可以在期望的时间段中选择性地评价表达。 Similarly, if a polypeptide is expressed at a particular time (e.g., after a certain time or induction of development), the expression can be evaluated selectively at a desired time period.

[0212] V.植物育种 [0212] V. Plant Breeding

[0213] 遗传多态性是种群中离散的等位基因序列差异。 [0213] Genetic polymorphism is allelic sequence differences in discrete populations. 通常,以或更多存在的等位基因被认为是遗传多态性。 Generally, the presence of the allele or more is considered to be genetic polymorphism. 本文公开的多肽能够调控盐度耐性和/或氧化胁迫耐性含量的发现可用于植物育种,因为对这类多肽的基因座显示一定程度连锁的遗传多态性更可能与盐度耐性和/或氧化胁迫耐性性状的变异相关。 Polypeptide disclosed herein can modulate salinity tolerance and / or oxidative stress levels found tolerance may be useful in plant breeding, since the display a certain degree of genetic polymorphism linked loci such polypeptides are more likely with a salinity tolerance and / or oxidative variation-related stress tolerance traits. 例如,与这类多肽基因座关联的遗传多态性更可能用于标记物辅助的育种程序中,产生在盐度耐性和/或氧化胁迫耐性性状中具有期望调控的品系。 For example, genetic polymorphism associated with a locus such polypeptides are more likely to be used in marker-assisted breeding program to produce strains having the desired regulation in the salinity tolerance and / or oxidative stress tolerance traits in.

[0214] 因此,本发明的一个方面包括鉴定一种或多种遗传多态性是否与盐度耐性和/或氧化胁迫耐性性状的变异相关的方法。 [0214] Accordingly, one aspect of the present invention comprises identifying one or more genetic polymorphisms is associated with variation salinity tolerance and / or oxidative stress tolerance trait method. 这类方法涉及测定给定种群内的遗传多态性是否与图1到6中所示多肽之一和/或其功能性同源物(例如但不限于序列表中所示功能性同源物)的基因座显示连锁。 Such methods relates to the determination of genetic polymorphisms in a given population to FIGS whether one of the polypeptides and / or functional homologue thereof in FIG 16 (e.g., without limitation, a functional homolog shown in Sequence Listing ) locus display chain. 测量植物种群中盐度耐性性状和/或氧化胁迫耐性性状的变异和种群植物中一个或多个遗传多态性的存在之间的关联,从而鉴定一个或多个遗传多态性是否与性状的变异相关。 Measurement of salinity tolerance traits in plant populations and / or variations and plant populations oxidative stress tolerance trait association between a genetic polymorphism present in one or more, in order to identify whether one or more genetic polymorphisms and traits related variation. 如果具体等位基因的存在统计学显著地与盐度耐性和/或氧化胁迫耐性性状的期望调控相关,则等位基因与所述一种或两种性状的变异相关,并可用作一种或多种所述性状的标记物。 If a particular allele is statistically significantly correlated with a desired regulation of salinity tolerance and / or oxidative stress tolerance traits, the allele is associated with variation of one or both of the traits and used as an or more markers in the trait. 另一方面,如果具体等位基因的存在不与期望的调控显著相关, 则等位基因不与一种或多种所述性状的变异相关,并且不可用作标记物。 On the other hand, if there is not a desired allele specific regulatory significant correlation, the variant alleles are not associated with one or more of the traits and is not useful as a marker.

[0215] 一些方法可用于下述种群,所述种群含有天然存在的内源多肽而不是编码所述多肽的外源核酸,即所述种群对外源核酸而言不是转基因的。 [0215] The following methods may be used for population, said population comprising naturally occurring endogenous polypeptide rather than an exogenous nucleic acid encoding the polypeptide, i.e., populations of the exogenous nucleic acids are not transgenic. 然而,应当理解,适用于所述方法的种群可含有针对另一不同性状(例如除草剂抗性)的转基因。 However, it should be understood that the method is suitable for the population may contain a transgene for another, different traits (e.g. herbicide resistance).

[0216] 适用于这类方法中的遗传多态性包括简单序列重复(SSR,或微卫星)、多态DNA的快速扩增(RAPD)、单核苷酸多态性(SNP)、扩增的片段长度多态性(AFLP)和限制性片段长度多态性(RFLP)。 [0216] suitable for genetic polymorphism such methods include simple sequence repeats (the SSR, or microsatellites), rapid amplification of polymorphic DNA (of RAPD), single nucleotide polymorphism (the SNP), amplification the fragment length polymorphism (of AFLP) and restriction fragment length polymorphism (RFLP). 例如可以如下鉴定SSR多态性:制备序列特异性探针,并通过PCR从目的种群中的个体扩增模板DNA。 SSR polymorphisms can be identified, for example, as follows: Preparation of sequence specific probes and amplifying template DNA from individuals in the population of interest by PCR. 如果探针在种群中SSR的两侧,则会产生不同大小的PCR 产物。 If both sides of the probe populations in SSR will be generated PCR products of different sizes. 见例如美国专利5,766,847。 See, for example, US Patent 5,766,847. 或者,可以通过使用一个或多个PCR产物作为探针, 针对来自种群中不同个体的Southern印迹鉴定SSR多态性。 Alternatively, by using one or more PCR product as a probe for Southern from different individuals in the population blot SSR polymorphisms. 见UH Refseth等,(1997) Electrophoresis 18 : 1519。 See UH Refseth etc., (1997) Electrophoresis 18: 1519. RFLP 的鉴定例如在Alonso-Blanco 等(Methods in Molecular Biology,第82 卷,"ArabidopsisProtocols,,,第137-146 页,JM Martinez-Zapater 禾口J.Salinas 编,c. 1998, Humana Press, Totowa, NJ) ;Burr( "Mapping Genes with Recombinantlnbreds,,,第249-254页,Freeling,M.禾口V. Walbot (编),The MaizeHandbook, c.1994, Springer-Verlag New York, Inc. :New York, NY, USA ;Berlin Germany ;Burr 等Genetics (1998) 118 :519 ;和Gardiner,J. (1993) Genetics 134:917)中讨论。 Identification of RFLP for example, in Alonso-Blanco et (Methods in Molecular Biology, Vol. 82, "ArabidopsisProtocols ,,, pp. 137-146, JM Martinez-Zapater Hekou J.Salinas ed, c. 1998, Humana Press, Totowa, NJ); Burr ( "Mapping Genes with Recombinantlnbreds ,,, pp. 249-254, Freeling, M Wo mouth V. Walbot (ed.), The MaizeHandbook, c.1994, Springer-Verlag New York, Inc.: New York , NY, USA; Berlin Germany; Burr et Genetics (1998) 118: 519; (1993) Genetics 134 and Gardiner, J:. 917) discussed. AFLP 的鉴定在例如EP 0 534 858和美国专利5,878,215中讨论。 AFLP Identification example discussed in EP 0 534 858 and in U.S. Patent 5,878,215.

[0217] 在一些实施方案中,该方法涉及对植物品系育种。 [0217] In some embodiments, the method involves breeding plant line. 这类方法在标记物辅助的育种程序中使用如上文所述鉴定的遗传多态性以便于开发下述品系,所述品系在一种或多种盐度耐性和/或氧化胁迫耐性性状中具有期望的改变。 Such methods use genetic polymorphisms identified as described above in a marker assisted breeding program to facilitate the development of the following line, the line having one or more of salinity tolerance and / or oxidative stress tolerance traits in desired change. 一旦合适的遗传多态性被鉴定为与性状的变异相关,则鉴定具有与期望的变异相关的多态等位基因的一株或多株个体植物。 Once a suitable genetic polymorphism is identified as being associated with variation characteristics, the identification of polymorphic alleles associated with a desired variation of one or more individual plant lines. 然后在育种程序中使用这些植物,将多态等位基因和与期望变异相关的其他基因座上的大量其他等位基因组合。 Plants are then used in a breeding program, a number of other combinations of alleles and polymorphic alleles of other loci related to the desired variation. 适用于植物育种程序中的技术是本领域已知的,并且包括但不限于, 回交、混合选择(mass selection)、系谱育种、混合选择(bulkselection)、与其他种群杂交和轮回选择。 Applicable to plant breeding program techniques are known in the art, and include, without limitation, backcrossing, mass selection (mass selection), pedigree breeding, hybrid selection (bulkselection), and other populations hybridization and recurrent selection. 这些技术可以单独使用或者与育种程序中的一种或多种其他技术组合使用。 These techniques can be used alone or in a breeding program in one or more in combination with other techniques. 因此,使每株鉴定的植物自交或与不同的植物杂交产生种子,随后使所述种子萌发形成 Thus, identification of plants per selfed to produce seed, or crossing a plant with a different, subsequently forming the seed germination

37后代植物。 37 progeny plants. 然后使至少一株这类后代植物自交或与不同的植物杂交形成后继的后代世代(subsequent progeny generation)。 Then at least one of such selfed progeny plant or progeny of succeeding generations formed (subsequent progeny generation) with different plant hybridization. 育种程序可以适当地将自交或异型杂交再重复0到5个世代,从而在得到的植物品系中实现期望的均一性和稳定性,所述得到的植物品系保留多态等位基因。 The breeding program can be suitably selfing or outcrossing repeated 0-5 generations to achieve the desired uniformity and stability in the resulting plant line, the retention plant lines obtained polymorphic alleles. 在大部分育种程序中,应当在每个世代中进行具体的多态等位基因分析,尽管需要时可以在交替的世代中进行分析。 Should be specifically polymorphic alleles analyzed in each generations in most breeding programs, analysis can be carried out in alternate generations although required.

[0218] 在一些情况下,还进行对其他有用性状到选择,例如选择真菌抗性或细菌抗性。 [0218] In some cases, also to the selection of other useful traits, such as selection of fungal resistance or bacterial resistance. 针对这类其他性状的选择可以在鉴定具有期望多态等位基因的个体植物之前、期间或之后进行。 Before an individual plant may be desirable in the identification of polymorphic alleles selected for such other traits, during or after.

[0219] VI.制造物品(article of manufacture) [0219] VI. Manufactured article (article of manufacture)

[0220] 本文提供的转基因植物在农业和能源生产工业中具有多种用途。 [0220] Transgenic plants provided herein have various uses in the agricultural and energy production industries. 例如,本文所述转基因植物可用于制造动物饲料和食品。 For example, the transgenic plants described herein can be used in the manufacture of animal feed and food. 然而这类植物通常尤其可用作能源生产的原料。 However, such plants are typically used as raw materials for energy production in particular.

[0221] 本文所述转基因植物相对于缺乏外源核酸的对照植物而言,通常每公顷产生更高的谷物和/或生物量产量。 [0221] As used herein the transgenic plant lacking the exogenous nucleic acid with respect to control plants, typically produce higher grain and / or biomass production per hectare. 在一些实施方案中,在降低的投入(例如肥料和/或水)的条件下生长时,这类转基因植物相对于对照植物而言每公顷提供相等或甚至提高的谷物和/ 或生物量产量。 In some embodiments, when grown in reduced investment under conditions (fertilizers and / or water, for example), such transgenic plants relative to control plants provide equivalent per hectare or even improved grain and / or biomass production. 因此,可以使用这类转基因植物在更低的投入成本和/或环境胁迫的条件例如干旱下提供产量稳定性。 Thus, such transgenic plants may be used to provide yield stability at a lower input cost conditions and / or under environmental stresses such as drought. 在一些实施方案中,本文所述植物具有下述组成,所述组成允许被更有效地加工成游离糖随后加工成醇,用于能源生产。 In some embodiments, plants described herein have the following composition, allowing the composition to be more efficient processing into free sugars, then processed into an alcohol, for energy production. 在一些实施方案中,这类植物相对于对照植物而言每千克植物材料提供更高的乙醇、丁醇、其他生物燃料分子和/或糖衍生的副产物的产量。 In some embodiments, such plants relative to control plants per kg of plant material provides higher ethanol, butanol, other biofuel molecules yield and / or sugar-derived byproducts. 通过在相等或甚至降低的生产成本下提供相对于对照更高的产量,本文所述的转基因植物对农民和加工者而言提高了收益率,并且对消费者而言降低了成本。 By providing equal or even at a reduced production cost higher yield relative to control transgenic plant as described herein for farmers and processors in terms of increased yield and reduced costs for consumers.

[0222] 可以通过本领域已知的手段使来自本文所述转基因植物的种子保持良好状态并装袋在包装材料中,形成制造物品。 [0222] described herein can be made from the seeds of transgenic plants in good condition and bagged in packaging material by means known in the art to form an article of manufacture. 包装材料例如纸和布是本领域公知的。 Packaging materials such as paper and cloth are well known in the art. 种子的包装可具有描述其中种子特质的标记,例如缝在包装材料上的标签或标记、印制在包装材料上的标记、或插入包装内的标记。 Seed package may have the characteristics described in which the seed marker, for example on the packaging material sewn tag or label, printed indicia on the packaging material, or insertion marker within the package.

[0223] 增强的盐和/或氧化胁迫耐性提供了下述机会:在盐或氧化胁迫条件下培养作物而没有盐诱导的离子失衡、水内稳态的破坏、代谢的抑制、对膜的损害和/或细胞死亡导致的矮化生长和减少的产量。 [0223] Enhanced salt and / or oxidative stress tolerance offers the following opportunities: growing crops under salt or oxidative stress conditions without salt-induced ion imbalance, disruption within the water homeostasis, inhibition of metabolism, damage to the film stunted growth and reduced production and / or cell death caused. 在盐或氧化胁迫条件下培养植物的能力会导致可耕田地的全面扩张,和目前由于提高的盐度或氧化胁迫条件而具有较低生产力的土地的提高的产出。 Capacity-building plant in the salt or oxidative stress conditions can lead to overall expansion of farming land, and is currently due to the salinity or oxidative stress conditions have improved and increased land less productive output.

[0224] 种子或幼苗活力是能够大幅影响植物(例如作物植物)成功生长的一个重要特征。 [0224] seed or seedling vigor is capable of significantly affecting an important feature of the plant (such as crop plants) successful growth. 不利的环境条件例如盐和/或氧化条件能够影响植物生长周期、种子萌发和幼苗活力(即在这类条件下成功和失败的植物生长之间生活力和强度会有差异)。 Adverse environmental conditions such as salt and / or oxidative conditions can affect a plant growth cycle, germination and seedling vigor (Viability and Growth i.e. strength between the success and failure of the plant under such conditions will vary). 幼苗活力通常被定义为包括下述种子特性,所述种子特性确定“在大范围大田条件下正常幼苗快速、均一发生和发育的潜能”。 Seedling vigor is typically defined as comprising a seed characteristic, said characteristic determining seed "in a wide range of field conditions of normal seedlings fast, occurrence and development of a uniform potential." 因此,开发下述植物种子是有利的,所述植物种子具有提高的活力,尤其是在提高的盐度和/或氧化胁迫条件下。 Therefore, following the development of the plant seed is advantageous, the plant having increased seed vigor, particularly in salinity and / or oxidative stress conditions is improved.

[0225] 例如,对于谷物植物例如稻、玉米、小麦等等的产生而言,提高的种子活力会是有利的。 [0225] For example, for a plant to produce cereals such as rice, corn, wheat, etc., increased seed vigor would be advantageous. 对这些作物而言,盐化和/或氧化通常会减缓或终止萌发和生长。 These crops, salified and / or oxidation generally reduced or terminated germination and growth. 因此寻找与盐和/或氧化胁迫条件下提高的种子活力相关的基因来产生改进的植物变种(Walia等(2005) Plant Physiology 139 :822_835)。 Thus looking to improve the correlation and / or oxidative stress conditions salts of seed vigor gene to produce improved plant varieties (Walia et (2005) Plant Physiology 139: 822_835).

