CN101817879A - Metallothionein and encoding gene and application thereof - Google Patents

Metallothionein and encoding gene and application thereof Download PDF

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CN101817879A
CN101817879A CN 200910078200 CN200910078200A CN101817879A CN 101817879 A CN101817879 A CN 101817879A CN 200910078200 CN200910078200 CN 200910078200 CN 200910078200 A CN200910078200 A CN 200910078200A CN 101817879 A CN101817879 A CN 101817879A
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gene
osmtla
protein
plant
plants
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CN 200910078200
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储成才
吴耀荣
杨昭
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中国科学院遗传与发育生物学研究所
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/825Metallothioneins
    • CCHEMISTRY; METALLURGY
    • 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
    • CCHEMISTRY; METALLURGY
    • 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 discloses a metallothionein and encoding genes and applications thereof. The protein is composed of the amino acid sequence shown in sequence 2 in the sequence table; and the protein can be used to cultivate the plants with higher drought resistance and/or higher zinc content. The invention also discloses a method for cultivating the plants with higher drought resistance and/or higher zinc content. The method is to introduce the genes in plants to obtain the transgenic plants with higher drought resistance and/or higher zinc content. The metallothionein and encoding genes thereof of the invention has great significance in agricultural production.

Description

金属硫蛋白及其编码基因与应用 Metallothionein its coding gene and application

技术领域 FIELD

[0001] 本发明涉及金属硫蛋白及其编码基因与应用。 [0001] The present invention relates to a metallothionein gene and its coding applications. 背景技术 Background technique

[0002]金属硫蛋白(metallothioneins,MT)是一类低分子量(6000 〜7000Da)、高Cys 含量,具有金属结合能力的多肽,广泛存在于生物界。 [0002] metallothionein (metallothioneins, MT) is a low molecular weight (6000 ~7000Da), a high content of Cys, metal binding polypeptide having the ability to widely present in the biosphere. 它具有独特的氨基酸排列顺序,即多肽的N端和C端具有两个富含Cys的金属结合结构域。 It has a unique amino acid sequence alignment, i.e., the polypeptide having the N-terminal and C-terminal two Cys-rich metal binding domain. Cyc残基与许多金属离子以硫醇键形式结合形成金属硫的四面体单位。 Cyc residues with many metal ions to form a thiol bond binding tetrahedral units formed metallothionein. 由于MT特有的氨基酸组成与分子结构,它在生物体内担负着重要的生理功能。 Since the amino acid composition and molecular structure of the specific MT, which bears the important physiological functions in vivo. 目前对植物MT功能的研究还处于起步状态,尚无十分明确的结论。 Current research on plant MT function still in its initial state, there is no very clear conclusion. 一般认为MT在金属离子的储存和运输、重金属的解毒、维持金属离子浓度稳态等方面起重要作用。 MT is generally believed that storage and transportation of metal ions, heavy metal detoxification, and maintenance of homeostasis and other metal ion concentration play an important role. 另外,MT还与植物的生长发育、胚胎发生、果实成熟、衰老等过程有关。 In addition, MT also with the growth and development of plants, embryogenesis, fruit ripening, aging and other processes. 越来越多的研究表明MT是植物体内重要的氧自由基清除剂之一,能提高植物的抗氧化能力。 More and more studies show that MT is one of the important plants of oxygen free radical scavenger, can increase the antioxidant capacity of the plant.

[0003] 根据氨基酸尤其是Cys的排列方式,可以把植物中的MT分为四类。 [0003] The particular arrangement of amino acid Cys may be in the plant MT divided into four categories. I型在根中表达量远远高于地上部分,II型则刚好相反,集中在叶中表达。 Type I expression in roots is much higher than the amount of the ground portion, II, is exactly the opposite, concentrated expression in leaves. III型主要在成熟果实中表达。 Type III is predominantly expressed in mature fruits. 而IV型迄今为止只在种子中发现,比如小麦胚芽和芝麻籽。 And type IV so far only found in seeds, such as wheat germ and sesame seeds. 水稻的MT家族包含11个成员,分别属于四类。 MT rice contains 11 family members belong to four categories. 除了被各种金属离子诱导外,其他一些刺激比如蔗糖饥饿、双氧水和盐胁迫也能提高水稻MT合成增加。 In addition to being induced by various metal ions, a number of other stimuli such as sucrose starvation, hydrogen peroxide can be improved, and salt stress in rice MT synthesis. 初步研究表明,水稻MT参与了重金属代谢、种子发育和抗氧化过程。 Preliminary studies indicate that MT of rice involved in the metabolism of heavy metals, seed development and anti-oxidation process.

[0004] 水稻是一种重要的粮食作物,是世界上约1/3人口的主要粮食来源。 [0004] Rice is an important food crop, is the world's main source of food for about one-third of the population. 现今世界范围内水稻的发展受到水资源匮乏以及地区性、季节性干旱的严重限制,增产潜力受到明显制约。 Today the development of rice by water scarcity worldwide and regional, seasonal drought severely limited yield potential was significantly restricted. 中国作为发展中国家,飞速发展的工业化和城市化进程加剧了水资源危机。 China as a developing country, industrialization and urbanization process of rapid development exacerbated the water crisis. 另外,由于农药的广泛使用,大量的汞、镉、铅等重金属造成土壤污染日趋严重。 In addition, due to the widespread use of pesticides, significant amounts of mercury, cadmium, lead and heavy metals cause soil pollution is worsening. 旱灾及各种污染影响了水稻的质量和产量。 Drought and various forms of pollution affect the quality and yield of rice. 因此,对水稻抗旱、抗金属毒害等生理进行研究,以期提高其抗旱等抗逆能力,显得十分必要。 Therefore, the physiology of rice resistance to drought, toxic metals and other research, in order to improve its drought resilience, etc., is very necessary.

[0005] 作物抗旱等非生物逆境研究是植物研究领域最具挑战性的工作之一。 Abiotic stress study [0005] drought and other crop plants is one of the working areas of study most challenging. 选择合适的研究材料是关键的第一步。 Select the appropriate study materials is a critical first step. 陆稻原产于巴西,又称巴西旱稻。 Upland native to Brazil, also known as Brazil upland rice. 一般认为,旱稻是由水稻演变而来的适于旱地栽培的“土地生态型”,因而陆稻的抗旱性比水稻强。 Generally it believed that upland rice is rice evolved from dry land suitable for the cultivation of "ecological land" and thus stronger than the drought resistance of upland rice. 陆稻与水稻的亲缘性很近。 Close affinity with upland rice. 比起远缘的双子叶植物比如拟南芥等,从陆稻研究中得到的功能基因应用于水稻抗旱实践更具效果。 Compared to distant dicots such as Arabidopsis, functional genes resulting from drought upland rice research in applied practice more effective.