[0226] 本发明将在以下实施例中进一步描述,所述实施例不限制权利要求书中所述本发 [0226] The present invention will be further described in the following embodiments, the embodiments do not limit the claims of the invention

38明的范围。 38 out of range.

[0227] VII.实施例 [0227] VII. Example

[0228] 实施例1 :农杆菌介导的拟南芥转化 [0228] Example 1: Agrobacterium-mediated transformation of Arabidopsis thaliana

[0229] 宿主植物和转基因:用含有下述克隆的Ti质粒独立转化野生型拟南芥ffassilewskija(WS)植物,所述克隆编码SEQ ID NO :43、44、45、86、136、138、140、141、 142、143、144和SEQ ID NO :140的氨基酸等同物1到135的多肽。 [0229] and a transgenic host plant: the Ti plasmid clone containing the following independent transformation wild-type Arabidopsis ffassilewskija (WS) plants, the clone encodes SEQ ID NO: 43,44,45,86,136,138,140 , 141, 142,143,144, and SEQ ID NO: 1 to 135 amino acid polypeptide equivalents 140. 实例包括Ceres克隆ID no. 1792354、Ceres SEEDLINE IDno. ME06748、Ceres SEEDLINE ID no. ME08768、Ceres SEEDLINE IDno. ME19173 和Ceres 克隆ID no. 375578。 Examples include Ceres CLONE ID no. 1792354, Ceres SEEDLINE IDno. ME06748, Ceres SEEDLINE ID no. ME08768, Ceres SEEDLINE IDno. ME19173 and Ceres CLONE ID no. 375578. 除非另有说明,每个Ceres 克隆和/ 或来自克隆的幼苗处于相对于Ti质粒中任一35S启动子而言有义的方向上。 Unless otherwise indicated, each Ceres Clone and / or seedling from clones in any direction with respect to a Ti plasmid 35S promoter in terms of a sense. 适用于这类构建体的Ti质粒载体CRS 338含有Ceres-构建的植物可选择标记基因膦丝菌素乙酰转移酶(PAT),其赋予被转化的植物除草剂抗性。 Ti plasmid vector construct suitable for such plants contain CRS 338 constructed Ceres- selectable marker gene phosphinothricin acetyltransferase (PAT), which confers herbicide resistance to transformed plants.

[0230] 土壤混合物的制备:将24L SunshineMix#5 土壤(Sun Gro Horticulture, Ltd., Bellevue, WA)与16L Therm-0-Rock 蛭石(Therm-0-Rock West, Inc.,Chandler, AZ)在水泥混合机(cement mixer)中混合,制备60 : 40的土壤混合物。 [0230] Preparation of Soil Mixture: 24L SunshineMix # 5 the soil (Sun Gro Horticulture, Ltd., Bellevue, WA) with 16L Therm-0-Rock vermiculite (Therm-0-Rock West, Inc., Chandler, AZ) cement mixer (cement mixer) were mixed preparation 60: 40 mixture of soil. 向该土壤混合物中添加2 Tbsp Marathon 颗粒(Hummert,EarthCity,M0)、3Tbsp 0SM0C0TE® 14-14-14 (Hummert, Earth City, M0)和lTbsp Peters 肥料20-20-20 (JR Peters, Inc.,Allentown, PA),这些被添加的成分首先被添加进3加仑水中,然后添加进土壤中并充分混合。 Add 2 Tbsp Marathon granules (Hummert, EarthCity, M0) to the soil mixture, 3Tbsp 0SM0C0TE® 14-14-14 (Hummert, Earth City, M0) and lTbsp Peters fertilizer 20-20-20 (JR Peters, Inc., allentown, PA), were added to these ingredients are first added to 3 gallons of water, soil and mixed well and then add. 一般用土壤混合物填充4-英寸直径的花盆。 4- inch diameter pots are typically filled with soil mixture. 然后用8-英寸的尼龙膜方块覆盖花盆。 Pots then covered with 8-inch squares of nylon membrane.

[0231] 种植:使用60mL注射器抽吸35mL的种子混合物。 [0231] planting: seed using a 60mL syringe pump a mixture of 35mL. 向每盆中添加25滴。 Add 25 drops to the pot. 在花盆顶部放置透明的繁殖盖子(propagation dome),然后将盆置于55%阴影的编织物下并通过添加1英寸水进行地下灌溉(subirrigated)。 A transparent cover disposed under propagation (propagation dome) at the top of the pots, and the pots placed in the shadow of the braid 55% by adding 1 inch of water subirrigated (subirrigated).

[0232] 植物培养:种植后3到4天,去除盖子和阴影编织物。 [0232] Plant Culture: 3-4 days after planting, lids and removing shadows braid. 根据需要给植物浇水。 According to water the plants need. 7-10 天后,使用镊子将花盆间苗至每盆20株植物。 After 7-10 days, pots thinning using forceps to 20 plants per pot. 2周后,用Peters肥料以每加仑水ITsp的比率对所有的植物进行地下灌溉。 After 2 weeks, all subirrigated with Peters fertilizer at a rate of plants per gallon of water ITsp. 当花芽(bolt)约5-10厘米长时,将其在第一节和茎底部之间修剪以诱导次生花芽(secondary bolt)。 When the flower buds (Bolt) about 5-10 cm long, they are trimmed between the first and the bottom stem to induce secondary flower buds (secondary bolt). 修剪后6到7天进行浸泡浸润(Dipping infiltration)。 6-7 days after pruning soaking infiltration (Dipping infiltration).

[0233] 农杆菌的制备:向150mL新鲜的YEB中添加各0. lmL的羧苄西林、壮观霉素和利福平(各为100mg/ml的储存浓度)。 [0233] Preparation of Agrobacterium: adding 0. lmL of each of carbenicillin, spectinomycin and rifampicin to 150mL of fresh YEB (each of 100mg / ml in storage). 获得农杆菌起子板(starter block)(含有生长至0D_ 约1. 0的农杆菌培养物的96-孔板)并通过从起子板的适当孔中转移lmL来接种每种构建体一个培养瓶。 Agrobacterium starter plate obtained (starter block) (containing about 0D_ grown Agrobacterium culture to 1.0 of the 96-well plates) and by transferring the appropriate wells lmL starter plate was used to inoculate a culture flask for each construct. 然后将培养物在27°C振荡培养。 The culture was then cultured with shaking at 27 ° C. 达到约1. 0的0D6TO(约24小时)后将培养物离心。 Up to about 1.0 of 0D6TO (about 24 hours) after culture was centrifuged. 向重悬的农杆菌沉淀物中添加200mL浸润培养基。 Was added to 200mL infiltration resuspended Agrobacterium culture precipitate. 浸润培养基通过向900mL水中添加2. 2g MS盐、50g蔗糖和5 u 1 2mg/ml苄氨基嘌呤制备。 Infiltration by 2. 2g MS salts Medium 50g of sucrose and 5 u 1 2mg prepared, / ml benzylaminopurine was added to 900mL water.

[0234] 浸泡浸润:将花盆倒置并浸入水中5分钟,从而植物的地上部分被置于农杆菌悬浮液中。 [0234] soaking infiltration: The pots were inverted and immersed in water for 5 minutes to ground parts of plants are placed in an Agrobacterium suspension. 允许植物正常生长并收集种子。 Allow normal growth of plants and collecting seeds.

[0235] 实施例2 :盐条件筛选 Salt screening conditions: [0235] Example 2

[0236] 盐条件筛选:筛选常规地通过高盐琼脂平板测定以及高盐土壤测定进行。 [0236] Filter salt conditions: screening routinely performed by high-salt agar plate assay and determination of high soil salt. 在高盐条件下评价的性状包括:幼苗面积、光合效率、盐生长指数和再生能力。 Evaluation under high salt conditions traits include: area, photosynthesis efficiency, salt growth index and regeneration ability seedlings.

[0237] 幼苗面积:约2周龄幼小植物的总叶面积。 [0237] Seedling area: the total leaf area of ​​a young plant about 2 weeks old.

[0238] 光合效率(Fv/Fm):通过最大荧光信号Fm和可变荧光Fv之间的关系评价幼苗光合效率或通过光系统II的电子传递。 [0238] Photosynthesis efficiency (Fv / Fm): Seedling photosynthetic efficiency was evaluated by the relationship between Fm, the maximum fluorescence signal Fv variable fluorescence or transmitted through an electronic optical system II. 本文中,最适量子产率(Fv/Fm)的降低表示胁迫,因此可以被用于监测盐胁迫条件下与非转基因植物相比转基因植物的性能。 As used herein, reducing the optimum quantum yield (Fv / Fm) indicates stress, and therefore can be used for the performance of transgenic plants compared to non-transgenic plants under salt stress monitoring.

[0239] 盐牛长指数=幼苗面积x光合效率(Fv/Fm)。 [0239] Bovine salt-index = seedling area x photosynthesis efficiency (Fv / Fm).

[0240] 再牛能力:将茎切除后植物在盐土壤中再生枝条的能力,土壤用200mM NaCl溶液灌溉。 [0240] and then cattle capability: the ability of the plant regenerated shoots in saline soil after stems are cut, the soil was irrigated with 200mM NaCl.

[0241] 转化体鉴定:使用PCR在一个随机选择的T2植物中扩增cDNA插入片段。 [0241] transformants were identified: cDNA insert was amplified by PCR in a randomly chosen T2 plant. 然后测序该PCR产物来证实植物中的序列。 The PCR product was then sequenced to confirm the sequence in a plant.

[0242] 鉴定盐胁迫耐件棺物:在种子的超级池(superpool)中筛选对SA(如下文详述)和高盐显示增强耐性的转基因植物。 [0242] Identification of resistance to salt stress was member Coffin: screening SA (as detailed below) and show enhanced high salt tolerance in transgenic plant seeds super pool (superpool) in. 对三个独立的候选植物测序,并且转基因序列与ME02064 匹配。 Three independent candidate plants for sequenced and the transgene sequence matched ME02064.

[0243] 评价对盐胁迫的耐件:通常选择四到十个独立转化的植物品系并定性评价它们在1\世代中对盐胁迫的耐性。 [0243] Evaluation of resistance to salt stress member: selecting plant lines typically four to ten independently transformed qualitatively evaluated for their tolerance to salt stress in a \ generations. 选择两个或三个在世代中定性地显示最强盐胁迫耐性的转化品系,在T2和T3世代中进行进一步评价。 Choose two or three qualitatively show the strongest line in the conversion generations salt stress tolerance for further evaluation in the T2 and T3 generations. 该评价涉及将来自所选择的转化植物品系的种子播种在含lOOmM或150mM NaCl的MS琼脂平板上,并将种子孵育5到14天,允许其萌发和生长。 This evaluation involves sowing seeds from the selected transformed plant lines on MS agar plates containing lOOmM or 150mM NaCl, and the seeds were incubated for 5-14 days to allow germination and growth. 例如,对ME02064而言,在盐平板上比较五个T2事件与野生型Ws的盐胁迫。 For example, in terms of ME02064 compared with wild-type event T2 five Ws in the salt flat plate salt stress. 与对照Ws相比,基于对每个事件36株植物幼苗面积的测量选择三个事件ME02064-01、-03和-04。 Compared with the control Ws, for each event based on measurement of seedling area of ​​36 plants selected three events ME02064-01, -03 and -04. 用T2和T3世代在ME02064-01、-03和-04中进行盐耐性的进一步评价。 Further evaluation of salt tolerance in ME02064-01, -03 and -04 with T2 and T3 generations.

[0244] 计算SGI :在萌发和牛长后,测定转化品系和野牛型对照的幼苗面积和光合效率。 [0244] Calculation SGI: After germination and cattle long conversion area and photosynthetic efficiency was measured strains and bison type control seedlings. 从这些测量中计算盐生长指数(SGI),并在野生型和转化的幼苗之间比较。 Salt Growth Index is calculated (the SGI) from these measurements, and comparison between wild-type and transformed seedlings. 通过用每个转化品系和野生型对照的36株幼苗一式两份获得的幼苗面积乘以光合效率测量(结果)来计算SGI,并进行t-检验。 SGI is calculated by using the area of ​​each 36 seedlings Seedlings transformed lines and wild type controls in duplicate measurements obtained by multiplying the photosynthetic efficiency (result), and t- test.

[0245] 测定转基因拷贝数:在BASTA™平板上测试T2世代转化植物,从而测定每个转化品系的转基因拷贝数。 [0245] Determination of Transgene Copy Number: T2 generation transformed plants are tested on BASTA ™ plates to thereby determine each transgenic line transformed copy number. 15 : 1的BASTA™抗性:BASTA™敏感分离比通常表明两个拷贝的转基因,3 : 1的这类分离比通常表明一个拷贝的转基因。 15: BASTA ™ 1 resistant: BASTA ™ sensitive segregation ratio usually indicates two copies of the transgene, 3: 1 ratio of such separations usually indicates one copy of the transgene.

[0246] 实施例3 :氧化胁迫条件筛选 [0246] Example 3: Oxidation Stress Screening

[0247] 在正常的生长条件下,拟南芥莲座包含约0. g/g鲜重的游离SA。 [0247] Under normal growth conditions, Arabidopsis rosette contains about 0. g / g fresh weight of free SA. 应答胁迫条件或病原体攻击时,游离SA水平可达到高达10 ug/g鲜重,大约等于60 u M。 When the response stress conditions or pathogen attacks, the free SA levels can reach as high as 10 ug / g fresh weight, about equal to 60 u M. 通过喷雾向拟南芥叶上外源应用100-500 yM SA能够诱导强防御应答而不引发明显的坏死损害形成。 Application 100-500 yM SA can induce strong defense responses without incurring significant necrotic lesions formed by spray to exogenous Arabidopsis leaves. 一旦SA浓度提高至5mM或更高,坏死损害形式的细胞死亡会出现在被喷雾的叶上。 Once the SA concentration increased to 5mM or more, damage in the form of necrotic cell death appears to be sprayed on the leaves. 如果通过生长培养基应用SA,则拟南芥对SA-诱导的氧化胁迫更加敏感,可能归因于持续的吸收。 If the growth medium by application of SA, Arabidopsis is more sensitive to stress-induced oxidative SA-, likely due to continued absorption. 向生长培养基中添加100-150 yM SA足以降低植物生长,但是不杀死野生型拟南芥Ws中的植物。 Added to the growth medium 100-150 yM SA sufficient to reduce plant growth, but does not kill the plant in the wild-type Arabidopsis Ws. 因此,我们使用这一范围的SA筛选增强的氧化胁迫耐性。 Therefore, we use SA screening this range of enhanced oxidative stress tolerance.

[0248] 水杨酸筛选:常规地,使用100 ii M或150 u M外源水杨酸钠,通过琼脂平板测定进行筛选。 [0248] Salicylic Acid Screening: Conventionally, using 100 ii M or 150 u M exogenous sodium salicylate, screened by agar plate assay. 培养基含有1/2X MS (Sigma)、150 iiM水杨酸钠(Sigma) ,0. 5g MES水合物(Sigma) 和0. 7% phytagar (EM Science),使用ION K0H 调节至pH 5. 7。 Medium containing 1 / 2X MS (Sigma), 150 iiM sodium salicylate (Sigma), 0. 5g MES hydrate (Sigma) and 0. 7% phytagar (EM Science), using ION K0H was adjusted to pH 5. 7 .

[0249] 为了筛选超级池,将种子在30%漂白溶液中表面灭菌5分钟,然后用无菌水重复冲洗。 [0249] To screen super cell, the seeds were surface sterilized in 30% bleach solution for 5 minutes, and then rinsed repeatedly with sterile water. 以每个平板850粒种子的密度,将约2500粒种子单层播种在培养基平板上。 At a density of 850 seeds per plate, approximately 2500 sown on media plates in a monolayer seed. 在相当的平板上培养野生型和阳性对照。 Wild-type positive control cultures and on comparable plates. 用通气带缠绕平板,并在4°C暗中放置三天使其成层。 With aeration tape winding plates and dark place three days so as to layer 4 ° C. 这段时间结束时,将平板转移至Conviron培养箱中,所述培养箱设定为22°C、16 : 8小时的 At the end of this time, the plates were transferred to a Conviron incubator, the incubator set at 22 ° C, 16: 8 hours

40光:暗循环、70%湿度、具有发射〜100 y爱因斯坦光强度的白炽灯和银光灯的组合。 40 light: dark cycle, 70% humidity, a combination of the light emission intensity Einstein ~100 y silver and incandescent lamps.

[0250] 从第6天开始每天筛选幼苗。 [0250] Screening starts on day 6 day seedlings. 选择与WS对照植物相比生长得更大并且保持更绿的幼苗作为阳性候选者,并转移至土壤中进行恢复和结籽。 Select grow larger as compared to WS control plants and seedlings held greener as positive candidates and transferred to soil for recovery and seed set.

[0251] 通过将来自每个候选者的36粒种子与WS对照一起置于相同的水杨酸钠平板上, 对候选植物进行再筛选。 [0251] placed in the same sodium salicylate plate together by the WS 36 seeds from each candidate on the control, plants were re-screened for candidate. 如上文所述处理植物,并在萌发后4天开始幼苗筛选。 As described above, the treatment of plants, seedlings and screening began 4 days after germination. 从确认有耐性的候选者中收获叶组织用于DNA提取,并通过PCR扩增转基因。 Patience candidates confirmed from harvested leaf tissue used for DNA extraction, and the transgene was amplified by PCR.

[0252] 或者,将超级池直接播种在土壤上并用10mM SA喷雾。 [0252] Alternatively, a super pool sown directly in the soil and spray with 10mM SA. 从耐性候选植物中收获叶组织以分离用于PCR扩增转基因的DNA,随后对PCR产物测序。 Harvested from tolerant candidate plants to isolate leaf tissue used for PCR amplification of the transgenic DNA, followed by sequencing PCR products.

[0253] 在水杨酸钠条件下评价的性状包括:幼苗面积、光合效率、水杨酸生长指数(SAG) 和再生能力。 [0253] Evaluation of Characteristics under sodium salicylate conditions include: area, photosynthesis efficiency, salicylic acid growth index (the SAG) and regeneration ability seedlings.

[0254] 幼苗面积:约2周龄的幼小植物的总叶面积。 [0254] Seedling area: the total leaf area of ​​a young plant about 2 weeks old.

[0255] 光合效率(Fv/Fm):通过最大荧光信号Fm和可变荧光Fv之间的关系评价幼苗光合效率或通过光系统II的电子传递。 [0255] Photosynthesis efficiency (Fv / Fm): Seedling photosynthetic efficiency was evaluated by the relationship between Fm, the maximum fluorescence signal Fv variable fluorescence or transmitted through an electronic optical system II. 本文中,最适量子产率(Fv/Fm)的降低表示胁迫,因此可以被用于监测氧化胁迫条件下与非转基因植物相比转基因植物的性能。 As used herein, reducing the optimum quantum yield (Fv / Fm) indicates stress, and therefore can be used for the performance of transgenic plants compared to non-transgenic plants under oxidative stress conditions monitored.

[0256] 水杨酸牛长(SAG)指数=幼苗面积(cm2) x光合效率(Fv/Fm)。 [0256] salicylic acid A cow (the SAG) Index = seedling area (cm2) x photosynthesis efficiency (Fv / Fm).

[0257] 在一株随机选择的T2植物中使用PCR扩增cDNA插入片段。 [0257] PCR amplification using cDNA insert in one randomly chosen T2 plant. 然后对该PCR产物测序以验证植物中的序列。 PCR products were then sequenced to verify the sequence of plants.

[0258] 评价对氧化胁迫的耐性:最初针对所有可获得的独立转化的T2植物品系定性评价它们与野生型对照相比对氧化胁迫的耐性。 [0258] Evaluation of resistance against oxidative stress: their initial tolerance to oxidative stress as compared to wild type control for all available independently transformed T2 plant lines qualitative evaluation. 选择定性显示对氧化胁迫最强耐性的阳性转基因品系,用于使用内部非转基因分离体作为对照在T2和T3世代中进一步评价。 Qualitative oxide selected transgenic lines displaying the strongest positive stress tolerance, for internal use as a non-transgenic controls separator further evaluation in the T2 and T3 generations. 该评价涉及将来自所选择的转化植物品系的种子播种在含lOOyM或150PM水杨酸钠的MS琼脂平板上,并将种子孵育至少4天,允许萌发和生长以及转基因状态分析。 This evaluation involves sowing seeds from the selected transformed plant lines on MS agar plates containing lOOyM 150PM or sodium salicylate and incubating the seeds for at least 4 days to allow germination and growth and transgene status analysis.

[0259] 计算SAG :萌发和生长后,测定转化品系和野生型对照的幼苗面积和光合效率。 [0259] Calculation SAG: After germination and growth, and photosynthetic efficiency measurement area transformed lines and wild type control seedlings. 从这些测量结果中计算水杨酸生长指数(SAG)并在野生型和转化幼苗之间比较。 And comparing the calculated salicylic acid growth index (the SAG) from these measurements between wild-type and transformed seedlings. 通过用每个转化品系和野生型对照的36株幼苗一式两份获得的幼苗面积乘以光合效率测量结果来计算SAG,并进行t-检验。 Measurements to calculate SAG, t- test and by multiplying seedling area with photosynthesis efficiency of 36 seedlings for each transformed line and a wild-type control in duplicate obtained.

[0260] 测定转基因拷贝数:在BASTA™平板上测试T2世代转化植物,从而测定每个转化品系的转基因拷贝数。 [0260] Determination of Transgene Copy Number: T2 generation transformed plants are tested on BASTA ™ plates to thereby determine each transgenic line transformed copy number. 15 : 1的BASTA™抗性:BASTA™敏感分离比通常表明两个拷贝的转基因,3 : 1的这类分离比通常表明一个拷贝的转基因。 15: BASTA ™ 1 resistant: BASTA ™ sensitive segregation ratio usually indicates two copies of the transgene, 3: 1 ratio of such separations usually indicates one copy of the transgene.

[0261] 在一些情况下,使用还补充了100 u M SNP的培养基进行验证。 [0261] In some cases, the medium is also supplemented with 100 u M SNP verified.

[0262]实施例 4 :ME02064 (Ceres 克隆375578 ;SEQ ID No. 138) [0262] Example 4: ME02064 (Ceres clone 375578; SEQ ID No. 138)

[0263] 用带有35S启动子和Ceres克隆375578的Ti质粒转化野生型拟南芥Wassilewskija。 [0263] with the wild-type Arabidopsis thaliana Wassilewskija 35S promoter and Ceres Clone 375578 Ti plasmid transformation. 在预验证实验中,三个转化品系ME02064-01 和ME02064-03、ME02064-04 显示对盐胁迫最强的定性耐性(表4-1)。 In the pre-verification experiment, three transformed lines ME02064-01 and ME02064-03, ME02064-04 displaying the strongest qualitative tolerance to salt stress (Table 4-1). 在两代验证实验中进一步评价它们对150mM NaCl 的耐性。 Further evaluated for their resistance to 150mM NaCl in two generations of the verification experiment. 分离比(BASTA™ 抗性:BASTA™ 敏感)表明ME02064-01 和ME02064-03、ME02064_04 转化品系各自带有一个拷贝的转基因。 Split ratio (BASTA ™ resistant: BASTA ™ sensitive) indicated ME02064-01 and ME02064-03, ME02064_04 transformed lines each carry one copy of the transgene.

[0264] 表4-1 :与野生型Ws相比ME02064盐耐性的预验证实验 [0264] Table 4-1: pre-verification experiment ME02064 salt tolerance as compared with the wild-type Ws

[0265] [0265]

41 41

Figure CN101981191AD00421

[0266] *在含150mM NaCl的MS琼脂平板上生长14天的36株植物的平均幼苗面积 [0266] * Average seedling area on MS agar plates containing 150mM NaCl for 14 days growth 36 of the plant

[0267]在含 150mM NaCl 的MS 琼脂平板上生长时,ME02064-01 和ME02064-03、ME02064_04 转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0267] When grown on MS agar plates of containing 150mM NaCl, ME02064-01 and ME02064-03, ME02064_04 transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI. 如表4-2中所示,与非转基因对照幼苗相比时,ME02064-01幼苗的T2-世代SGI值提高110%,ME02064-03幼苗提高131%,ME02064-04 幼苗提高72%。 As shown in Table 4-2 and non-transgenic control seedlings when compared, T2- generation SGI value ME02064-01 seedlings increased 110%, ME02064-03 seedlings increased 131%, ME02064-04 seedlings increased 72%. 在T3 世代中,ME02064-01 的SGI 提高43%,ME02064_03 提高47%,ME02064-04提高64%。 In T3 generations, ME02064-01 SGI increase of 43%, ME02064_03 increase 47%, ME02064-04 64% increase. 转基因和非转基因幼苗之间的差异是统计学显著的,并且清楚地证明ME02064转化体品系中增强的对盐胁迫的耐性是Ceres克隆375578的异位表达的结果。 The difference between the transgenic and non-transgenic seedlings are statistically significant, and clearly demonstrate ME02064 transformant lines in enhanced tolerance to salt stress that ectopic expression of Ceres Clone 375578 results.

[0268] 表4-2 :两世代中ME02064对盐胁迫耐性的验证实验 [0268] Table 4-2: Experimental verification two generations ME02064 salt stress tolerance

[0269] [0269]

Figure CN101981191AD00422

[0270] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0270] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0271] 结果总结: [0271] The results are summarized:

[0272] 处于35S启动子控制下的Ceres克隆375578的异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0272] Ceres is under control of the 35S promoter was cloned 375578 Ectopic expression enhances tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0273]实施例 5 :ME03140 :克降375578 :SEQ ID No. 142 [0273] Example 5: ME03140: down g 375578: SEQ ID No. 142

[0274] 用带有与Ceres克隆375578 (SEQ ID NO : 142)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewskija,并研究五个转基因品系ME03140-01、ME03140-02、 ME03140-03、ME03140-04 和ME03140-05 对盐胁迫的耐性。 [0274] and with a Ceres CLONE 375578: Ti plasmid (SEQ ID NO 142) 35S promoter operably linked to the transformed wild-type Arabidopsis thaliana Wassilewskija, research and five transgenic lines, ME03140-01, ME03140-02, ME03140- 03, patience ME03140-04 and ME03140-05 to salt stress. 在含有150mM NaCl 的MS 琼脂平板上生长时,在与非转基因对照幼苗相比的定量实验中,这些转基因品系显示提高的对盐胁迫的耐性。 When grown on MS agar plates containing 150mM NaCl in quantitative experiments and compared to non-transgenic control seedlings of these transgenic lines showed increased tolerance to salt stress.

[0275]在含 150mM NaCl 的MS 琼脂平板上生长时,ME03140-01、ME03140-02、ME03140-03、 ME03140-04和ME03140-05转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0275] When grown on MS agar plates containing the 150mM NaCl, ME03140-01, ME03140-02, ME03140-03, ME03140-04 and ME03140-05 transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI . 如表5中所示,与非转基因对照幼苗相比时,ME03140-01幼苗的T2-世代SGI值提高102. 18%, ME03140-02 幼苗提高60. 78%, ME03140-03 幼苗提高120. 32%, ME03140-04 幼苗提高45. 07%, ME03140-05幼苗提高90. 53%。 As shown in Table 5, when compared to non-transgenic control seedlings, T2- generation SGI value ME03140-01 seedlings increased 102. 18%, ME03140-02 seedlings increased 60. 78%, ME03140-03 seedlings increased 120.32 %, ME03140-04 seedlings increased 45.07%, increase 90. 53% ME03140-05 seedlings. 转基因和非转基因幼苗之间SGI值的差异对所有转基因品系而言具有统计学显著的P值,并且这些定量实验清楚地证明Ceres克隆375578的异位表达赋予转基因幼苗中增强的对盐胁迫的耐性。 Differences in transgene and SGI values ​​between seedlings non-transgenic significant P value significant for all transgenic lines concerned, and these quantitative experiments clearly demonstrate Ceres CLONE ectopic 375578 expression confers transgenic seedlings in enhanced tolerance to salt stress .

[0276] 表5 :—世代中ME03140盐胁迫耐性的验证实验 [0276] Table 5: - Verification experiment ME03140 salt stress tolerance generations

[0277] [0277]

Figure CN101981191AD00431

[0278] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0278] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0279] 结果总结: [0279] The results are summarized:

[0280] 处于35S启动子控制下的Ceres克隆375578的异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0280] Ceres is under control of the 35S promoter was cloned 375578 Ectopic expression enhances tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0281]实施例 6 :ME08732 :克降560066 :SEQ ID No. 44 [0281] Example 6: ME08732: down g 560066: SEQ ID No. 44

[0282] 用带有与Ceres克隆560066 (SEQ ID NO :44)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewskija,并研究三个转基因品系ME08732-01、ME08732-02和ME08732-03对盐胁迫的耐性。 [0282] and with a Ceres CLONE 560066: Ti plasmid 35S promoter operably linked to (SEQ ID NO 44) transformed wild type Arabidopsis thaliana Wassilewskija, research and three transgenic lines ME08732-01, ME08732-02 and ME08732- 03 tolerance to salt stress. 在含有150mM NaCl的MS琼脂平板上生长时,在与非转基因对照幼苗相比的定量实验中,这些转基因品系显示提高的对盐胁迫的耐性。 When grown on MS agar plates containing 150mM NaCl in quantitative experiments and compared to non-transgenic control seedlings of these transgenic lines showed increased tolerance to salt stress.

[0283]在含 150mM NaCl 的MS琼脂平板上生长时,ME08732-01、ME08732_02和ME08732-03 转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0283] When grown on MS agar plates containing the 150mM NaCl, ME08732-01, ME08732_02 and ME08732-03 transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI. 如表6中所示,与非转基因对照幼苗相比时,ME08732-01幼苗的T2-世代SGI值提高88. 35%, ME08732-02幼苗提高41. 72%, ME08732-03幼苗提高26. 23%。 As shown, the non-transgenic control seedlings compared in Table 6, T2- generation SGI value ME08732-01 seedlings increased 88. 35%, ME08732-02 seedlings increased 41. 72%, ME08732-03 seedlings increased 26.23 %. 转基因和非转基因幼苗之间SGI值的差异对ME08732-01和ME08732-02转基因品系而言具有统计学显著的P值,并且这些定量实验清楚地证明Ceres克隆560066的异位表达赋予转基因幼苗中增强的对盐胁迫的耐性。 Differences in SGI values ​​transgenic and non-transgenic seedlings between a pair ME08732-01 and ME08732-02 transgenic lines in terms of statistically significant P values, and these quantitative experiments clearly demonstrate that ectopic Ceres Clone 560066 confers enhanced expression in transgenic seedlings tolerance to salt stress.