发明内容 SUMMARY

[0006] 本发明的目的是提供一种金属硫蛋白及其编码基因与应用。 [0006] The object of the present invention is to provide a metallothionein gene and its coding applications.

[0007] 本发明所提供的金属硫蛋白,命名为OsMTla,来源于巴西旱稻,是如下a)或b)的蛋白: [0007] The present invention provides metallothionein, named OsMTla, upland rice from Brazil, is the following a) or b) protein:

[0008] a)序列表中序列2所示的氨基酸序列组成的蛋白质; 2 protein consisting of amino acid sequence shown in [0008] a) sequence table;

[0009] b)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸由a)衍生的蛋白质。 [0009] b) the amino acid sequence 2 by substitution and / or deletion and / or addition of one or several amino acids of a) a protein derived in the Sequence Listing.

[0010] 为了使a)的OsMTla蛋白质便于纯化,可在由序列表中序列2所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。 [0010] In order to a) is OsMTla proteins facilitate purification, the tag shown in Table 1 may be attached at the amino terminus or the carboxy-terminal sequence of the sequence shown in Table 2 proteins.

[0011] 表1.标签的序列 [0011] Table 1. Sequence tags

[0012] [0012]

<table>table see original document page 4</column></row> <table> <Table> table see original document page 4 </ column> </ row> <table>

[0013] 上述b)中的OsMTla蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。 OsMTla proteins can be synthesized [0013] b) above can also be synthesized first gene encoding, biological expression then obtained. 上述b)中的OsMTla蛋白质的编码基因可通过将序列表中序列1所示的DNA序列中缺失一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变,和/或在其5'端和/或3'端连上表1所示的标签的编码序列得到。 OsMTla gene encoding a protein b) above may be prepared by the deletion of a missense or several amino acid residues in the sequence shown in Sequence Listing 1 codon DNA sequence, and / or one or a few base pairs mutations, and / or obtained at its 5 'end and / or 3' of the coding sequence shown in table 1, the label on the terminal is connected.

[0014] 所述蛋白的编码基因也属于本发明的保护范围。 [0014] The gene encoding the protein are also within the scope of the present invention.

[0015] 所述蛋白的编码基因是如下1)或2)或3): [0015] The gene encoding the protein is the following 1) or 2) or 3):

[0016] 1)其核苷酸序列是序列表中序列1 ; [0016] 1) a nucleotide sequence which is a sequence table;

[0017] 2)在严格条件下与1)限定的DNA片段杂交且编码金属硫蛋白的DNA分子; [0017] 2) 1) defined DNA fragments and hybrid DNA molecule encoding metallothionein under stringent conditions;

[0018] 3)与1)或2)的基因具有90%以上的同源性,且编码金属硫蛋白的DNA分子。 [0018] 3) 1) or 2) a gene having a homology of 90% or more, and a DNA molecule encoding metallothionein.

[0019] 所述步骤3)中的基因,与1)的基因最好有95%以上的同源性。 In [0019] step 3) gene, a gene) is preferably more than 95% homology.

[0020] 上述严格条件可为在6XSSC,0. 5% SDS的溶液中,在68 °C下杂交,然后用2XSSC,0. 1% SDS 和1XSSC,0. SDS 各洗膜一次。 [0020] The stringency conditions may be in 6XSSC, 0. 5% SDS solution, hybridization at 68 ° C, followed by 2XSSC, 0. 1% SDS and 1XSSC, 0. SDS Each membrane is washed again.

[0021] 扩增OsMTla基因全长或任一片段的引物对也属于本发明的保护范围。 [0021] The full-length gene was amplified OsMTla range, or any fragment primer protection also belong to the present invention.

[0022] 含有上述OsMTla基因的重组载体、转基因细胞系和重组菌也属于本发明的保护范围。 [0022] recombinant vector containing the above-described OsMTla gene, transgenic cell lines and recombinant strain also belong to the scope of the present invention.

[0023] 可用现有的植物表达载体构建含有OsMTla基因的重组表达载体。 [0023] with available plant expression vector containing the recombinant expression vector OsMTla gene. 所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等,如pCAMBIA3301、pCAMBIA1300、 pBI121、pBinl9、pCAMBIA2301、pCAMBIA1301_UbiN或其它衍生植物表达载体。 The binary plant expression vector comprising Agrobacterium vector and plants can be used to support microprojectile bombardment and the like, such as pCAMBIA3301, pCAMBIA1300, pBI121, pBinl9, pCAMBIA2301, pCAMBIA1301_UbiN or other plant-derived expression vectors. 携带有本发明的OsMTla基因的植物表达载体可通过Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、 显微注射、电导、农杆菌介导等常规生物学方法转化到植物细胞或组织中。 OsMTla carrying the gene of the present invention can be obtained by plant expression vector Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection conventional biological methods, conductivity, etc. Agrobacterium-mediated transformation into plant cells or tissues.

[0024] 使用OsMTla基因构建重组植物表达载体时,在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子,如花椰菜花叶病毒(CaMV) 35S启动子、 泛生素基因Ubiquitin启动子(pUbi)等,它们可单独使用或与其它的植物启动子结合使用;此外,使用本发明的OsMTla基因构建植物表达载体时,还可使用增强子,包括翻译增强子或转录增强子,这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等,但必需与编码序列的阅读框相同,以保证整个序列的正确翻译。 [0024] When constructing a recombinant expression vector OsMTla plant gene, in which a transcription initiation nucleotides can be added before any enhancement, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CaMV ) 35S promoter, the Ubiquitin pangenesis gene promoter (pUbi) and the like, which may be used alone or in combination with other plant promoters; in addition, when the plant expression vector used according to the present invention OsMTla gene, an enhancer may be used , including translation enhancer or transcription enhancers, the enhancer region may be adjacent to the ATG start codon or the start codon and other regions, but required the same reading frame of the coding sequence to ensure proper translation of the entire sequence. 所述翻译控制信号和起始密码子的来源是广泛的,可以是天然的,也可以是合成的。 The source of translational control signals and initiation codons is extensive, it may be natural or may be synthetic. 翻译起始区域可以来自转录起始区域或结构基因。 Translation initiation region may be derived from the transcriptional initiation region or structural gene.

[0025] 为了便于对转基因植物细胞或植物进行鉴定及筛选,可对所用植物表达载体进行加工,如加入可在植物中表达可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除莠剂基因)等。 Gene [0025] To facilitate a transgenic plant cell or plant identification and screening, may be of the plants for processing an expression vector, such as the addition can be expressed in plants may produce a color change of the enzyme or luminescent compound (GUS gene, luciferase gene, etc.), antibiotic resistant markers (markers gentamycin, kanamycin marker, and the like) or chemical agents marker gene (e.g., herbicide resistance gene) and the like.