[0284] 表6 :—世代中ME08732盐胁迫耐性的验证 [0284] Table 6: - Verify ME08732 salt stress tolerance generations

[0285] [0285]

Figure CN101981191AD00441

[0286] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0286] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0287] 结果总结: [0287] The results are summarized:

[0288] 处于35S启动子控制下的Ceres克隆560066的异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0288] Ceres is under control of the 35S promoter was cloned 560066 Ectopic expression enhances tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0289]实施例 7 :ME08768 :克降539458 :SEQ ID No. 86 [0289] Example 7: ME08768: down g 539458: SEQ ID No. 86

[0290] 用带有与Ceres克隆539458 (SEQ ID NO :86)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewskija,并研究五个转基因品系ME08768-01、ME08768-02、 ME08768-03、ME08768-04 和ME08768-05 对盐胁迫的耐性。 [0290] and with a Ceres CLONE 539458 (SEQ ID NO: 86) Ti plasmid operably linked to the 35S promoter transformed wild-type Arabidopsis thaliana Wassilewskija, research and five transgenic lines, ME08768-01, ME08768-02, ME08768- 03, patience ME08768-04 and ME08768-05 to salt stress. 在含有150mM NaCl 的MS 琼脂平板上生长时,在与非转基因对照幼苗相比的定量实验中,这些转基因品系显示提高的对盐胁迫的耐性。 When grown on MS agar plates containing 150mM NaCl in quantitative experiments and compared to non-transgenic control seedlings of these transgenic lines showed increased tolerance to salt stress.

[0291]在含 150mM NaCl 的MS 琼脂平板上生长时,ME08768-01、ME08768-02、ME08768_03、 ME08768-04和ME08768-05转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0291] When grown on MS agar plates containing the 150mM NaCl, ME08768-01, ME08768-02, ME08768_03, ME08768-04 and ME08768-05 transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI. 如表7中所示,与非转基因对照幼苗相比时,ME08768-01幼苗的T2代SGI值提高80. 04%, ME008768-02 幼苗提高111. 63%,ME008768-03 幼苗提高22. 62%, ME008768-04 幼苗提高115. 40%, ME008768-05幼苗提高74. 41%。 When as shown in Table, as compared to non-transgenic control seedlings. 7, T2-generation SGI value ME08768-01 seedlings increased 80. 04%, ME008768-02 seedlings increased 111. 63%, ME008768-03 seedlings increased 22.62% , ME008768-04 seedlings increased 115. 40%, ME008768-05 seedlings increased 74.41%. 转基因和非转基因幼苗之间SGI值的差异对ME08768-01、ME08768-02、ME08768-04和ME08768-05转基因品系而言具有统计学显著的P值,并且这些定量实验清楚地证明Ceres克隆539458的异位表达导致转基因幼苗中增强的对盐胁迫的耐性。 Transgenic and differences in SGI values ​​between non-transgenic seedlings have statistically significant P values ​​for ME08768-01, ME08768-02, ME08768-04 and ME08768-05 transgenic lines concerned, and these quantitative experiments clearly demonstrate the Ceres clone 539458 ectopic expression results in transgenic seedlings enhanced tolerance to salt stress.

[0292] 表7 :—世代中ME08768盐胁迫耐性的验证实验 [0292] Table 7: - Verification experiment ME08768 salt stress tolerance generations

[0293] [0293]

Figure CN101981191AD00442

[0294] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率)[0295] 结果总结: [0294] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency) [0295] The results are summarized:

[0296] 处于35S启动子控制下的Ceres克隆539458的异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0296] Ceres is under control of the 35S promoter was cloned 539458 Ectopic expression enhances tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0297]实施例 8 :ME10681 :克降335348 :SEQ ID No. 141 [0297] Example 8: ME10681: down g 335348: SEQ ID No. 141

[0298] 用带有与Ceres克隆335348 (SEQ ID NO : 141)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewskija,并研究六个转基因品系ME10681-01-T2,ME10681-01-T3, ME10681-02-T2, ME10681-02-T3, ME10681-04_T2 和ME10681-05_T2 对盐胁迫的耐性。 [0298] and with a Ceres CLONE 335348: Ti plasmid (SEQ ID NO 141) 35S promoter operably linked to the transformed wild-type Arabidopsis thaliana Wassilewskija, research and six transgenic lines ME10681-01-T2, ME10681-01- T3, ME10681-02-T2, ME10681-02-T3, ME10681-04_T2 ME10681-05_T2 and tolerance to salt stress. 在含有150mM NaCl的MS琼脂平板上生长时,在与非转基因对照幼苗相比的定量实验中,这些转基因品系显示提高的对盐胁迫的耐性。 When grown on MS agar plates containing 150mM NaCl in quantitative experiments and compared to non-transgenic control seedlings of these transgenic lines showed increased tolerance to salt stress.

[0299]在含 150mM NaCl 的MS 琼脂平板上生长时,ME10681-01_T2,ME10681-01_T3, ME10681-02-T2, MEIO68I-O2-T3, ME10681-04-T2 和ME10681-05_T2 转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0299] When grown on MS agar plates containing the 150mM NaCl, ME10681-01_T2, ME10681-01_T3, ME10681-02-T2, MEIO68I-O2-T3, ME10681-04-T2 and ME10681-05_T2 transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI. 如表8中所示,与非转基因对照幼苗相比时, ME010681-01-T2 幼苗的T2-世代SGI 值提高39. 17%, ME010681-01-T3 幼苗提高19. 77%, ME10681-02-T2 幼苗提高119. 17% , ME10681-02-T3 幼苗提高6. 21 %, ME010681-04-T2 幼苗提高113. 51%和ME010681-05-T2幼苗提高103. 98%。 As shown in Table 8, and when compared to non-transgenic control seedlings, ME010681-01-T2 T2- generation SGI value seedlings increased 39.17%, improve 19. 77% ME010681-01 T3-seedlings, ME10681-02- T2 seedlings increased 119.17% increase 6. 21% ME10681-02-T3 seedlings, ME010681-04-T2 seedlings increased 113.51% and ME010681-05-T2 seedlings increased 103.98%. 转基因和非转基因幼苗之间SGI值的差异对ME10681-01-T3、ME10681-02-T2、ME10681-04_T2 和ME10681-05_T2 转基因品系而言具有统计学显著的P值,并且这些定量实验清楚地证明Ceres克隆335348的异位表达导致转基因幼苗中增强的对盐胁迫的耐性。 Differences in SGI values ​​transgenic and non-transgenic seedlings between a pair ME10681-01-T3, ME10681-02-T2, ME10681-04_T2 ME10681-05_T2 and P values ​​in terms of transgenic lines having statistically significant, and these quantitative experiments clearly demonstrate that ectopic expression of Ceres cLONE 335348 results in transgenic seedlings in enhanced tolerance to salt stress.

[0300] 表8 :两世代中ME10681盐胁迫耐性的验证实验 [0300] Table 8: Experimental verification two generations of ME10681 salt stress tolerance

[0301] [0301]

Figure CN101981191AD00451

[0302] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0302] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0303] 结果总结: [0303] The results are summarized:

[0304] 处于35S启动子控制下的Ceres克隆335348的异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0304] Ceres is under control of the 35S promoter was cloned 335,348 Ectopic expression enhances tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0305]实施例 9 :ME18973 :Ceres cDNA ID 23457556 :SEQ ID No. 43 [0305] Example 9: ME18973: Ceres cDNA ID 23457556: SEQ ID No. 43

[0306]用带有与 Ceres cDNA ID 23457556 (SEQ ID NO :43)有效连接的35S 启动子的Ti质粒转化野生型拟南芥Wassilewskija,并研究六个转基因品系ME18973_01_T2、 ME18973-02-T2、ME18973-02-01-T3、ME18973-03-T2、ME18973-05-T2 和ME18973-05-03_T3 对 [0306] with the use of Ceres cDNA ID 23457556 (SEQ ID NO: 43) 35S promoter operably linked to a wild-type Ti plasmid transformed Arabidopsis Wassilewskija, and six transgenic lines studied ME18973_01_T2, ME18973-02-T2, ME18973 -02-01-T3, ME18973-03-T2, ME18973-05-T2 and to ME18973-05-03_T3

45盐胁迫的耐性。 45 salt stress tolerance. 在含有150mMNaCl的MS琼脂平板上生长时,在与非转基因对照幼苗相比的定量实验中,这些转基因品系显示提高的对盐胁迫的耐性。 When grown on MS agar plates containing 150mMNaCl in quantitative assays as compared to non-transgenic control seedlings of these transgenic lines showed increased tolerance to salt stress.

[0307]在含 150mM NaCl 的MS 琼脂平板上生长时,ME18973-01、ME18973_02_T2、 ME18973-02-01-T3、ME18973_03_T2、ME18973_05_T2 和ME18973-05-03_T3 转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0307] When grown on MS agar plates containing the 150mM NaCl, ME18973-01, ME18973_02_T2, ME18973-02-01-T3, ME18973_03_T2, ME18973_05_T2 ME18973-05-03_T3 and transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI. 如表9中所示,与非转基因对照幼苗相比时,ME018973-01-T2 幼苗的T2&T3-世代SGI 值提高230. 01 %, ME18973_02_T2 幼苗提高22. 44%, ME18973-02-01-T3 幼苗提高14. 96%, ME18973-05_T2 幼苗提高16. 12%, 1^18973-05-03-113幼苗提高13. 97%。 As shown in Table 9 and in the non-transgenic control seedlings when compared, T2 ME018973-01 T2-generation SGI Seedlings & T3- value increased 230.01%, 22. 44% ME18973_02_T2 increase seedling, ME18973-02-01 T3-Seedlings increase 14.96%, 16. 12% ME18973-05_T2 increase seedling increase 13.97% 1 ^ 18973-05-03-113 seedlings. 转基因和非转基因幼苗之间SGI值的差异对ME18973 转基因品系而言具有统计学显著的P值,并且这些定量实验清楚地证明Ceres cDNA ID23457556的异位表达导致转基因幼苗中增强的对盐胁迫的耐性。 Differences in transgene and SGI values ​​between seedlings non-transgenic for ME18973 transgenic lines in terms of P values ​​statistically significant, and these quantitative experiments clearly demonstrate that ectopic Ceres cDNA ID23457556 expression results in transgenic seedlings in enhanced tolerance to salt stress .

[0308] 表9 :两世代中ME18973盐胁迫耐性的验证实验 [0308] Table 9: Experimental verification of ME18973 salt stress tolerance in two generations

[0309] [0309]

Figure CN101981191AD00461

[0311] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0311] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0312] 结果总结: [0312] The results are summarized:

[0313] 处于35S启动子控制下的Ceres cDNA ID 23457556异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0313] Ceres cDNA is under control of the 35S promoter ID 23457556 Ectopic expression of enhanced tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0314]实施例 10 :ME19657 ;cDNA ID 23621377 ;SEQ ID No. 45 [0314] Example 10: ME19657; cDNA ID 23621377; SEQ ID No. 45

[0315]用带有与 Ceres cDNA ID 23621377 (SEQ ID NO :45)有效连接的35S 启动子的Ti质粒转化野生型拟南芥Wassilewskija,并研究两个转基因品系ME19657_01_T2、 ME19657-01-05-T3、ME19657-01-08_T3、ME19657_02_T2、ME19657_03_T2、ME19657_04_T2 和ME19657-04-01-T3对盐胁迫的耐性。 [0315] with the use of Ceres cDNA ID 23621377 (SEQ ID NO: 45) Ti plasmid 35S promoter operably linked to the transformed wild-type Arabidopsis thaliana Wassilewskija, research and two transgenic lines ME19657_01_T2, ME19657-01-05-T3 , resistance ME19657-01-08_T3, ME19657_02_T2, ME19657_03_T2, ME19657_04_T2 ME19657-04-01-T3, and salt stress. 在含有150mM NaCl的MS琼脂平板上生长时,在与非转基因对照幼苗相比的定量实验中,这些转基因品系显示提高的对盐胁迫的耐性。 When grown on MS agar plates containing 150mM NaCl in quantitative experiments and compared to non-transgenic control seedlings of these transgenic lines showed increased tolerance to salt stress.

[0316]在含 150mM NaCl 的MS 琼脂平板上生长时,ME19657_01_T2、ME19657-01-05_T3、 ME19657-01-08-T3、ME19657_02_T2、ME19657_03_T2、ME19657_04_T2 和ME19657-04-01_T3 转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0316] When grown on MS agar plates containing the 150mM NaCl, ME19657_01_T2, ME19657-01-05_T3, ME19657-01-08-T3, ME19657_02_T2, ME19657_03_T2, ME19657_04_T2 ME19657-04-01_T3 and transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI. 如表10中所示,与非转基因对照幼苗相比时,ME19657-01-T2幼苗的T2&T3-世代SGI值提高82. 29 %,1^19657-01-05-113幼苗提高82. 29%,ME19657-01-08_T3 幼苗提高21. 90% ,ME19657-02-T2 幼苗提高39. 50%, ME19657-03-T2 幼苗提高98. 28%, ME19657-04_T2 幼苗提高4. 38%, ME19657-04-01-T2幼苗提高7. 44 %。 As shown, and when compared to non-transgenic control seedlings, T2 & T3- ME19657-01 T2-generation SGI value seedlings increased 82.29%, 1 ^ 19657-01-05-113 seedlings increased 82.29% Table 10, ME19657-01-08_T3 seedlings increased 21.90%, improve 39. 50% ME19657-02 T2-seedlings, improve 98. 28% ME19657-03 T2-seedlings, ME19657-04_T2 seedlings increased 4. 38%, ME19657-04- 01-T2 seedlings increased 7.44%. 转基因和非转基因幼苗之间SGI值的差异对ME19657-01-T2、ME19657-01-05_T3、ME19657-01-08_T3、ME19657_02_T2 和ME19657_03_T2 转基因品系而言具有统计学显著的P值,并且这些定量实验清楚地证明Ceres cDNA ID 23621377的异位表达导致转基因幼苗中增强的对盐胁迫的耐性。 Differences in SGI values ​​transgenic and non-transgenic seedlings between a pair ME19657-01-T2, ME19657-01-05_T3, ME19657-01-08_T3, ME19657_02_T2 ME19657_03_T2 and P values ​​in terms of transgenic lines having statistically significant, and these quantitative experiments clearly demonstrate that ectopic Ceres cDNA ID 23621377 results in expression in transgenic seedlings enhanced tolerance to salt stress.

[0317] 表10 :两世代中ME19657盐胁迫耐性的验证实验 [0317] Table 10: Experimental verification two generations of ME19657 salt stress tolerance

[0318] [0318]

Figure CN101981191AD00471

[0319] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0319] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0320] 结果总结: [0320] The results are summarized:

[0321] 处于35S启动子控制下的Ceres cDNA ID 23621377的异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 Ectopic expression of [0321] is in Ceres cDNA ID 23621377 35S promoter enhances tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0322]实施例 U :ME24076 :克降229668 :SEQ ID No. 143 [0322] Example U: ME24076: down g 229668: SEQ ID No. 143

[0323] 用带有与Ceres Clone =229668 (SEQ ID NO :143)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewski ja,并研究两个转基因品系ME24076-01和ME24076-02对盐胁迫的耐性。 [0323] with the use of Ceres Clone = 229668: Ti plasmid promoter 35S (SEQ ID NO 143) linked transformed wild-type Arabidopsis Wassilewski ja, and research and two transgenic lines, ME24076-01 ME24076-02 of tolerance to salt stress. 在含有150mM NaCl的MS琼脂平板上生长时,在与非转基因对照幼苗相比的定量实验中,这些转基因品系显示提高的对盐胁迫的耐性。 When grown on MS agar plates containing 150mM NaCl in quantitative experiments and compared to non-transgenic control seedlings of these transgenic lines showed increased tolerance to salt stress.