[0026] 本发明的另一个目的是提供一种培育抗旱能力提高和/或锌含量提高的植物的方法。 [0026] Another object of the present invention is to provide an improved cultivation drought tolerance and / or increased content of zinc plant.

[0027] 本发明所提供的培育抗旱能力提高和/或锌含量提高的植物的方法,是将所述的OsMTla基因导入植物中,得到抗旱能力提高和/或锌含量提高的转基因植物。 [0027] The present invention provides improved drought resistance of a plant and / or increase the zinc content of the cultivation, the gene is introduced into a plant OsMTla obtain improved drought tolerance and / or increase the zinc content of the transgenic plants.

[0028] 所述植物可以为各种单子叶或双子叶植物,如棉花、小麦或者水稻等。 [0028] The plant may be a variety of monocotyledonous or dicotyledonous plants, such as cotton, wheat or rice.

[0029] 本发明通过SSH(抑制消减杂交)的方法从盐胁迫的巴西旱稻中克隆得到一个I 型的金属硫蛋白OsMTla。 [0029] The present invention SSH (suppression subtractive hybridization) method of a cloned type I metallothionein OsMTla from Brazil salt stress in upland rice. 转OsMTla基因酵母和水稻中的微量元素锌的累积提高,并且转OsMTla基因水稻的抗氧化及耐逆性能提高。 Gene transfer in yeast and rice OsMTla cumulative increase in zinc, and gene transfer OsMTla rice oxidation resistance and inverse improved. 本发明的金属硫蛋白OsMTla可用来培育抗旱能力提高和/或锌含量提高的植物,对农业生产具有重要的意义。 Metallothionein OsMTla the present invention may be used to improve the ability of developing drought and / or increase the zinc content of the plant has important implications for agricultural production.

附图说明 BRIEF DESCRIPTION

[0030] 图1为Northern杂交分析日本晴中OsMTla的表达。 [0030] FIG. 1 is a Northern blot analysis of expression of Nipponbare OsMTla.

[0031] 图2为金属处理的巴西旱稻中OsMTla的表达。 Expression [0031] FIG. 2 is a metallized OsMTla in the Brazilian Upland.

[0032] 图3为非生物胁迫处理的巴西旱稻中OsMTla的表达。 Expression [0032] FIG. 3 as abiotic stress treatment in OsMTla of Brazilian Upland.

[0033] 图4为300mM甘露醇处理的野生型日本晴(WT)和转OsMTla基因水稻表型变化及失水率测定。 [0033] FIG. 4 is a 300mM Mannitol treated wild type Nipponbare (WT) and transgenic rice transfected OsMTla phenotypic changes and loss rate measurement.

[0034] 图5为CAT、APX和POD活性测定结果。 [0034] FIG. 5 is a CAT, APX and POD activity measurement result.

[0035] 图6为转OsMTla基因株系中Ossiz的表达。 [0035] FIG. 6 is a transfected gene expression OsMTla strains of Ossiz.

具体实施方式 Detailed ways

[0036] 下述实施例中如无特殊说明所用方法均为常规方法,所用试剂均可从商业途径获得。 [0036] Examples Unless otherwise noted methods used are conventional methods the following examples are available from commercial sources the reagent. 下述实施例中如无特殊说明所述百分含量均为质量百分含量。 The following Examples Unless otherwise specified the percentages are percentages by mass.

[0037] 实施例1、金属硫蛋白(OsMTla)基因的应用 [0037] Application Example 1, metallothionein (OsMTla) Gene

[0038] DOsMTla基因的克隆 Cloning [0038] DOsMTla Gene

[0039] 以盐处理和未处理的巴西旱稻为材料,利用消减抑制杂交的方法,直接从旱稻中分离了94个盐诱导基因。 [0039] In the treated and untreated salt is Brazilian Upland material, a method of inhibiting the use of subtractive hybridization, isolated directly from the 94 Upland in salt-induced genes. 其中一个基因的cDNA全长222bp,编码74个氨基酸,在N端和C端分别有六个半胱氨酸位于保守的位置,还有大约40氨基酸的spacer区,其中包含芳香族氨基酸酪氨酸和苯丙氨酸,这是I型植物MT蛋白的典型特征,将其命名为OsMTla。 Wherein a full-length cDNA of 222bp gene, encoding the 74 amino acids at the N-terminal and C-terminal positioned cysteine ​​respectively six conserved positions, there is a spacer region of about 40 amino acids, which comprises an aromatic amino acid tyrosine and phenylalanine, which is typical of type I MT vegetable protein, named as OsMTla. OsMTla蛋白与其他单子叶植物I型MT蛋白都有较高的相似性,比如玉米(73. 7% ),大麦(67. 6% )。 OsMTla protein and other monocots Type I MT protein has high similarity, such as corn (73.7%), barley (67.6%).

[0040] 分别取日本晴植株根、叶、花以及幼苗部分,按文献(Chomczynski,P. andSacchi, N. (1987)Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 162,156-159.)方法提取RNA,以OsMTla全长cDNA为探针进行Northern杂交分析。 [0040] were collected from Nipponbare plant roots, leaves, flowers and seedlings portion, according to the literature (Chomczynski, P. AndSacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 162, 156-159.) method for extracting RNA, to OsMTla full length cDNA as a probe for Northern blot analysis.

[0041] Northern杂交结果如图1所示,表明OsMTla在水稻的根、花、叶、幼苗均有表达,其中在根中表达量最高。 [0041] Northern hybridization results shown in Figure 1, show OsMTla expressed in roots, flowers, leaves, seedlings of rice, wherein the highest expression in roots.

[0042] 图1中,“1”代表花、“2”代表叶、“3”代表幼苗、“4”代表根。 In [0042] FIG. 1, "1" flower, "2" leaves, "3" represents the seedlings, "4" for the root.

[0043] 巴西旱稻种子在37°C浸种两天,露白后铺在放有两层纱布的平皿上培养,培养条件为28°C,持续光照强度为25001UX,光周期为16/8h。 [0043] Brazilian Upland Soaking the seeds in 37 ° C for two days, after grin spread on gauze placed two culture plates, culture conditions were 28 ° C, continuous light intensity 25001UX, photoperiod of 16 / 8h. 培养两周后取幼苗进行处理。 After two weeks in culture seedling taking process.

[0044] 金属处理:分别用10 μ M ZnCl2, CuCl2^MnCl2和FeCl3溶液处理24小时。 [0044] Metal Processing: respectively 10 μ M ZnCl2, CuCl2 ^ MnCl2 and FeCl3 solution for 24 hours.

[0045] 非生物胁迫处理:分别用150mM NaCl,7% PEG6000和10 μ M ABA溶液处理24小时。 [0045] The abiotic stress treatment: respectively 150mM NaCl, 7% PEG6000 solution and 10 μ M ABA for 24 h.

[0046] 检测金属处理1、6、12和24小时和非生物胁迫处理0、0. 5、6和24小时OsMTla的表达。 [0046] Metal detector 6, 12 and 24 h treatment and abiotic stress treatment Expression 0,0. 5,6, and 24 hours of OsMTla.

[0047] 提取各个处理不同时间的巴西旱稻的总RNA,用Promega公司的MMLV逆转录酶反转录后利用实时定量荧光PCR技术,根据厂家(Bio-Rad)提供的使用方法,在PCR体系中加入SYBR green I荧光染料,在荧光定量PCR仪(Bio-Rad)上检测OsMTla的表达模式。 [0047] Total RNA Brazilian Upland of respective different time, by the company Promega using MMLV reverse transcriptase PCR, real-time quantitative fluorescence after reverse transcription, according to the manufacturer (Bio-Rad) using the method provided in the PCR system fluorescent dye SYBR green I was added to detect the expression pattern OsMTla in quantitative PCR instrument (Bio-Rad). 用水稻ACTim基因表达水平做内参。 To do with the internal reference gene expression levels in rice ACTim. 扩增OsMTla的引物分别为:上游引物5,-GAAGATGTCTTGCAGCTGTGGAT-3,,下游引物5,-AGATGGTAGATGCAGGCAGGC-3,。 OsMTla amplification primers were: Forward primer 5, -GAAGATGTCTTGCAGCTGTGGAT-3 ,, downstream primer 5, -AGATGGTAGATGCAGGCAGGC-3 ,.

[0048] 同时,以OsMTla全长cDNA为探针进行Northern杂交分析。 [0048] Meanwhile, OsMTla full length cDNA as a probe for Northern blot analysis.

[0049] 金属处理的实时定量荧光PCR结果如图2所示,表明10μ M的Zn能诱导OsMTla 的表达上调,同样浓度的Mn、Fe、Cu不能诱导OsMTla的表达上调。 Real-time quantitative fluorescence PCR results [0049] The metal-treated as shown in FIG 2, show that the Zn 10μ M induces upregulation OsMTla, the same concentrations of Mn, Fe, Cu OsMTla not induce upregulation.

[0050] 图2 中,A 为10 μΜ Mn2+、Fe3+、Cu2+ 处理O 〜24 小时OsMTla 表达变化;B 为10 μ M Zn2+处理0〜24小时OsMTla表达变化。 In [0050] FIG. 2, A is 10 μΜ Mn2 +, Fe3 +, Cu2 + treatment O ~24 hours OsMTla expression; B is 10 μ M Zn2 + treatment 0~24 hours OsMTla expression.

[0051] 非生物胁迫处理的实时定量荧光PCR结果如图3所示,OsMTla受到渗透胁迫、盐胁迫以及ABA的诱导表达,其中渗透胁迫的诱导最为明显。 [0051] Real-time quantitative fluorescent PCR abiotic stress treatment results shown in Figure 3, OsMTla expression is induced by osmotic stress, salt stress and ABA, wherein osmotic stress induced by the most obvious.

[0052] 设计正反向引物,利用PCR方法从水稻基因组DNA中扩增出OsMTla基因,并在正反向引物末端分别添加限制性酶EcoRI和BamHI的酶切位点。 [0052] The forward and reverse primers were designed, was amplified by PCR from the rice gene OsMTla genomic DNA, and positive and negative adding restriction enzymes EcoRI and BamHI restriction sites, respectively, to the terminal primers.

[0053]正向引物为 5,-GAATTCGAAGATGTCTTGCAGCTGTG-3 ; [0053] The forward primer was 5, -GAATTCGAAGATGTCTTGCAGCTGTG-3;

[0054]反向引物为 5,-GGATCCGCAGGCAGGCATCTTA-3,。 [0054] reverse primer 5, -GGATCCGCAGGCAGGCATCTTA-3 ,.

[0055] PCR产物回收后连接入pMD18-T(TAKARA,大连)中进行测序,测序结果表明,PCR产物的核苷酸序列如序列表中序列1所示,编码序列表中序列2所示的蛋白。 [0055] The PCR product was ligated into the sequencing of pMD18-T (TAKARA, Dalian), sequencing results showed that the nucleotide sequence of the PCR product sequence table as shown in FIG. 1, the coding sequence shown in Sequence Listing 2 protein.

[0056] 2)培育抗旱和/或锌含量提高的水稻 [0056] 2) developing drought and / or increase the zinc content in rice

[0057] 用EcoRI与BamHI双酶切PCR产物,与同样双酶切的双元表达载体pCAMBIA2300-pUbiquitin-0CS (购自Cambia,澳大利亚,http://www. cambia. org/daisy/ cambia/home. html)连接,得重组表达载体pCAMBIA2300-pUbiquitin-0sMTla_0CS。 [0057] with EcoRI and BamHI double digested PCR product was double digested with the same binary expression vector pCAMBIA2300-pUbiquitin-0CS (available from Cambia, Australia, http:.. // www cambia org / daisy / cambia / home . html) is connected, a recombinant expression vector pCAMBIA2300-pUbiquitin-0sMTla_0CS.

[0058]将载体 pCAMBIA2300-pUbiquitin-0sMTla_0CS 通过农杆菌AGLl (购自ATCC(American Type Culture Collection))导入到水稻日本晴愈伤组织中。 [0058] The vector pCAMBIA2300-pUbiquitin-0sMTla_0CS by Agrobacterium AGLl (purchased from ATCC (American Type Culture Collection)) introduced into the calli of rice Nipponbare. 转化筛选方法参考如下文献:易自力,曹守云,王力,何锶洁,储成才,唐祚舜,周朴华,田文忠(2001)提高农杆菌转化水稻频率的研究,遗传学报,28 (4) :352-358)。 Transformation screening methods refer to the following document: Yi independent, Agrobacterium transformation of rice frequency Caoshou Yun, Wang, He strontium Jie, Chu taught, Tang Zuo Shun, Zhou Pu China, TIAN Wen-Zhong (2001) increase, Genetics, 28 (4): 352-358) . 获得抗性筛选阳性转OsMTla基因水稻Ttl代株系共10株。 Screening of positive transformants acquired resistance gene OsMTla Ttl rice mutant lines were 10.

[0059] 分别取营养生长同一时期的野生型日本晴(WT) 6株和转OsMTla基因植株(L6和L7各6株)叶片及收割后去掉谷壳的种子进行金属含量测定,实验重复3次,对测定结果进行统计学分析。 [0059] were collected from vegetative growth of wild type Nipponbare (the WT) 6 and transfected OsMTla transgenic plants (L6 and L7 each 6) during the same period of leaves and removing chaff after harvest seed metal content measurement, the experiment was repeated three times, the measurement results were analyzed statistically.