[0324] 在含150mM NaCl的MS琼脂平板上生长时,仅ME024076_01_T2和转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0324] When grown on MS agar plates containing 150mM NaCl, just ME024076_01_T2 and transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI. 如表11中所示,与非转基因对照幼苗相比时,ME24076-01-T2 幼苗的T2-世代SGI 值提高65. 57%,ME24076_02_T2 幼苗降低1. 12%。 As shown in Table 11, when compared to non-transgenic control seedlings, T2- generation SGI value ME24076-01-T2 seedlings increased 65. 57%, ME24076_02_T2 seedlings decreased 1.12%. 转基因和非转基因幼苗之间SGI值的差异对转基因品系ME24076-01而言具有统计学显著的P值,并且这些定量实验清楚地证明Ceres克隆229668的异位表达导致转基因幼苗中增强的对盐胁迫的耐性。 Transgenic and differences in SGI values ​​between non-transgenic seedlings have statistically significant P values ​​for transgenic lines ME24076-01 concerned, and these quantitative experiments clearly demonstrate that ectopic Ceres Clone 229668 of expression results in enhanced transgenic seedlings to salt stress patience.

[0325] 表11 :一世代中ME24076盐胁迫耐性的验证实验 [0325] Table 11: Experimental verification of a generation ME24076 salt stress tolerance

[0326] [0326]

Figure CN101981191AD00481

[0327] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0327] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0328] 结果总结: [0328] The results are summarized:

[0329] 处于35S启动子控制下的Ceres克隆229668的异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0329] Ceres is under control of the 35S promoter was cloned 229668 Ectopic expression enhances tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0330]实施例 12 :ME24217 :克降375578 :SEQ ID No. 144 [0330] Example 12: ME24217: down g 375578: SEQ ID No. 144

[0331] 用带有与Ceres克隆375578 (SEQ ID NO : 144)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewski ja,并研究两个转基因品系ME24217_07_T2和ME24217_09_T2 对盐胁迫的耐性。 [0331] and with a Ceres CLONE 375578 (SEQ ID NO: 144) Ti plasmid 35S promoter operably linked to a wild-type Arabidopsis transformed Wassilewski ja, and research and two transgenic lines ME24217_07_T2 ME24217_09_T2 tolerance to salt stress. 在含有150mM NaCl的MS琼脂平板上生长时,在与非转基因对照幼苗相比的定量实验中,这些转基因品系显示提高的对盐胁迫的耐性。 When grown on MS agar plates containing 150mM NaCl in quantitative experiments and compared to non-transgenic control seedlings of these transgenic lines showed increased tolerance to salt stress.

[0332]在含 150mM NaCl 的MS 琼脂平板上生长时,ME24217_07_T2 和ME24217_09_T2 转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0332] When grown on MS agar plates of containing 150mM NaCl, ME24217_07_T2 ME24217_09_T2 and transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI. 如表12中所示,与非转基因对照幼苗相比时,ME24217-07幼苗的T2-世代SGI值提高30. 41%, ME24217-09幼苗提高134.46%。 When as shown in Table, as compared to non-transgenic control seedlings. 12, T2- generation SGI value ME24217-07 seedlings increased 30. 41%, ME24217-09 seedlings increased 134.46%. 转基因和非转基因幼苗之间SGI值的差异对ME24217-07-T2*ME24217-09-T2 转基因品系而言具有统计学显著的P值,并且这些定量实验清楚地证明Ceres克隆375578 的异位表达导致转基因幼苗中增强的对盐胁迫的耐性。 Transgenic and differences in SGI values ​​between non-transgenic seedlings have statistically significant P values ​​for ME24217-07-T2 * ME24217-09-T2 transgenic lines concerned, and these quantitative experiments clearly demonstrate that expression of Ceres Clone 375578 results in ectopic transgenic seedlings enhanced tolerance to salt stress.

[0333] 表12 :—世代中ME24217盐胁迫耐性的验证实验 [0333] Table 12: - Verification experiment ME24217 salt stress tolerance generations

[0334] [0334]

Figure CN101981191AD00482

[0335] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0335] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0336] 结果总结: [0336] The results are summarized:

[0337] 处于35S启动子控制下的Ceres克隆375578的异位表达增强对盐胁迫的耐性,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0337] Ceres is under control of the 35S promoter was cloned 375578 Ectopic expression enhances tolerance to salt stress, salt stress caused by the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0338]实施例 13 :ME02064C :克隆375578C ;SEQ ID No. 140 [0338] Example 13: ME02064C: clone 375578C; SEQ ID No. 140

[0339] 用带有与Ceres克隆375578 (SEQ ID NO :140)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewski ja,并研究六个转基因品系ME02064C_01_T2、 ME02064C-02-T2、ME02064C-03_T2、ME02064C-04_T2、ME02064C-05-T2 和ME02064C-06_T2 对盐胁迫的耐性。 [0339] and with a Ceres CLONE 375578: Ti plasmid (SEQ ID NO 140) 35S promoter operably linked to a wild-type Arabidopsis transformed Wassilewski ja, and six transgenic lines studied ME02064C_01_T2, ME02064C-02-T2, ME02064C -03_T2, ME02064C-04_T2, ME02064C-05-T2 and ME02064C-06_T2 tolerance to salt stress. 在含有150mMNaCl的MS琼脂平板上生长时,在与非转基因对照幼苗相比的 When grown on MS agar plates containing 150mMNaCl in non-transgenic control seedlings compared to

48定量实验中,大部分这些转基因品系不显示对盐胁迫的耐性。 48 quantitative experiments, most of these transgenic lines do not show tolerance to salt stress.

[0340] 表13显示在含lOOmM NaCl的MS琼脂平板上生长时,与非转基因对照幼苗相比,ME02064C-01-T2幼苗的T2-世代SGI值为0. 55 % ;与非转基因对照幼苗相比, ME02064C-02-T2幼苗为1. 31 % ;与非转基因对照幼苗相比,ME02064C_03_T2幼苗为9. 67%; 与非转基因对照幼苗相比,ME02064C-04-T2幼苗为-7. 78% ;与非转基因对照幼苗相比, ME02064C-05-T2幼苗为-15. 77 % ;与非转基因对照幼苗相比,ME02064C_06_T2幼苗为17. 78%。 [0340] Table 13 shows the growth on MS agar plates containing lOOmM NaCl, and compared to non-transgenic controls, T2- generation SGI value of 0.55% seedlings ME02064C 01 T2--seedlings; non-transgenic control seedlings with ratio, ME02064C 02-T2-seedlings was 1.31%; compared with non-transgenic control seedlings, ME02064C_03_T2 seedlings was 9.67%; compared with non-transgenic control seedlings, ME02064C-04-T2 seedlings -7 78% ; compared with non-transgenic control seedlings, ME02064C-05-T2 seedlings -15 77%; compared with non-transgenic control seedlings, ME02064C_06_T2 seedlings was 17.78%.

[0341] 表13 :—世代中ME02064C盐胁迫耐性的验证实验 [0341] Table 13: - Verification experiment ME02064C salt stress tolerance generations

[0342] [0342]

Figure CN101981191AD00491

[0343] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0343] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0344] 结果总结: [0344] The results are summarized:

[0345] 处于35S启动子控制下的Ceres克隆375578的异位表达可能不促进对盐胁迫耐性的增强,所述盐胁迫在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0345] Ceres is under control of the 35S promoter cloned 375578 Ectopic expression of growth may not promote enhanced tolerance to salt stress, salt stress caused by the necrotic lesions and stunted in the wild-type Ws seedlings.

[0346]实施例 14 ! [0346] Example 14! ME02064P1 :克降375578P1-SEQ ID No. 140 的氨基酸1 到135 ME02064P1: g drop 375578P1-SEQ ID No. 140 of the amino acids 1-135

[0347] 用带有与编码Ceres克隆375578P1 (SEQ ID NO :140的氨基酸1到135)的核酸有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewski ja,所述核酸是上文实施例1中所述Ceres克隆375578的3'截短变体。 [0347] use with encoding Ceres CLONE 375578P1 (SEQ ID NO: 140 amino acids 1 through 135) Ti plasmid nucleic acid is operably linked to the 35S promoter in wild-type Arabidopsis transformed Wassilewski ja, the above embodiment is a nucleic acid Example 1 Ceres cLONE 375578 3 'truncated variant. 研究五个转基因品系ME02064P1_03_T2、 ME02064Pl-07-T2、ME02064P1-09_T2、ME02064P1-10_T2 和ME02064P1_15_T2 对盐胁迫的耐性。 Study of five transgenic lines ME02064P1_03_T2, ME02064Pl-07-T2, ME02064P1-09_T2, ME02064P1-10_T2 ME02064P1_15_T2 and tolerance to salt stress. 在与非转基因对照幼苗相比的定量实验中,所有这些五个转基因品系显示对盐胁迫的耐性。 In quantitative assays as compared to non-transgenic control seedlings, all five of these transgenic lines showed tolerance to salt stress. 如表10中所示,与非转基因的对照幼苗相比,ME02064P1幼苗的T2-世代SGI提高32. 57%,89. 52%,66. 84%,25. 43%,36. 95%。 As shown in the table, as compared to non-transgenic control seedlings, T2- generation SGI ME02064P1 10 seedlings increased 32.57%, 89.52%, 66.84%, 25.43%, 36.95%.

[0348]在含 150mM NaCl 的MS 琼脂平板上生长时,ME02064P1-03、ME02064P1-07、 ME02064P1-09、ME02064P1-10和ME02064P1-15转基因植物相对于非转基因植物显示显著更大的幼苗面积和SGI。 [0348] When grown on MS agar plates containing the 150mM NaCl, ME02064P1-03, ME02064P1-07, ME02064P1-09, ME02064P1-10 and ME02064P1-15 transgenic plants relative to non-transgenic plants showed significantly greater seedling area and SGI . 如表14中所示,与非转基因对照幼苗相比时,ME02064P1-03幼苗的T2-世代SGI 值提高32. 57%, ME02064P1-07 幼苗提高89. 52%, ME02064P1-09 幼苗提高66. 84%, ME02064P1-10 幼苗提高25. 43%, ME02064P1-15 幼苗提高36. 95%。 As shown in Table 14 when compared with non-transgenic control seedlings, T2- generation SGI value ME02064P1-03 seedlings increased 32.57%, 89. 52% ME02064P1-07 seedlings increase, ME02064P1-09 seedlings increased 66.84 %, ME02064P1-10 seedlings increased 25.43%, 36. 95% ME02064P1-15 seedlings increase. 转基因和非转基因幼苗之间SGI值的差异对转基因品系ME02064Pl-03-T2、ME02064P1_07_T2、ME02064Pl-09-T2、ME02064Pl-10-T2 和ME02064P1-15_T2 而言具有统计学显著的P 值,并且这些定量实验清楚地证明Ceres克隆37558P1的异位表达导致转基因幼苗中增强的对盐胁迫的耐性。 Differences in SGI values ​​transgenic and non-transgenic seedlings between transgenic lines ME02064Pl-03-T2, ME02064P1_07_T2, ME02064Pl-09-T2, ME02064Pl-10-T2 and ME02064P1-15_T2 terms of statistically significant P values, and these quantitative Ceres cLONE experiments clearly demonstrate that ectopic expression leads 37558P1 transgenic seedlings in enhanced tolerance to salt stress.

[0349] 表14 :一世代中ME02064P1盐胁迫耐性的验证实验 [0349] Table 14: Experimental verification ME02064P1 salt stress tolerance in one generation

[0350] [0350]

Figure CN101981191AD00501

[0351 ] *SGI(盐生长指数)=幼苗面积xFv/Fm (光合效率) [0351] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0352] 结果总结: [0352] The results are summarized:

[0353] 处于35S启动子控制下的克隆375587P1的异位表达增强对盐胁迫的耐性。 [0353] Ectopic expression of clones in 375587P1 under the control of the 35S promoter enhances tolerance to salt stress.

[0354]实施例 15 :ME02064P2 :克降375578P2-SEQ ID No. 140 的氨基酸188 到498 [0354] Example 15: ME02064P2: g drop 375578P2-SEQ ID No. 140 of the amino acid 188-498

[0355] 用带有35S启动子和编码Ceres克隆375578P2(SEQ ID NO : 140的氨基酸188 到498)的核酸的Ti质粒转化野生型拟南芥Wassilewski ja,所述核酸是上文实施例中所述Ceres克隆375578的5'截短变体。 [0355] with a 35S promoter and the coding Ceres CLONE 375578P2: Ti plasmid nucleic acid (SEQ ID NO 140 amino acids 188-498) of the wild-type Arabidopsis transformed Wassilewski ja, the nucleic acid is as described above in Example embodiments said Ceres cLONE 375578 5 'truncated variant. 研究八个ME02064P2转基因品系对盐的耐性。 Research ME02064P2 eight transgenic lines tolerance to salt. 在与非转基因对照幼苗相比的定量实验中,与非转基因对照幼苗相比,四个转基因品系ME02064P2-01-T2、ME02064CP2-04_T2、ME02064P2-05_T2、ME02064P2-06_T2、 ME02064P2-07-T2,ME02064P2-T2-08 和ME02064P2-09_T2 不显示统计学显著的盐耐性;一个转基因品系ME02064P2-10-T2显示盐耐性的统计学显著降低。 In quantitative assays as compared to non-transgenic control seedlings in comparison to non-transgenic control seedlings, four transgenic lines, ME02064P2-01-T2, ME02064CP2-04_T2, ME02064P2-05_T2, ME02064P2-06_T2, ME02064P2-07-T2, ME02064P2 -T2-08 and ME02064P2-09_T2 not show statistically significant salt tolerance; a statistically ME02064P2-10-T2 transgenic lines display a significant reduction in salt tolerance.