[0060] 测定方法如下: [0060] The measuring method is as follows:

[0061] 材料洗净后,经过烘箱80°C彻底干燥24小时后,磨碎,进行微波消解。 [0061] After washing the material, through an oven at 80 ° C after thorough drying for 24 hours, milling, microwave digestion. 每200mg加9ml HNO3>2ml H2O2在180°C消解20min,冷却后开盖,转移至25mL容量瓶中加超纯水定容, 摇勾。 Each 200mg plus 9ml HNO3> 2ml H2O2 digestion at 180 ° C 20min, cooled after opening the cover, transferred to a 25mL flask was added ultrapure water volume, shake hook. 禾U用Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES)电感耦合等离子发生光谱(0ptima-2000DV,Perkin Elemer, USA)进行相关重金属含量的测定。 Wo U was measured spectrum related to the occurrence of heavy metal content (0ptima-2000DV, Perkin Elemer, USA) by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) Inductively Coupled Plasma. 叶片及种子中金属含量的测定结果如表1所示。 The measurement results in leaf and seed metal content as shown in Table 1.

[0062] 表1.叶片及种子中金属的含量 [0062] Table 1. The content of metal leaves and seeds

[0063]<table>table see original document page 7</column></row> <table>[0064] 表1中金属含量的单位为(yg/g干重),**代表通过t检验得出转基因株系和野生型有显著性差异的置信概率为99%。 [0063] <table> table see original document page 7 </ column> </ row> <table> [0064] Table 1 Unit of metal content (yg / g dry weight), obtained by t-test ** indicates wild-type and transgenic lines have confidence probability significant difference was 99%.

[0065] 表1表明,两个转OsMTla基因株系L6和L7中,Mn和Zn的积累都有所提高,而Zn 的提高是显著性的,比野生型分别提高了45. 38%和54. 15%。 [0065] Table 1 shows two lines transfected genes OsMTla L6 and L7 are, Mn and Zn has increased accumulation, and Zn improve the significant nature, than the wild-type increased by 45.38% and 54 15%. 在种子中也有相同的趋势, Zn的积累最为明显,L6和L7分别比野生型提高44. 43%和53. 93%。 In seeds also have the same tendency, Zn most obvious accumulation, L6 and L7 increased by 44.43% and 53.93% than the wild type. 因此,OsMTla基因可用于提高作物中的微量元素锌的累积。 Thus, OsMTla gene can be used to improve the accumulation of trace elements zinc crops.

[0066] 将生长两周的野生型日本晴(WT) 10株和转OsMTla基因(L6和L7各10株)水稻幼苗转移至含有300mM甘露醇的MS培养基中培养10天,观察对比表型变化,实验重复3次。 [0066] The growth of wild type Nipponbare two weeks (the WT) and 10 OsMTla gene transfer (L6 and L7 each 10) Rice seedlings were transferred to MS medium containing 300mM mannitol for 10 days, were observed phenotypic comparison ,The experiment was repeated three times.

[0067] 表型变化结果如图4A所示,对水稻幼苗进行300mM甘露醇处理两周后,可以看到野生型叶片极度卷曲、发黄、萎缩,几近枯死;而转OsMTla基因(L6和L7)表现较好的生长状态,叶片微卷、叶尖发黄,主体仍颜色鲜绿。 [0067] The results shown in FIG phenotypic change, the rice seedlings were 300mM mannitol weeks of treatment, the wild-type 4A can be seen is extremely curly leaves, yellow, atrophy, almost dead; OsMTla the transfected gene (L6 and L7) showed better growth state, slightly curled leaves, yellow tip, the body is still bright green color.

[0068] 取在温室中于大小体积相等的花盆中生长两个月的野生型日本晴(WT)5株和转OsMTla基因(L6和L7各5株)约IcM长样段迅速称取0. 5g置于室内桌面,室温下每隔5-10分钟称重一次,直至一个小时。 [0068] taken up in a greenhouse in the same size volume pots grown wild type Nipponbare months (the WT) and 5 OsMTla gene transfer (L6 and L7 each 5) long sample section of about IcM 0 quickly weighed. 5g placed indoors table, weighted once every 5-10 minutes at room temperature, up to one hour. 利用以下公式计算失水率,实验重复3次: Water loss rate is calculated using the following equation, the experiment was repeated three times:

[0069] [0069]

<formula>formula see original document page 7</formula> <Formula> formula see original document page 7 </ formula>

[0070] 失水率结果如图4B所示,转OsMTla基因(L6和L7)的叶片一小时后失水率滞后于野生型3.75%。 [0070] The results shown in Figure 4B rate of water loss, leaf OsMTla gene transfer (L6 and L7) one hour after the hysteresis loss rate of 3.75% in the wild-type.

[0071] 上述实验结果表明OsMTla过表达株系比野生型具有更好的抗旱性。 [0071] The above results show that overexpression lines OsMTla drought resistance better than the wild type.

[0072] 用EcoRI与BamHI双酶切PCR产物,与同样双酶切的ρ 18IAINE载体(ρ 18IAINE载体构建方法参照文献Vernet,T.,Dignard,D. and Thomas,DV (1987),A family of yeast expression vectors containing the phage f1 intergenic region, Gene52,225-233 ; Riesmeier JW, Willmitzer L, Frommer WB (1992), Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast. EMBO J 11:4705-4713 ;Daram P, Brunner S, Persson BL, Amrhein N, Bucher Μ. (1998), Functional analysis and cell-specific expression of a phosphate transporter from tomato. Planta 206 :225_233.)(中国科学院遗传与发育生物学研究所)连接, 获得重组载体P181AINE: :OsMTla,将pl81AINE: :OsMTla和pl81AINE分别转化酵母BY4741 (MATA his3 leu2 metl5ura3)(购自Euroscarf,目录号Y00000)。 [0072] with EcoRI and BamHI double digested PCR product, with the same double-digested vector ρ 18IAINE construct (ρ 18IAINE vector according to the literature method Vernet, T., Dignard, D. And Thomas, DV (1987), A family of yeast expression vectors containing the phage f1 intergenic region, Gene52,225-233; Riesmeier JW, Willmitzer L, Frommer WB (1992), Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast EMBO J 11: 4705-. . 4713; Daram P, Brunner S, Persson BL, Amrhein N, Bucher Μ (1998), Functional analysis and cell-specific expression of a phosphate transporter from tomato 206 Planta: 225_233) (CAS genetic and developmental biology studies. the) is connected, to obtain a recombinant vector P181AINE:: OsMTla, the pl81AINE:: OsMTla were transformed into yeast and pl81AINE BY4741 (MATA his3 leu2 metl5ura3) (available from Euroscarf, Cat. No. Y00000). 在SD/Leu(_)缺陷固体培养基上于30°C生长2-3天,长出的克隆为带plSlAINE-leu标签的阳性克隆。 Clones on solid medium Growth at 30 ° C for 2-3 days, to grow with a tag plSlAINE-leu positive clones SD / Leu (_) defects. 再以5,-GAAGATGTCTTGCAGCTGTGGAT-3,和5,-AGATGGTAGATGCAGGCAGGC-3,为引物进行PCR 扩增,扩增出253bp的片段鉴定转P181AINE: :OsMTla阳性克隆。 Then to 5, -GAAGATGTCTTGCAGCTGTGGAT-3, and 5, -AGATGGTAGATGCAGGCAGGC-3, PCR amplification as primers, 253bp amplified fragment identified turn P181AINE:: OsMTla positive clones.

[0073] 每升SD/leu (-)缺陷液体培养基:YNB酵母氮源(无氨基酸、硫酸铵)1. 7g、硫酸铵5g、营养缺陷混合物(Drop-out Mix) 1. 4g、葡萄糖20g、L-Trp, L-His 和Uracil 各20mg,加水至IL灭菌。 . [0073] liter of SD / leu (-) Defects liquid medium: YNB yeast nitrogen base (without amino acids, ammonium sulfate) 1 7g, ammonium sulfate 5g, auxotrophic mixture (Drop-out Mix) 1. 4g, 20g glucose , L-Trp, L-His and Uracil each 20mg, sterilized water was added to the IL. 每升SD/leu(-)缺陷液体培养基中加入20g琼脂粉得到SD/leu(-)缺陷固体培养基。 Per liter of SD / leu (-) defects in liquid medium was added to give 20g of agar powder SD / leu (-) Defects solid medium. 其中YNB、Drop-out Miχ、L-Trp、L-His、Uracil均购自sigma公司,货号分别为Y1251、Y2001、T0254、H8000、U0750。 Wherein YNB, Drop-out Miχ, L-Trp, L-His, Uracil were purchased from sigma Company, Catalog respectively Y1251, Y2001, T0254, H8000, U0750.

[0074] 将pl81AINE: : OsMTla阳性克隆与空载体pl81AINE阳性克隆于SD/Leu_缺陷液体培养基中30°C培养到指数生长期,然后加入ImM ZnCl2溶液,经过10小时处理后收菌、干燥后进行金属含量测定,每个样品取三次独立重复实验的测定结果并进行统计学分析。 [0074] The pl81AINE:: OsMTla positive clones and empty vector in the positive clone pl81AINE SD / Leu_ defects in the liquid culture medium 30 ° C to exponential growth phase, followed by addition of ImM ZnCl2 solution, after 10 hours of treatment received bacteria, dried after the metal content is measured, the measurement result of each sample is repeated three independent experiments and statistical analysis. 金属含量的测定结果如表2所示。 Metal content measurement results shown in Table 2.

[0075] 表2.酵母中金属的含量 [0075] Table 2. The metal content of yeast

[0076]<table>table see original document page 8</column></row> <table> [0076] <table> table see original document page 8 </ column> </ row> <table>

[0077] 表2中星号**代表通过t检验得出转基因酵母和对照有显著性差异的置信概率为99%,表中金属含量的单位为(yg/g干重)。 [0077] Table 2. Representative asterisks ** t test results and control transgenic yeast with a confidence level of significant difference was 99% in terms of units of metal content (yg / g dry weight).

[0078] 表2的结果表明,过量表达OsMTla的酵母细胞在非Zn处理的普通状态下,能够比对照多吸收19. 2%的Zn ;而在ImM Zn处理条件下,能比对照多积累1. 4倍的Zn,显著地提高对Zn的吸收。 [0078] The results in Table 2 show that overexpression OsMTla yeast cells in the normal state of the non-treated Zn can absorb 19.2% of Zn than the control; ImM Zn and in process conditions can accumulate more than the control 1 4 times Zn, significantly improves the absorption of Zn. OsMTla蛋白可能参与了水稻体内Zn离子的代谢过程。 OsMTla protein may be involved in metabolic processes Zn ions rice plant.

[0079] 3)转OsMTla基因植株抗氧化酶活测定 [0079] 3) transgenic plants transfected OsMTla antioxidant activity assay

[0080] 生长两周的野生型日本晴(WT)IO株和转OsMTla基因(L6和L7各10株)水稻幼苗用ImM双氧水处理5小时,分别取0. 5g叶片,加入预冷的酶提取液(IOOmMTris HCl (ρΗ7· 0),20%甘油,PVP),研磨至勻浆;4°C, IOOOOg离心30分钟;上清为粗酶提取液。 [0080] Growth weeks wild type enzyme Nipponbare (WT) IO lines and gene transfer OsMTla (L6 and L7 each of 10) for 5 hours with rice seedlings ImM hydrogen peroxide, 0. 5g leaves were taken, prechilled extract (IOOmMTris HCl (ρΗ7 · 0), 20% glycerol, PVP), ground to a homogenate; 4 ° C, IOOOOg centrifuged for 30 min; the supernatant as a crude enzyme extract.

[0081] 测定APX时,在上述提取液中加ImM AsA。 [0081] measurement APX, the above plus ImM AsA extract.

[0082] CAT,APX 和POD 活性参照Rahnama 和Ebrahimzadeh (2005)的方法测定(Rahnama H and Ebrahimzadeh H(2005),The effect of NaCl on antioxidant enzyme activities in potato seedlings,Biol Plant 49,pp. 93—97.)。 [0082] CAT, APX and POD and reference Rahnama Ebrahimzadeh (2005) determined by the method (Rahnama H and Ebrahimzadeh H (2005), The effect of NaCl on antioxidant enzyme activities in potato seedlings, Biol Plant 49, pp. 93-97 .). CAT 活性用Δ A240mg-lproteinmin_l 表示;APX 活性用AA265mg-lproteinmin-l 表示;POD 活性用Δ A53ciIiig-1Proteinmirr1 表示。 CAT activity was expressed by Δ A240mg-lproteinmin_l; APX activity represented by AA265mg-lproteinmin-l; POD activity was expressed by Δ A53ciIiig-1Proteinmirr1. 不同波长下的吸光度变化用美卡西斯0PTIZEN-2120UV分光光度计连续测定。 Change in absorbance was measured continuously at different wavelengths using methylene Cassis 0PTIZEN-2120UV spectrophotometer. 蛋白质含量测定参照(Bradford,Μ. Μ. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254.)的方法。 Referring methods for protein determination (Bradford, Μ. Μ. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254.) Of. 每个样品取三次独立重复实验的测定结果,并进行统计学分析。 Each sample was repeated three independent measurement results of experiments, and statistical analysis.