[0356] 表15显示在含lOOmM NaCl的MS琼脂平板上生长时,与非转基因的对照幼苗相比,ME02064P2-01-T2幼苗的T2-世代SGI值为1.62% ;与非转基因的对照幼苗相比,ME02064P2-04-T2幼苗为20. 31% ;与非转基因的对照幼苗相比,ME02064P2_05_T2幼苗为31.24% ;与非转基因的对照幼苗相比,ME02064P2-06-T2幼苗为41. 14% ;与非转基因的对照幼苗相比,ME02064P2-07-T2幼苗为15.91% ;与非转基因的对照幼苗相比, ME02064P2-08-T2幼苗为40. 82% ;与非转基因的对照幼苗相比,ME02064P2_09_T2幼苗为135. 79% ;与非转基因的对照幼苗相比,ME02064P2-10-T2为-12. 36%。 [0356] Table 15 shows when grown on MS agar plates containing lOOmM NaCl, compared with non-transgenic control seedlings, ME02064P2-01 T2-generation SGI value seedlings T2- 1.62%; and non-transgenic control seedlings with ratio, ME02064P2-04 T2-seedlings was 20.31%; compared with non-transgenic control seedlings, ME02064P2_05_T2 seedlings was 31.24%; compared with non-transgenic control seedlings, ME02064P2-06 T2-seedlings was 41.14%; compared to non-transgenic control seedlings, ME02064P2-07-T2 seedlings was 15.91%; compared with non-transgenic control seedlings, ME02064P2-08 T2-seedlings was 40.82%; compared with non-transgenic control seedlings, ME02064P2_09_T2 seedlings was 135.79%; compared with non-transgenic control seedlings, ME02064P2-10-T2 -12 36%. [0357] 在含lOOmM NaCl的MS琼脂平板上生长时,相对于非转基因植物而言,ME02064P2-01-T2, ME02064P2-04-T” ME02064P2-05-T” ME02064P2-06-T” ME02064P2-07-T2、ME02064P2_08_T2 和ME02064P2_09_T2 转基因植物显示显著更大的幼苗面积和SGI。然而如表3中所示,ME02064P2-10-T2幼苗的T2-世代SGI值与非转基因对照幼苗相比显示SGI的降低。 [0357] When grown on MS agar plates containing lOOmM NaCl with respect to the non-transgenic plants, ME02064P2-01-T2, ME02064P2-04-T "ME02064P2-05-T" ME02064P2-06-T "ME02064P2-07 -T2, ME02064P2_08_T2 ME02064P2_09_T2 and transgenic plants showed significantly greater seedling area and SGI. However, as shown in the table, T2- generation SGI value of the non-transgenic control seedlings ME02064P2-10-T2 seedlings as compared to 3 in the SGI exhibit reduced.

[0358] 表15 :—世代中ME02064P2对盐耐性的验证实验 [0358] Table 15: - Verification experiment generations ME02064P2 salt tolerance

[0359] [0359]

Figure CN101981191AD00511

[0360] *SGI (盐生长指数)=幼苗面积xFv/Fm (光合效率) [0360] * SGI (Salt Growth Index) = seedling area xFv / Fm (photosynthesis efficiency)

[0361] 结果总结: [0361] The results are summarized:

[0362] •处于35S启动子控制下的克隆375587P2的异位表达增强对盐胁迫的耐性。 [0362] • in ectopic expression clones 375587P2 under the control of the 35S promoter enhances tolerance to salt stress.

[0363] • Ceres 克隆375578P2 保留位于SEQ ID NO : 140 氨基酸残基137-157 内的Ceres 克隆375578的a-0结构域,但是不保留SEQ ID NO : 140的Ceres克隆375578的6-r 结构域。 [0363] • Ceres Clone 375578P2 retains located SEQ ID NO: 140 amino acid residues 137-157 in the Ceres clone a-0 375578 domain, but does not retain SEQ ID NO: Ceres 140 clone 6-r domains 375578 .

[0364]实施例 16 :ME10681 :克隆335348 :SEQ ID No. 141 [0364] Example 16: ME10681: Clone 335348: SEQ ID No. 141

[0365] 用带有与Ceres cDNA 335348 (SEQ ID NO : 141)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewskija。 [0365] with the use of Ceres cDNA 335348: transform wild type Arabidopsis Wassilewskija Ti plasmid 35S promoter operably linked to (SEQ ID NO 141). 在含100或150yM SA的平板上生长时,野生型Ws幼苗显示坏死损害和矮化的生长,而转基因植物显示显著更好的生长。 When grown on plates containing 100 or 150yM SA wild type Ws seedlings show necrotic lesions and stunted growth, the transgenic plants showed significantly better growth.

[0366] 通过含100 ii M SA的MS琼脂平板上的生长,定量研究三个转基因品系ME10681-0UME10681-02和ME10681-05。14天后,使用EPSON彩色扫描仪或荧光扫描仪扫描平板,并针对每株植物计算SAGI。 [0366] by growth on MS agar plates containing 100 ii M SA quantitative study of three transgenic lines and ME10681-05.14 ME10681-0UME10681-02 days, using EPSON color scanner or fluorescence scanner plate, and for each plant is calculated SAGI. 数据概括于表16中。 The data is summarized in Table 16.

[0367] 在含100 uM SA的MS琼脂平板上生长时,ME10681-02_T2和ME10681-05_T2转基因植物相对于非转基因植物显示显著提高的幼苗面积和SAGI。 [0367] When grown on MS agar plates containing 100 uM SA's, ME10681-02_T2 ME10681-05_T2 and transgenic plants relative to non-transgenic plants showed significantly increased seedling area and SAGI. 然而,ME10681-01-T2相对于非转基因植物显示轻微的SAGI降低。 However, ME10681-01-T2 relative to non-transgenic plants showed a slight decrease in SAGI. 如表12中所示,ME10681-01-T2、ME10681-02-T2*ME10681-05-T2幼苗的T2代SAGI值分别为-3. 29%、17. 65%和51. 84%。 Such as, ME10681-01-T2, T2-generation SAGI value ME10681-02-T2 * ME10681-05-T2 seedlings as shown in Table 12, respectively -3. 29%, 17.65% and 51.84%. 转基因和非转基因幼苗之间的差异对品系ME10681-02-T2和ME10681-05-T2而言具有统计学显著的P值,并且清楚地证明增强的对氧化胁迫的耐性是ME10681转化体品系中Ceres cDNA 36505846异位表达的结果。 The difference between the gene transfer and non-transgenic seedlings have statistically significant P values ​​for lines ME10681-02-T2 ME10681-05-T2 and terms, and clearly demonstrate enhanced tolerance to oxidative stress is Ceres ME10681 transformant strains cDNA 36505846 ectopic expression results.

[0368] 表16 :—世代中ME10681的水杨酸验证实验 [0368] Table 16: - Generation of verification experiments salicylic acid ME10681

[0369] [0369]

Figure CN101981191AD00521

[0370] 结果总结: [0370] The results are summarized:

[0371] 总之,处于35S启动子控制下的Ceres克隆335348的异位表达增强氧化胁迫耐性,所述氧化胁迫耐性在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0371] In summary, in the Ceres clone the 35S promoter enhanced ectopic expression of 335,348 oxidative stress tolerance, oxidative stress tolerance due to the necrotic lesions and stunted growth in wild-type Ws seedlings.

[0372]实施例 17 :ME24091 :克降106263 :SEQ ID No. 136 [0372] Example 17: ME24091: down g 106263: SEQ ID No. 136

[0373] 用带有与Ceres cDNA 016263 (SEQ ID NO : 135)有效连接的35S启动子的Ti质粒转化野生型拟南芥Wassilewskija。 [0373] with the use of Ceres cDNA 016263 (SEQ ID NO: 135) transform wild type Arabidopsis Wassilewskija Ti plasmid 35S promoter operably linked. 在含100或150yM SA的平板上生长时,野生型Ws幼苗显示坏死损害和矮化的生长,而转基因植物显示显著更好的生长。 When grown on plates containing 100 or 150yM SA wild type Ws seedlings show necrotic lesions and stunted growth, the transgenic plants showed significantly better growth.

[0374] 通过含100 ii M SA的MS琼脂平板上的生长,定量研究十个转基因品系ME24091-01-T2、ME24091-02-T” ME24091-03_T2、ME24091-04_T2、ME24091-05_T2、 ME24091-06-T2、ME24091_07_T2、ME24091-08_T2、ME24091_09_T2 和ME24091_10_T2。14 天后,使用EPSON彩色扫描仪或荧光扫描仪扫描平板,并针对每株植物计算SAGI。 [0374] by growth on MS agar plates containing 100 ii M SA quantitative study of ten transgenic lines, ME24091-01-T2, ME24091-02-T "ME24091-03_T2, ME24091-04_T2, ME24091-05_T2, ME24091-06 -T2, ME24091_07_T2, ME24091-08_T2, ME24091_09_T2 and ME24091_10_T2.14 days, using EPSON color scanner or fluorescence scanner plate and SAGI calculated for each plant.

[0375]在含 100 ii M SA 的MS 琼脂平板上生长时,ME24091_01_T2、ME24091_02_T2、 ME2409卜03-T2、ME24091-04-01_T3、ME24091-04_T2、ME24091-05-01_T3、ME24091-05_T2、 ME24091-06-01、ME24091-06、ME24091-07-01、ME24091-07、ME24091-08-01、ME24091-08、 ME24091-09-01、ME24091-09、ME24091-10-01 和ME24091-10 转基因植物相对于非转基因植物显示显著提高的幼苗面积和SAGI。 [0375] When grown on MS agar plates containing 100 ii M SA's, ME24091_01_T2, ME24091_02_T2, ME2409 Bu 03-T2, ME24091-04-01_T3, ME24091-04_T2, ME24091-05-01_T3, ME24091-05_T2, ME24091-06 -01, ME24091-06, ME24091-07-01, ME24091-07, ME24091-08-01, ME24091-08, ME24091-09-01, ME24091-09, ME24091-10-01 and ME24091-10 transgenic plants with respect to non-transgenic plants showed significantly increased seedling area and SAGI. 如表17中所示,ME24091-01、ME24091-02、 ME24091-03、ME24091-04、ME24091-05、ME24091-06、ME24091-07、ME24091-08、ME24091-09 和ME24091-10 幼苗的T2 代SAGI 值分别提高119. 47%,198. 00%和133. 67%,241. 50%, 143. 70%和248. 12% ,186. 59%,188. 86%,285. 42%和180. 46%。 As shown in Table, ME24091-01, ME24091-02, ME24091-03, ME24091-04, ME24091-05, ME24091-06, ME24091-07, ME24091-08, ME24091-09 and ME24091-10 seedlings of the T2 generation 17 SAGI values ​​were increased by 119.47%, 198.00% and 133.67%, 241.50%, 143.70% and 248.12%, 186.59%, 188.86%, 285.42% and 180 46%. 转基因和非转基因幼苗之间的差异对品系ME24091-01、ME24091-02、ME24091-03、ME24091-04-01、ME24091-04、 ME24091-05-01、ME24091-05、ME24091-06-01、ME24091-06、ME24091-07-01、ME24091-07、 ME24091-08、ME24091-09-01、ME24091-09 和ME24091-10 而言具有统计学显著的P 值,并且清楚地证明增强的对氧化胁迫的耐性是ME24091转化体品系中Ceres克隆106263异位表达的结果。 Seedlings differences between transgenic and non-transgenic lines for ME24091-01, ME24091-02, ME24091-03, ME24091-04-01, ME24091-04, ME24091-05-01, ME24091-05, ME24091-06-01, ME24091 -06, ME24091-07-01, ME24091-07, ME24091-08, ME24091-09-01, ME24091-09 and ME24091-10 P values ​​in terms of statistically significant, and clearly demonstrate enhanced oxidative stress ME24091 transformant is resistant strains ectopic expression of Ceres clone 106263 results.

[0376] 表17 :两世代中ME24091的水杨酸验证实验 [0376] Table 17: ME24091 two generations verification experiment salicylic acid

52[0377] 52 [0377]

Figure CN101981191AD00531
Figure CN101981191AD00541

[0379] 结果总结: [0379] The results are summarized:

[0380] 总之,处于35S启动子控制下的Ceres cDNA克隆106263的异位表达增强氧化胁迫耐性,所述氧化胁迫耐性在野生型Ws幼苗中引起坏死损害和矮化的生长。 [0380] In summary, Ceres cDNA is under control of the 35S promoter was cloned 106263 Ectopic expression of oxidative stress tolerance enhancing the oxidative stress tolerance caused necrotic lesions and stunted growth in wild-type Ws seedlings.

[0381] 实施例18 :通过Reciprocal BLAST确定功能件同源物 [0381] Example 18: determining function member by Reciprocal BLAST homolog

[0382] 如果候选和参照序列编码具有相似功能和/或活性的蛋白质,则认为候选序列是参照序列的功能性同源物。 [0382] If the candidate having similar functions or protein and / or activity of the reference sequence coding, the reference candidate sequence was considered a functional homolog sequences. 使用已知为Reciprocal BLAST (Rivera等,Proc. Natl. Acad. Sci.USA,95 =6239-6244(1998))的方法从数据库中鉴定可能的功能同源序列,所述数据库由所有可获得的公开和专利肽序列组成,包括来自NCBI的NR和来自Ceres克隆的肽翻译。 Known as Reciprocal BLAST (Rivera et, Proc. Natl. Acad. Sci.USA, 95 = 6239-6244 (1998)) method from the database identify potential functional homolog sequences, a database of all available and Patent Publication peptide sequences, including NR from NCBI and peptide translations from Ceres and clones.

[0383] 开始Reciprocal BLAST方法之前,使用BLAST针对来自特定参照多肽来源物种的所有肽搜索该特定的参照多肽,从而鉴定下述多肽,所述多肽与参照多肽在85%的比对长度上具有80%或更大的BLAST序列同一性,或者在比对中更短的序列上具有更大的BLAST 序列同一性。 Prior methods Reciprocal BLAST, BLAST search using the [0383] start for all peptides from a particular species of origin of the reference polypeptide particular reference polypeptide, thereby identifying a polypeptide, said polypeptide and reference polypeptide than 85% of the length of 80 % or more sequence identity BLAST, BLAST or has a greater sequence identity in an alignment of a shorter sequence. 参照多肽和任何上述鉴定的多肽称为簇。 Reference polypeptide and any of the aforementioned identified polypeptides called a cluster.

[0384] 使用来自Washington University at Saint Louis,Missouri,USA 的2. 0 版BLASTP程序测定BLAST序列同一性和E-值。 [0384] using from Washington at Saint Louis, Missouri, USA version 2. 0 BLASTP program BLAST sequence identity and E- measured values ​​University. 2. 0版BLASTP程序包含以下参数:a)E_值截止为1.0e-5;2)字长为5;和3)-p0StSW选项。 Version 2. 0 BLASTP program includes the following parameters: a) E_ value cutoff of 1.0e-5; 2) a word size of 5; and 3) -p0StSW option. 以鉴定的潜在功能性同源物序列的第一BLAST HSP (High-scoring SegmentPairs)与特定参照多肽的比对为基础,计算BLAST序列同一性。 A first BLAST HSP (High-scoring SegmentPairs) polypeptide with a particular reference to specific potential functional homolog sequences identified calculated based on BLAST sequence identity. 用BLASTHSP比对中完全匹配的残基数除以HSP长度,然后乘以100,得到BLAST序列同一性。 Divided by the number of residues in the alignment BLASTHSP exact match HSP length, and then multiplied by 100 to give the BLAST sequence identity. HSP长度通常包含比对中的缺口,但是在一些情况下排除缺口。 HSP length typically comprise gaps in the alignment, but in some cases excluding gaps.

[0385] 主要的Reciprocal BLAST方法包括两轮BLAST搜索:正向搜索和反向搜索。 [0385] The main Reciprocal BLAST process comprises two BLAST searches: Forward search and reverse search. 在正向搜索步骤中,针对来自目的物种的所有蛋白质序列BLAST来自来源物种SA的参照多肽序列“多肽A”。 In the forward search step, BLAST against all protein sequences from a species of interest from a reference polypeptide sequence source species SA "polypeptide A". 使用10_5的E-值截止和35%的序列同一性截止确定顶极命中(top hit)。 E- value cutoff of use 10_5 and 35% sequence identity cutoff determination climax hit (top hit). 在顶极命中中,具有最低E-值的序列被称为最佳命中,并且被认为是潜在的功能性同源物或直系同源物。 In the climax hits, with potential functional homolog or ortholog sequences E- minimum value is referred to as the best hit, and is considered to be. 与最佳命中或与原始参照多肽具有80%或更大序列同一性的任何其他顶极命中同样被认为是可能的功能性同源物或直系同源物。 The best hit or to the original reference polypeptide of 80% or greater sequence identity to any other hit or a climax also is considered a possible functional homolog or ortholog. 针对所有目的物种重复该方法。 This process is repeated for all the species of interest.