[0083] ImM双氧水处理5小时前后水稻叶片内抗氧化物酶CAT、P0D和APX的酶活变化如图5所示,说明转OsMTla基因植株的CAT,POD, APX酶活相比野生型都有不同程度的提高; L7的CAT酶活比WT提高81 %,POD酶活比WT提高32 %,APX酶活比WT提高17 %。 [0083] ImM hydrogen peroxide treated rice leaves the antioxidant enzyme CAT, P0D and APX enzymatic change before and after 5 hours Figure 5, the transgenic plants described transfer OsMTla CAT, POD, APX compared with the wild-type enzyme has different degree; L7 of CAT activity higher than WT 81%, POD activity by 32% compared to WT, APX activity ratio of 17% WT.

[0084] 上述实验结果表明,过表达OsMTla能提高体内抗氧化系统的效率。 [0084] The above results show that overexpression OsMTla vivo antioxidant can improve the efficiency of the system.

[0085] 图5中星号*和**代表通过t检验得出转OsMTla基因株系与野生型之间存在显著性差异的置信概率分别为95%和99%。 [0085] FIG. 5 asterisks * and ** indicates the derived lines transfected with the gene OsMTla confidence probability of the significant difference between wild type and 95%, respectively 99%, by t-test.

[0086] 4) OsMTla提高植物抗逆性的机理 [0086] 4) OsMTla mechanism of increasing the resistance of plants

[0087] 为了获得准确的耐逆性机理信息,选取了报道已知受到干旱胁迫诱导的三个锌指转录因子ZFl (Accession No. AF332876)、WRKY71 (Accession No. NM_001052629) (Takatsuji, H. (1999). Zinc-finger proteins :the classical zinc finger emerges in contemporary plant science. Plant Mol. Biol. 39,1073-1078 ;Segal, DJ , Stege, J. Τ. , and Barbas, CF , III (2003). Zinc fingers and a green thumb !manipulating gene expression in plants. Curr. Opin. Plant Biol.6,163-168)禾口Ossiz (Accession No. AK108249) (ffu, YR,Wang, QY,Ma, YM,and Chu, CC (2005). Isolation and expression analysis of salt up-regulated ESTs in upland rice using PCR-based subtractive suppression hybridization method. Plant Sci 168,847—853.),比较它7[门在野生型与转基因水稻中的表达量差别。 [0087] In order to obtain accurate information about the mechanism of stress tolerance, the selected report known to be induced by drought stress three zinc finger transcription factor ZFl (Accession No. AF332876), WRKY71 (Accession No. NM_001052629) (Takatsuji, H. ( .... 1999) Zinc-finger proteins:. the classical zinc finger emerges in contemporary plant science Plant Mol Biol 39,1073-1078; Segal, DJ, Stege, J. Τ, and Barbas, CF, III (2003). Zinc fingers and a green thumb! manipulating gene expression in plants. Curr. Opin. Plant Biol.6,163-168) Wo mouth Ossiz (Accession No. AK108249) (ffu, YR, Wang, QY, Ma, YM, and Chu, CC (2005). Isolation and expression analysis of salt up-regulated ESTs in upland rice using PCR-based subtractive suppression hybridization method. Plant Sci 168,847-853.), comparator 7 which [the difference in the amount of door expression of wild-type and in transgenic rice .

[0088] 提取生长两周的野生型日本晴(WT)和转OsMTla基因(L6和L7)水稻幼苗的总RNA,反转录后进行real-time PCR分析。 [0088] Total RNA was extracted two weeks growth Nipponbare wild type (WT) and transfected genes OsMTla (L6 and L7) of the rice seedlings, real-time PCR analysis for reverse transcription.

[0089] 引物分别为: [0089] The primers were:

[0090] ZFl :上游引物5,-TTGTGAATTGCGGTGGAAGC-3 '; [0090] ZFl: upstream primer 5, -TTGTGAATTGCGGTGGAAGC-3 ';

[0091]下游引物 5,-GGCTTCTTGAAGGCGAGGG-3,。 [0091] The downstream primer 5, -GGCTTCTTGAAGGCGAGGG-3 ,.

[0092] WRKY71 :上游引物5,-CGAGGAGTGCAAGCCCAAGAT-3,;[0093]下游引物 5,-AATCCTTGGTCGGCGAGAGCT-3,。 [0092] WRKY71: upstream primer 5, -CGAGGAGTGCAAGCCCAAGAT-3,; [0093] The downstream primer 5, -AATCCTTGGTCGGCGAGAGCT-3 ,.

[0094] Ossiz :上游引物5,-GCACCATGGCGAGCCGAGAG-3,; [0094] Ossiz: upstream primer 5, -GCACCATGGCGAGCCGAGAG-3 ,;

[0095]下游引物 5,-AGGATCCCGGGTGCTTCTACATCACAAGC-3,。 [0095] The downstream primer 5, -AGGATCCCGGGTGCTTCTACATCACAAGC-3 ,.

[0096] 转OsMTla基因株系中三个锌指转录因子0ssiz、ZFl、WRKY71的相对表达量如图6A所示,在OsMTla过表达植株中几个锌指转录因子基因表达上调;CCCH家族的Ossiz,C2H2 家族的ZF1,WRKY家族的WRKY71分别上调了约10倍、5倍和3倍。 [0096] Gene transfer lines OsMTla three zinc finger transcription factor 0ssiz, ZFl, the relative expression of WRKY71 6A, in the plant several zinc finger transcription factor gene expression regulated overexpression OsMTla; Ossiz CCCH family , the C2H2 family ZF1, WRKY family WRKY71 were raised by about 10 times, 5 times and 3 times.

[0097] 选择Ossiz上调最明显的OsMTla过表达植株进行Northern杂交。 [0097] The choice of the most upregulated Ossiz OsMTla overexpressing plants by Northern blot. 以OsMTla和Ossiz全长cDNA为双探针进行Northern杂交分析。 In OsMTla Ossiz full-length cDNA and a double probe Northern blot analysis.

[0098] 图6B所示是Northern检测转OsMTla基因株系中Ossiz的表达结果。 [0098] Figure 6B is a Northern expression results in the detection of genetically OsMTla Ossiz the transgenic lines. 在过表达OsMTla的植株中检测到了Ossiz的积累。 Plants overexpressing OsMTla detected in the accumulation of Ossiz. 这说明Ossiz位于OsMTla的下游。 This shows Ossiz located downstream of OsMTla. 而且Ossiz 同OsMTla —样,也能受到Zn的诱导。 Further Ossiz same OsMTla - like, can be induced by the Zn.

[0099] 图6C所示是10 μ M Zn2+处理0〜24小时Ossiz表达变化。 [0099] Figure 6C is a 10 μ M Zn2 + treatment 0~24 hours Ossiz expression.

[0100] 上述结果表明,金属硫蛋白OsMTla —方面可能通过感应Zn浓度变化来响应外界胁迫,通过提高体内抗氧化系统的效率来清除自由基,并提高植物的抗旱功能。 [0100] The above results show, metallothionein OsMTla - aspects is possible to respond to external stress by sensing changes in concentration of Zn to remove free radicals by antioxidation improving efficiency of the system, and to improve the function of plant drought resistance. 此外在对外界胁迫的应答过程中,OsMTla能够向一些锌指的转录因子,比如Ossiz直接或间接地提供Zn离子,协同作用来传导抗逆信号,调节下游基因表达,最终提高植物抗逆性。 In addition, in response to environmental stresses in the process, OsMTla is possible to a number of zinc finger transcription factors, such as directly or indirectly Ossiz Zn ions, synergy resilience to conduct signals downstream regulation of gene expression, and ultimately improve plant resistance.

[0101] 序列表 [0101] SEQUENCE LISTING

[0102] <110>中国科学院遗传与发育生物学研究所 [0102] Institute of Genetics and Developmental Biology <110>

[0103] <120>金属硫蛋白及其编码基因与应用 [0103] <120> metallothionein its coding gene and application

[0104] <130>CGGNARW92095 [0104] <130> CGGNARW92095

[0105] <160>2 [0105] <160> 2

[0106] <210>1 [0106] <210> 1

[0107] <211>222 [0107] <211> 222

[0108] <212>DNA [0108] <212> DNA

[0109] <213> 稻属陆稻(Oryza glaberrima) [0109] <213> Oryza upland rice (Oryza glaberrima)

[0110] <400>1 [0110] <400> 1

[0111] atgtcttgca gctgtggatc tagctgcagc tgcggctcaa actgctcctg cggaaagaag 60 [0111] atgtcttgca gctgtggatc tagctgcagc tgcggctcaa actgctcctg cggaaagaag 60

[0112] taccctgacc tggaagagaa gagcagcagc accaaggcca ccgtcgtgct gggtgtggcg 120 [0112] taccctgacc tggaagagaa gagcagcagc accaaggcca ccgtcgtgct gggtgtggcg 120

[0113] ccggagaaga aggcgcagca gtttgaggcg gccgcagagt ccggcgagac cgcccatggc 180 [0113] ccggagaaga aggcgcagca gtttgaggcg gccgcagagt ccggcgagac cgcccatggc 180

[0114] tgcagctgcg gttccagctg caggtgcaac ccttgcaact gt 222 [0114] tgcagctgcg gttccagctg caggtgcaac ccttgcaact gt 222

[0115] <210>2 [0115] <210> 2

[0116] <211>74 [0116] <211> 74

[0117] <212>PRT [0117] <212> PRT

[0118] <213> 稻属陆稻(Oryza glaberrima) [0118] <213> Oryza upland rice (Oryza glaberrima)

[0119] <400>2 [0119] <400> 2

[0120] Met Ser Cys Ser Cys Gly Ser Ser Cys Ser Cys Gly Ser Asn Cys Ser [0120] Met Ser Cys Ser Cys Gly Ser Ser Cys Ser Cys Gly Ser Asn Cys Ser

[0121] 15 10 15 [0121] 151015

[0122] Cys Gly Lys Lys Tyr Pro Asp Leu Glu Glu Lys Ser Ser Ser Thr Lys [0122] Cys Gly Lys Lys Tyr Pro Asp Leu Glu Glu Lys Ser Ser Ser Thr Lys

[0123] 20 25 30[0124] Ala Thr Val Val Leu Gly Val Ala Pro Glu Lys Lys Ala Gln Gln Phe [0123] 20 25 30 [0124] Ala Thr Val Val Leu Gly Val Ala Pro Glu Lys Lys Ala Gln Gln Phe

[0125] 35 40 45 [0125] 35 40 45

[0126] Glu Ala Ala Ala Glu Ser Gly Glu Thr Ala His Gly Cys Ser Cys Gly [0126] Glu Ala Ala Ala Glu Ser Gly Glu Thr Ala His Gly Cys Ser Cys Gly

[0127] 50 55 60 [0128] Ser Ser Cys Arg Cys Asn Pro Cys Asn Cys [0127] 50 55 60 [0128] Ser Ser Cys Arg Cys Asn Pro Cys Asn Cys

[0129] 65 70 [0129] 65 70

Claims (8)

  1. 一种蛋白质,是如下a)或b)的蛋白:a)序列表中序列2所示的氨基酸序列组成的蛋白质;b)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸由a)衍生的蛋白质。 A protein, is the following a) or b) protein: protein consisting of the amino acid sequences shown in a) of sequence table; b) the amino acid sequence 2 by substitution and / or deletion and / or in the Sequence Listing addition of one or several amino acids of a) derived protein.
  2. 2.权利要求1所述蛋白的编码基因。 The gene encoding a protein of claim 1.
  3. 3.根据权利要求2所述的基因,其特征在于:所述基因是如下1)或2)或3):1)其核苷酸序列是序列表中序列1 ;2)在严格条件下与1)限定的DNA片段杂交且编码金属硫蛋白的DNA分子;3)与1)或2)的基因具有90%以上的同源性,且编码金属硫蛋白的DNA分子。 The gene according to claim 2, wherein: the gene is as follows 1) or 2) or 3): 1) a nucleotide sequence which is the sequence table 1; 2) under stringent conditions with 1) defined DNA fragments and hybrid DNA molecule encoding metallothionein; 3) with 1) or 2) a gene having a homology of 90% or more, and a DNA molecule encoding metallothionein.
  4. 4.含有权利要求2或3所述基因的重组表达载体。 4. containing 23 or the recombinant expression vector as claimed in claim.
  5. 5.含有权利要求2或3所述基因的转基因细胞系或重组菌。 The gene contains 23 or the transgenic cell lines or recombinant bacterium as claimed in claim.
  6. 6. 一种培育抗旱能力提高和/或锌含量提高的植物的方法,是将权利要求2或3所述基因导入植物中,得到抗旱能力提高和/或锌含量提高的转基因植物。 A drought resistance of a plant to improve and / or increase the zinc content of the cultivation, or is the gene of claim 23 into a plant, to give improved drought resistance and / or increase the zinc content of the transgenic plants.
  7. 7.扩增权利要求2或3所述基因的全长及其任意片段的引物对。 7. A primer pair or the 23 full-length gene and amplification of any fragment of claim.
  8. 8.权利要求1所述蛋白、权利要求2或3所述基因、权利要求4所述重组表达载体、权利要求5所述转基因细胞系或重组菌在培育抗旱能力提高和/或锌含量提高的植物中的应用。 The protein of claim 1, claim 2 or claim 3 gene, the recombinant expression vector as claimed in claim 4, 5 or the transgenic cell lines as claimed in claim recombinant strain increase and / or zinc content increased drought resistance in cultivating application of plants.
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