[0386] 在反向搜索轮中,针对来自来源物种SA的所有蛋白质序列BLAST来自所有物种的正向搜索中鉴定的顶极命中。 [0386] In the reverse search round, against all protein sequences from the source species SA BLAST hits climax forward search from all species identification. 来自正向搜索的下述顶极命中也被认为是可能的功能性同源物,所述顶极命中返回来自上述簇中的多肽作为其最佳命中。 The following climax hit from the forward search that may also be considered a functional homologue thereof, said polypeptide from said top pole return hit cluster as its best hit.

[0387] 通过可能的功能性同源物序列的手检鉴定功能性同源物。 [0387] Hand subject through the identification of functional homologs of the possible functional homolog sequences. 图1-6和序列表中展示了SEQ ID NO. 2、35、41、43、44、45、86、109、135、136、138、140、141、142、143 和SEQ ID NO: 140的氨基酸XY和SEQ ID NO :140的氨基酸X_Y的代表性功能性同源物。 Figure 1-6 shows the Sequence Listing and SEQ ID NO 2,35,41,43,44,45,86,109,135,136,138,140,141,142,143 and SEQ ID NO:. 140 of XY and amino acid SEQ ID NO: representative amino acid X_Y functional homologues 140.

[0388] ti^l丨19 :诵过遍蔽马尔禾斗夫1草型石角定能t牛同源夺勿 [0388] ti ^ l Shu 19: chant through all over Malta shield Wo-fu fighting grass type 1 t Shek Kok will be able to seize the cattle do not homologous

[0389] 通过程序HMMER 2. 3. 2产生隐蔽马尔科夫模型(HMM)。 [0389] generating Hidden Markov Model (HMM) by the program HMMER 2. 3. 2. 为了产生每个HMM,使用默认HMMER 2. 3. 2程序参数,用于全局比对的conFigured。 To produce each of the HMM, the default HMMER 2. 3. 2 program parameters, conFigured for global alignment.

[0390] 使用图1中所示序列作为输入产生HMM。 1 HMM generated input sequence as shown in [0390] FIG. 将这些序列输入模型中,每个序列的HMM 二进制值示于序列表中。 These sequences were input model, HMM for each sequence of binary values ​​are shown in the Sequence Listing. 将额外的序列输入模型中,序列表中展示了额外序列的HMM 二进制值。 The additional model input sequence, the sequence table shows the binary value of the additional sequences of the HMM. 结果表明这些额外的序列是SEQ ID NO :86的功能性同源物。 The results indicate that these additional sequences are SEQ ID NO: 86 functional homolog.

[0391] 还使用图2-6中所示序列作为输入产生HMM。 [0391] As a further input HMM was generated using the sequence illustrated in Figures 2-6. 将这些序列输入各个模型中,序列表中展示了每个序列相应的HMM 二进制值。 The various models of these input sequences in the Sequence Listing shows the sequence corresponding to each of the HMM bit score. 将额外的序列输入模型中,序列表中展示了额外序列的HMM 二进制值。 The additional model input sequence, the sequence table shows the binary value of the additional sequences of the HMM. 结果表明这些额外的序列是图2-6中组的功能性同源物。 The results indicate that these additional sequences are functional groups in Figures 2-6 homologs.

[0392] 在一个替代性实施方案中产生HMM,前题是不使用PCT/US2007/06544中明确描述的氨基酸序列。 [0392] In an HMM generated alternative embodiment, the premise is not used in the amino acid sequence PCT / US2007 / 06544 explicitly described. 具体地,排除PCT/US2007/06544序列表中展示的以下氨基酸:SEQ ID NO: 99、SEQ IDNO :100、SEQ ID NO :102、SEQ ID NO :103、SEQ ID NO :104、SEQ IDN0 :180、SEQ ID NO : 252,SEQ ID NO : 298、SEQ ID NO : 300、SEQ IDNO : 301、SEQ ID N0:306 和SEQ ID NO: 312。 Specifically excluded PCT / US2007 / 06544 in the Sequence Listing shows the amino acid: SEQ ID NO: 99, SEQ IDNO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ IDN0: 180 , SEQ ID NO: 252, SEQ ID NO: 298, SEQ ID NO: 300, SEQ IDNO: 301, SEQ ID N0: 306 and SEQ ID NO: 312.

[0393] 参考文献 [0393] Reference

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Claims (38)

  1. 产生具有提高的盐度耐性或提高的氧化胁迫耐性的植物的方法,所述方法包括培养包含外源核酸的植物细胞,所述外源核酸包含与编码多肽的核苷酸序列有效连接的调节区,并且其中编码所述多肽的氨基酸序列的HMM二进制值大于约30,所述HMM以图1‑6之一中所示的氨基酸序列为基础,并且其中所述植物与不包含所述核酸的对照植物对盐度或氧化胁迫的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 A method of producing a plant having increased oxidative stress tolerance or increased salinity tolerance, the method comprising culturing a plant cell comprising an exogenous nucleic acid, said exogenous nucleic acid comprises a regulatory region nucleotide sequence encoding a polypeptide operably linked to and wherein the HMM bit score of the amino acid sequence encoding the polypeptide is greater than about 30, said HMM amino acid sequence shown in FIG. 1-6 is based, and wherein said plant does not comprise the nucleic acid control plants respective levels of tolerance to salinity or oxidative stress as compared to the level difference of tolerance to salinity or oxidative stress.
  2. 2.根据权利要求1的方法,其中编码所述多肽的氨基酸序列的HMM 二进制值大于约400,所述HMM以图1中所示氨基酸序列为基础。 2. The method according to claim 1, wherein the HMM bit score of the amino acid sequence encoding the polypeptide is greater than about 400, said HMM in the amino acid sequence shown in Figure 1 is based.
  3. 3.根据权利要求1的方法,其中编码所述多肽的氨基酸序列的HMM 二进制值大于约30,所述HMM以图2中所示氨基酸序列为基础。 3. The method according to claim 1, wherein the HMM bit score of the amino acid sequence encoding the polypeptide is greater than about 30, said HMM in the amino acid sequence shown in FIG. 2 is based.
  4. 4.根据权利要求1的方法,其中编码所述多肽的氨基酸序列的HMM 二进制值大于约120,所述HMM以图3中所示氨基酸序列为基础。 4. A method according to claim 1, wherein the HMM bit score of the amino acid sequence encoding the polypeptide is greater than about 120, said HMM in the amino acid sequence shown in FIG. 3 based.
  5. 5.根据权利要求1的方法,其中编码所述多肽的氨基酸序列的HMM 二进制值大于约150,所述HMM以图4中所示氨基酸序列为基础。 The method according to claim 1, wherein the HMM bit score of the amino acid sequence encoding the polypeptide is greater than about 150, said HMM in the amino acid sequence shown in Figure 4 is based.
  6. 6.根据权利要求1的方法,其中编码所述多肽的氨基酸序列的HMM 二进制值大于约425,所述HMM以图5中所示氨基酸序列为基础。 6. The method according to claim 1, wherein the HMM bit score of the amino acid sequence encoding the polypeptide is greater than about 425, said HMM in the amino acid sequence shown in FIG. 5 is based.
  7. 7.根据权利要求1的方法,其中编码所述多肽的氨基酸序列的HMM 二进制值大于约550,所述HMM以图6中所示氨基酸序列为基础。 7. A method according to claim 1, HMM binary value wherein the amino acid sequence encoding the polypeptide is greater than about 550, said HMM in the amino acid sequence shown in FIG. 6 is based.
  8. 8.产生具有提高的盐度耐性或提高的氧化胁迫耐性的植物的方法,所述方法包括培养包含外源核酸的植物细胞,所述外源核酸包含与编码下述多肽的核苷酸序列有效连接的调节区,所述多Jft与选自SEQ ID NO :2、4、6、8、9、11、13、14、15、17、19、20、22、23、24、25、27、 29、30、31、33、35、36、37、38、39、41、42、43、44、45、47、49、50、52、54、56、58、60、62、63、64、 66、68、69、71、73、74、76、78、80、81、83、84、86、88、90、91、93、94、96、98、100、101、102、104、 106、107、109、110、112、114、116、118、119、121、122、123、125、126、127、128、129、130、132、 134、136、138、140、141、142、143、144、145、147、149、151、153、154、156、158、160、162、163、 165、166、167、168 JPSEQ ID NO :140的氨基酸等同物1到135的氨基酸序列具有至少85% 或更高的序列同一性,其中由所述植物细胞产生的所 The method 8. generating plant having increased oxidative stress tolerance or increased salinity tolerance of a plant comprising culturing cells comprising exogenous nucleic acid, said exogenous nucleic acid comprising a nucleotide sequence encoding a polypeptide with an effective regulatory region connected to the plurality Jft selected from SEQ ID NO: 2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27, 29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64, 66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104, 106, 107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132, 134,136,138,140,141,142,143, 144,145,147,149,151,153,154,156,158,160,162,163, 165,166,167,168 JPSEQ ID NO: 140 amino acid equivalents 1-135 amino acid sequence having at least 85% or more sequence identity, wherein said plant cell produced by the 植物与不包含所述核酸的对照植物中的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 Plant compared to a control plant not comprising the nucleic acid respective levels of tolerance, the difference of the level of tolerance to salinity or oxidative stress.
  9. 9.产生植物的方法,所述方法包括培养包含外源核酸的植物细胞,所述外源核酸包含与下述核苷酸序列有效连接的调节区,所述核苷酸序列与选自SEQ ID N0:l、3、5、7、10、 12、16、18、21、26、28、32、34、40、46、48、51、53、55、57、59、61、65、67、70、72、75、77、79、82、 85、87、89、92、95、97、99、103、105、108、111、113、115、117、120、124、131、133、135、137、139、 146、148、150、152、155、157、159、161和164的核苷酸序列具有85%或更高的序列同一性, 其中由所述植物细胞产生的植物与不包含所述核酸的对照植物对盐度或氧化胁迫的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 9. A method of producing a plant, said method comprising culturing a plant cell comprising an exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence, said nucleotide sequence selected from SEQ ID N0: l, 3,5,7,10, 12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67, 70,72,75,77,79,82, 85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135, 137, 139, and 164 146,148,150,152,155,157,159,161 nucleotide sequence having 85% or more sequence identity, wherein a plant produced from said plant cell which does not comprise the said control plant a nucleic acid of the corresponding level of salinity or oxidative stress tolerance as compared to the level difference of tolerance to salinity or oxidative stress.
  10. 10.调控植物对盐度或氧化胁迫耐性水平的方法,所述方法包括向植物细胞中引入外源核酸,所述外源核酸包含与编码多肽的核苷酸序列有效连接的调节区,其中编码所述多肽的氨基酸序列的HMM 二进制值大于约30,所述HMM以图1-6之一中所示的氨基酸序列为基础,并且其中由所述植物细胞产生的植物与不包含所述外源核酸的对照植物对盐度或氧化胁迫的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 10. The method of regulation of plant to salinity or oxidative stress tolerance levels, said method comprising introducing into a plant cell an exogenous nucleic acid, said exogenous nucleic acid comprising a nucleotide sequence encoding a regulatory region operably linked polypeptide, wherein the coding HMM bit score of the amino acid sequence of the polypeptide is greater than about 30, said HMM amino acid sequence shown in FIG. 1-6 basis, and wherein a plant produced from the plant cell does not comprise the exogenous nucleic acid corresponding control plant salinity or oxidative stress tolerance levels compared, the difference of levels of tolerance to salinity or oxidative stress.
  11. 11.调控植物对盐度或氧化胁迫耐性水平的方法,所述方法包括向植物细胞中引入外源核酸,所述外源核酸包含与编码下述多肽的核苷酸序列有效连接的调节区,其中所述多肽与选自SEQ ID NO :2、4、6、8、9、11、13、14、15、17、19、20、22、23、24、25、27、29、30、31、33、 35、36、37、38、39、41、42、43、44、45、47、49、50、52、54、56、58、60、62、63、64、66、68、69、71、 73、74、76、78、80、81、83、84、86、88、90、91、93、94、96、98、100、101、102、104、106、107、109、 110、112、114、116、118、119、121、122、123、125、126、127、128、129、130、132、134、136、138、 140、141、142、143、144、145、147、149、151、153、154、156、158、160、162、163、165、166、167、 168,和SEQ ID NO :140的氨基酸等同物1到135的氨基酸序列具有85%或更高的序列同一性,其中由所述植物细胞产生的植物与不包含所述 11. The method of regulation of plant to salinity or oxidative stress tolerance levels, said method comprising introducing into a plant cell an exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region and a nucleotide sequence encoding a polypeptide operably linked, wherein said polypeptide selected from SEQ ID NO: 2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31 , 33, 35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69 , 71, 73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109, 110 , 112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138, 140,141,142,143,144,145,147 , 149,151,153,154,156,158,160,162,163,165,166,167, 168, and SEQ ID NO: 140 amino acid equivalents 1-135 amino acid sequence having 85% or more sequence identity, wherein a plant produced from said plant cell not comprising said 酸的对照植物对盐度或氧化胁迫的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 Acid corresponding control plant salinity or oxidative stress tolerance levels compared, the difference of levels of tolerance to salinity or oxidative stress.
  12. 12.权利要求1-8、10或11中任一项的方法,其中所述多肽选自SEQID NO :43、44、45、 86、140、141、142、143、144和SEQ ID NO :140的氨基酸等同物1到135,并且所述植物与不包含所述核酸的对照植物对盐度的相应耐性水平相比,对盐度的耐性水平有差异。 1-8,10 or 11 12. A method according to any one of claims, wherein said polypeptide is selected from SEQID NO: 43,44,45, 86,140,141,142,143,144 and SEQ ID NO: 140 amino acids 1 to 135 equivalents, and the plant does not comprise the nucleic acid as compared to a corresponding control plant salinity tolerance level, the level difference of salinity tolerance.
  13. 13.权利要求1-8、10或11中任一项的方法,其中所述多肽选自SEQID N0:136和141, 并且所述植物与不包含所述核酸的对照植物对氧化胁迫的相应耐性水平相比,对氧化胁迫的耐性水平有差异。 1-8,10 or 11 13. A method according to any one of claims, wherein said polypeptide is selected from SEQID N0: 136 and 141, and a corresponding plant not comprising said nucleic acid control plant resistance to oxidative stress compared level, there are differences in levels of tolerance to oxidative stress.
  14. 14.调控植物对盐度或氧化胁迫耐性水平的方法,所述方法包括向植物细胞中引入外源核酸,所述外源核酸包含与下述核苷酸序列有效连接的调节区,其中所述核苷酸序列与选自SEQ ID NO :1、3、5、7、10、12、16、18、21、26、28、32、34、40、46、48、51、53、55、57、59、61、 65、67、70、72、75、77、79、82、85、87、89、92、95、97、99、103、105、108、111、113、115、117、120、 124、131、133、135、137、139、146、148、150、152、155、157、159、161 和164 的核苷酸序列具有85%或更高的序列同一性,其中由所述植物细胞产生的植物与不包含所述核酸的对照植物对盐度或氧化胁迫的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 14. A method of regulation of plant to salinity or oxidative stress tolerance levels, said method comprising introducing into a plant cell an exogenous nucleic acid, said exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence, wherein the a nucleotide sequence selected from SEQ ID NO: 1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57 , 59, 61, 65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120 , 124,131,133,135,137,139,146,148,150,152,155,157,159,161 and 164 nucleotide sequence having 85% or more sequence identity, wherein the said plant or plant cells with the nucleic acid does not contain a corresponding control plant tolerance to salinity or oxidative stress levels compared, the difference of levels of tolerance to salinity or oxidative stress.
  15. 15.包含外源核酸的植物细胞,所述外源核酸包含与编码多肽的核苷酸序列有效连接的调节区,其中所述多肽的氨基酸序列的HMM二进制值大于约30,所述HMM以图1-6之一中所示的氨基酸序列为基础,并且其中所述植物与不包含所述核酸的对照植物对盐度或氧化胁迫的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 15. A plant cell comprising an exogenous nucleic acid, said exogenous nucleic acid comprising a nucleotide sequence encoding a regulatory region operably linked polypeptide, wherein the HMM bit score of the amino acid sequence of the polypeptide is greater than about 30, said HMM in FIG. the amino acid sequence shown in 1-6 basis, and wherein said plant does not comprise the nucleic acid as compared to a corresponding control plant tolerance to salinity or oxidative stress level, the level of tolerance to salinity or oxidative stress Differences.
  16. 16.包含外源核酸的植物细胞,所述外源核酸包含与编码多肽的核苷酸序列有效连接的调节区,其中所述多肽与选自SEQ ID NO :2、4、6、8、9、11、13、14、15、17、19、20、22、23、 24、25、27、29、30、31、33、35、36、37、38、39、41、42、43、44、45、47、49、50、52、54、56、58、60、 62、63、64、66、68、69、71、73、74、76、78、80、81、83、84、86、88、90、91、93、94、96、98、100、101、 102、104、106、107、109、110、112、114、116、118、119、121、122、123、125、126、127、128、129、 130、132、134、136、138、140、141、142、143、144、145、147、149、151、153、154、156、158、160、 162、163、165、166、167、168和SEQ ID NO :140的氨基酸等同物1到135的氨基酸序列具有至少85%或更高的序列同一性,其中由所述植物细胞产生的植物与不包含所述核酸的对照植物中对盐度或氧化胁迫的相应耐性水平相比,对盐 16. A plant cell comprising an exogenous nucleic acid, said exogenous nucleic acid comprising a nucleotide sequence encoding a regulatory region operably linked to a polypeptide, wherein said polypeptide is selected from SEQ ID NO: 2,4,6,8,9 , 11,13,14,15,17,19,20,22,23, 24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44 , 45,47,49,50,52,54,56,58,60, 62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86 , 88,90,91,93,94,96,98,100,101, 102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126 , 127,128,129, 130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160, 162,163,165 , 166,167,168, and SEQ ID nO: amino acid sequence of amino acids 1 to 135 was equivalent to 140 having at least 85%, or more sequence identity, wherein a plant produced from said plant cell with the nucleic acid does not comprise compared to a corresponding control plant tolerance to salinity or oxidative stress level, salt 或氧化胁迫的耐性水平有差异。 There are differences or oxidative stress tolerance levels.
  17. 17.包含外源核酸的植物细胞,所述外源核酸包含与下述核苷酸序列有效连接的调节区,所述核苷酸序列与选自SEQ ID NO :1、3、5、7、10、12、16、18、21、26、28、32、34、40、46、48、 51、53、55、57、59、61、65、67、70、72、75、77、79、82、85、87、89、92、95、97、99、103、105、108、`111、113、115、117、120、124、131、133、135、137、139、146、148、150、152、155、157、159、161和`164的核苷酸序列具有85%或更高的序列同一性,其中由所述植物细胞产生的植物与不包含所述核酸的对照植物对盐度或氧化胁迫的相应耐性水平相比,对盐度或氧化胁迫的耐性水平有差异。 17. A plant comprising a cell exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence, said nucleotide sequence selected from SEQ ID NO: 1,3,5,7, 10,12,16,18,21,26,28,32,34,40,46,48, 51,53,55,57,59,61,65,67,70,72,75,77,79, 82,85,87,89,92,95,97,99,103,105,108, `111,113,115,117,120,124,131,133,135,137,139,146,148,150 nucleotide sequence 152,155,157,159,161 and 164 'having 85% or more sequence identity, wherein a plant produced from said plant cells a control plant not comprising the nucleic acid of salinity or a corresponding level of oxidative stress tolerance as compared to the level difference of tolerance to salinity or oxidative stress.
  18. 18.包含权利要求15-17中任一项的植物细胞的转基因植物。 Claim 18. The transgenic plant comprising plant cells of any one of 15-17.
  19. 19.权利要求18的转基因植物,其中所述植物是选自以下的物种成员:柳枝稷、两色蜀黍(高粱,双色高粱)、巨芒草(芒属)、甘蔗属物种(能源蔗)、香脂白杨(白杨)、玉蜀黍(玉米)、大豆(黄豆)、欧洲油菜(卡诺拉油菜)、普通小麦(小麦)、陆地棉(棉花)、稻(水稻)、向日葵、紫苜蓿(苜蓿)、甜菜或御谷(珍珠粟)。 Balsam poplar switchgrass, Sorghum bicolor (sorghum, Sorghum bicolor), giant Miscanthus (miscanthus), Saccharum species (energy cane),: transgenic plant 18 to 19. The claim, wherein the plant is a species of a member selected from (poplar), Zea mays (maize), soybean (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), rice (rice), sunflower, alfalfa (Medicago sativa), beet or bajra (pearl millet).
  20. 20.包含来自根据权利要求15-17中任一项的转基因植物的种子或营养组织的制品。 20. The article of claim 15-17 comprising any one of a transgenic plant seed or vegetative tissue from.
  21. 21.权利要求20的制品,其中所述制品是食品或饲料制品。 21. The article of claim 20, wherein said article is a food or feed product.
  22. 22.包含编码下述多肽的核苷酸序列的分离的核酸,所述多肽与SEQID N0:2、4、6、22、 27、29、49、52、54、56、60、62、68、76、83、88、90、96、98、104、106、112、114、132、134、149、151或160中所示的氨基酸序列具有80%或更高的序列同一性。 Isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide of 22, the polypeptide SEQID N0: 2,4,6,22, 27,29,49,52,54,56,60,62,68, the amino acid sequence shown in 76,83,88,90,96,98,104,106,112,114,132,134,149,151 160 or 80% or more sequence identity.
  23. 23.鉴定多态性是否与性状变异相关的方法,所述方法包括:a)测定植物种群中一种或多种遗传多态性是否与下述多肽的基因座相关,所述多肽选自图1-6中所示多肽及其功能性同源物;和b)测量所述种群的植物中所述性状的变异与所述种群的植物中所述一种或多种多态性的存在的相关性,从而鉴定是否所述一种或多种多态性与所述性状的变异相关。 23. Identification of polymorphism is associated with the character variation, the method comprising: a) measured a population of plants whether one or more genetic polymorphisms in the polypeptide associated with the following loci, the polypeptide is selected from FIG. polypeptides and their functional homologues in Figure 1-6; mutant plants in the plant trait and b) measuring the population and the population of the one or more polymorphisms in the presence of correlation, thereby identifying whether the one or more polymorphisms variation associated with the trait.
  24. 24.权利要求23的方法,其中所述性状是提高的对盐度的耐性,提高的对氧化胁迫的耐性,或提高的生物量。 24. The method of claim 23, wherein said trait is an increased tolerance to salinity, an increased tolerance to oxidative stress, or increased biomass.
  25. 25.制备植物品系的方法,所述方法包括:a)测定植物种群中一种或多种遗传多态性是否与下述多肽的基因座相关,所述多肽选自图1-6中所示多肽及其功能性同源物;b)在所述种群中鉴定一种或多种植物,其中所述一种或多种多态性中至少一种等位基因的存在与盐耐性或氧化胁迫耐性的变异相关;c)将所述一种或多种鉴定的植物的每一种与其自身或不同的植物杂交,产生种子;d)将从所述种子生长的至少一株后代植物与其自身或不同的植物杂交;和e)将步骤c)和d)再重复0-5代来制备所述植物品系,其中所述至少一种等位基因存在于所述植物品系中。 The method for preparing a plant lines 25. comprising: a) measured a population of plants whether one or more genetic polymorphisms in the polypeptide associated with the following loci, the polypeptide is selected from 1-6 shown in FIG. polypeptides and their functional homologues; b) identifying one or more plants in said population, wherein the one or more polymorphisms of at least one allele is present in salt tolerance or oxidative stress Related tolerance variation; c) each one of said one or more identified plants with itself or a different hybrid plant, to produce seed; D) from the seed growth of at least one progeny plant with itself or different plant hybridization; and e) the steps c) and d) are repeated substituting 0-5 to prepare the plant line, wherein the at least one allele is present in said plant line.
  26. 26.权利要求24或25的方法,其中所述种群是柳枝稷植物的种群。 26. The method of claim 24 or claim 25, wherein said population is a population of switchgrass plants.
  27. 27.权利要求1、8、10或11中任一项的方法,前提是编码多肽的所述核苷酸序列不编码PCT/US2007/06544的序列表中明确描述的多肽。 The method of any one of claims 1,8,10 or 27. 11, provided that the polynucleotide encoding the polypeptide does not encode a polypeptide sequence shown in SEQ PCT / US2007 / 06544 is explicitly described.
  28. 28.权利要求1、8、10或11中任一项的方法,前提是编码多肽的所述核苷酸序列不编码PCT/US2007/06544 的以下多月太序列之一:SEQ IDNO : 99、SEQ ID NO : 100、SEQ ID NO: 102,SEQ ID NO : 103,SEQ IDNO : 104,SEQ ID NO : 180,SEQ ID NO :252,SEQ ID NO :298,SEQ IDNO :300, SEQ ID N0:301、SEQ ID NO :306 和SEQ ID NO :312。 28. The method of any one of claims 1,8,10 or 11, provided that the nucleotide sequence encoding a polypeptide does not encode one of the following month too PCT sequence / US2007 / 06544 of: SEQ IDNO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ IDNO: 104, SEQ ID NO: 180, SEQ ID NO: 252, SEQ ID NO: 298, SEQ IDNO: 300, SEQ ID N0: 301, SEQ ID NO: 306 and SEQ ID NO: 312.
  29. 29.权利要求9或14的方法,前提是所述核苷酸序列不明确描述于PCT/US2007/06544 的序列表中。 29. The method of claim 9 or claim 14, provided that the nucleotide sequence is not specifically described in the Sequence Listing PCT / US2007 / 06544 in.
  30. 30.权利要求9或14的方法,前提是所述核苷酸序列不是PCT/US2007/06544的以下核苷酸序列之一:SEQ ID NO :98、SEQ IDNO : 101、SEQ ID NO :225 和SEQ ID NO :299。 30. The method of claim 9 or claim 14, provided that one of the nucleotide sequences of the nucleotide sequence is not PCT / US2007 / 06544 of: SEQ ID NO: 98, SEQ IDNO: 101, SEQ ID NO: 225 and SEQ ID NO: 299.
  31. 31.权利要求15或16的植物细胞,前提是编码多肽的所述核苷酸序列不编码PCT/ US2007/06544的序列表中明确描述的多肽。 15 or 16, plant cell of claim 31, provided that the polypeptide is the nucleotide sequence Sequence Listing encoding the polypeptide does not encode PCT / US2007 / 06544 is explicitly described.
  32. 32.权利要求15或16的植物细胞,前提是编码多肽的核苷酸序列不编码PCT/ US2007/06544 的以下多肽序列之一:SEQ ID NO :99、SEQ IDNO :100、SEQ ID NO :102、SEQ ID NO : 103,SEQ ID NO : 104,SEQ IDNO : 180,SEQ ID N0:252、SEQ ID NO : 298,SEQ ID NO: 300、SEQ IDNO :30U SEQ ID NO :306 禾口SEQ ID NO :312。 15 or 16, plant cell of claim 32, provided that one of the nucleotide sequence encoding a polypeptide does not encode PCT / US2007 / 06544 of the following polypeptide sequences: SEQ ID NO: 99, SEQ IDNO: 100, SEQ ID NO: 102 , SEQ ID NO: 103, SEQ ID NO: 104, SEQ IDNO: 180, SEQ ID N0: 252, SEQ ID NO: 298, SEQ ID NO: 300, SEQ IDNO: 30U SEQ ID NO: 306 Wo port SEQ ID NO : 312.
  33. 33.权利要求17的植物细胞,前提是所述核苷酸序列不明确描述于PCT/US2007/06544 的序列表中。 33. The plant cell as claimed in claim 17, provided that the nucleotide sequence is not specifically described in the Sequence Listing PCT / US2007 / 06544 in.
  34. 34.权利要求17的植物细胞,前提是所述核苷酸序列不是PCT/US2007/06544的以下核苷酸序列之一:SEQ ID N0:98、SEQ IDNO =IOUSEQ ID NO :225 禾Π SEQ ID NO :299。 34. A plant cell as claimed in claim 17, provided that the nucleotide sequence is not one of PCT / US2007 / 06544 The nucleotide sequences: SEQ ID N0: 98, SEQ IDNO = IOUSEQ ID NO: 225 Wo Π SEQ ID NO: 299.
  35. 35.权利要求22的核酸序列,前提是所述核苷酸序列不明确描述于PCT/US2007/06544 的序列表中。 35. The nucleic acid sequence as claimed in claim 22, provided that the nucleotide sequence is not specifically described in the Sequence Listing PCT / US2007 / 06544 in.
  36. 36.权利要求22的核酸,前提是所述核苷酸序列不是PCT/US2007/06544的以下核苷酸序列之一:SEQ ID NO :98、SEQ IDNO =IOUSEQ ID NO :225 和SEQ ID NO :299。 36. The nucleic acid as claimed in claim 22, provided that one of the nucleotide sequences of the nucleotide sequence is not PCT / US2007 / 06544 of: SEQ ID NO: 98, SEQ IDNO = IOUSEQ ID NO: 225 and SEQ ID NO: 299.
  37. 37.权利要求23或25的方法,前提是所述多肽不明确描述于PCT/US2007/06544的序列表中。 37. The method of claim 23 or claim 25, provided that the polypeptide is not specifically described in the Sequence Listing PCT / US2007 / 06544 in.
  38. 38.权利要求23或25的方法,前提是所述多肽不是PCT/US2007/06544的以下多肽序列之一:SEQ ID NO :99, SEQ IDNO : 100, SEQ ID NO : 102, SEQ ID NO : 103, SEQ ID NO : 104、 SEQ IDNO :180、SEQ ID NO :252、SEQ ID NO :298、SEQ ID NO :300、SEQ IDNO :301、SEQ ID NO :306 和SEQ ID NO :312。 38. The method of claim 23 or claim 25, provided that the polypeptide is not one of PCT / US2007 / 06544 of the following polypeptide sequences: SEQ ID NO: 99, SEQ IDNO: 100, SEQ ID NO: 102, SEQ ID NO: 103 , SEQ ID NO: 104, SEQ IDNO: 180, SEQ ID NO: 252, SEQ ID NO: 298, SEQ ID NO: 300, SEQ IDNO: 301, SEQ ID NO: 306 and SEQ ID NO: 312.
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