CN100471954C - Cotton fiber specific promoter and its use - Google Patents

Cotton fiber specific promoter and its use Download PDF

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CN100471954C
CN100471954C CN 200610156823 CN200610156823A CN100471954C CN 100471954 C CN100471954 C CN 100471954C CN 200610156823 CN200610156823 CN 200610156823 CN 200610156823 A CN200610156823 A CN 200610156823A CN 100471954 C CN100471954 C CN 100471954C
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cotton
gene
promoter
fiber
plant
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CN 200610156823
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CN1995350A (en )
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磊 侯
灏 刘
李家宝
李德谋
霞 杨
明 罗
肖月华
炎 裴
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西南大学
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本发明涉及一种棉花纤维特异启动子,该启动子全长1037bp,-634至-543区域为核心区。 The present invention relates to a cotton fiber-specific promoter, the promoter the full length 1037bp, -634 to -543 region of the core area. 通过构建含有该启动子的植物表达载体,将其转化到转基因烟草和转基因棉花中,GUS组织化学染色、酶活性检测和GFP荧光纤维检测证实,该启动子在烟草中具有外种皮优势表达活性,在陆地棉中具有纤维表达特异性。 By constructing plant expression vector containing the promoter, was transformed into transgenic tobacco and transgenic cotton, the GUS histochemical staining, enzyme activity detection and GFP fluorescence fiber test confirmed, the promoter having testa advantage in tobacco expression activity , a fiber-specific expression in Gossypium hirsutum.

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棉花纤维特异启动子及其应用技术领域本发明属于植物基因工程领域,具体地说,涉及棉花纤维特异启动子,含有该启动子的植物表达载体,以及由该植物表达载体转化的转化体; 本发明还涉及含有上述启动子序列的转基因植物的制备方法。 Its cotton fiber specific promoter TECHNICAL FIELD The present invention belongs to the field of plant genetic engineering, particularly to cotton fiber-specific promoter, plant expression vector containing the promoter, and a transformant transformed with the expression vector from the plant; the present the invention also relates to a method of preparing a transgenic plant containing the promoter sequence. 背葦技水棉花纤维是重要的纺织工业原料,我国是棉纺织品消费和出口大国,因此, 棉花生产在我国国民经济中占有重要的地位,同时也是农民重要的经济来源。 Back reed water technology is an important cotton fiber textile industrial raw materials, China's cotton textile consumption and exporting countries, therefore, cotton production plays an important role in our national economy, but also an important source of income of farmers. 能否迅速提高我国棉花品种的纤维品质,直接关系到我国棉花产业的兴衰和纺织品生产加工、纺织设备制造、外贸出口等行业的生存与发展。 It can rapidly improve the fiber quality of cotton varieties in our country is directly related to the survival and development of the cotton industry and the rise and fall of China's textile production and processing, textile equipment manufacturing and export industries. 虽然利用传统的育种方法曾在棉花品种改良上取得了很大的成功,但近20年来,世界棉花品种在产量上已经达到一个平台期,利用现有的遗传资源和育种手段难以再大幅度提高棉花产量(Mered池WR Be""Oo/w 2000 84(4):6〜9)。 Although the use of traditional breeding methods have achieved great success in the cotton variety improvement, but over the past 20 years, world cotton varieties in production has reached a plateau, the use of existing genetic resources and breeding was difficult for them to greatly improve cotton yield (Mered pool WR Be "" Oo / w 2000 84 (4): 6~9). 单纯依靠常规育种改良棉花纤维品质相当困难。 Relying solely on conventional breeding improved cotton fiber quality is quite difficult. 这是因为:1)棉花纤维强力、细度等品质性状基因与皮棉产量存在负连锁,常规育种方法很难打破这种遗传上的负相关-,2)我国的棉花栽培品种主要是陆地棉,而优良纤维品质基因主要源于二倍体的瑟伯氏棉(纤维强度)、异常棉(纤维强度与细度)以及四倍体的海岛棉(纤维强度与细度)等,这些优良性状基因的利用在常规育种中受到诸多限帝ij。 This is because: 1) cotton fiber strength, fineness and quality trait lint yield a negative chain, conventional breeding methods are difficult to break the negative correlation of this genetic -, 2) cotton cultivars of mainly upland cotton, and mainly due to high quality of fiber gene diploids thurberi (fiber strength), abnormal cotton (fiber strength and fineness) and Gossypium barbadense (fiber strength and fineness) and the like, these fine trait gene tetraploid limited use by many emperor ij in conventional breeding. 瑟伯氏棉与异常棉为二倍体野生种,与四倍体陆地棉杂交后,存在远缘杂交带来的农艺性状差、后代难于稳定、周期太长等问题;即便是同为四倍体的海x陆杂交,同样存在后代分离强烈,产量-品质负连锁关系仍难打破等问题(潘家驹1998賴花,辨学99-104北京:中国农业出版社)。 Thurber's cotton and cotton abnormal diploid wild species, and the tetraploid upland cotton hybrid, the presence of distant hybridization caused by poor agronomic traits, and their offspring is difficult to stabilize, the cycle is too long and other issues; even with four times x land and sea of ​​hybrids, there are also future generations a strong separation, production - quality relationship is still difficult to break the chain of negative issues (1998 Panjia Ju Lai flower, identified learn 99-104 Beijing: China agriculture Press). 棉花纤维是由胚珠外珠被表层细胞经分化突起、伸长和细胞壁增厚而形成的,整个发育过程可分为:纤维原始细胞分化、纤维伸长(初生壁形成)、次生壁形成和纤维脱水成熟四个时期。 Cotton fibers are projecting from the outer integument of the ovule differentiated superficial cells, cell wall thickening and elongation is formed, the entire development process can be divided into: cell differentiation original fiber, fiber elongation (primary cell wall formation), and secondary wall formation fiber dehydration mature four periods. 棉花纤维的发育以单细胞形式出现,在其发育过程中,纤维细胞具有均一性和同步性,而且在表皮细胞突起后,纤维细 Cotton fiber development appears as single cells, in the course of its development, has a uniform fibroblast and synchronization, and projection in the epidermal cells, micronaire

胞迅速伸长,可以将细胞的分化与细胞的伸长过程完全分开。 Rapid cell elongation, elongation process can be completely separated from cells and cell differentiation. 随着纤维细胞生长发育的进行,细胞形态变化越来越大,最后其长/径比可达1,000-3,000。 As for the growth and development of fibroblasts, increasing cell morphology, finally the length / diameter ratio of up to 1,000 to 3,000. 成熟纤维主要由纤维素组成(约占87%),此外还含有少量的果胶、半纤维素和蛋白质等物质。 Mature fiber is mainly composed of cellulose (about 87%), also contains a small amount of pectin, hemicellulose and protein and other substances. 棉纤维的化学组成及其分子间的排列方式决定棉纤维的理化性质(品质)。 Arrangement between the chemical composition and molecular determinants of cotton fiber cotton fiber physicochemical properties (quality). 在棉纤维中引进新的生物大分子是改良棉纤维品质的重要策略之利用基因工程方法可以打破物种间的遗传障碍,实现优良目的基因的定向转移,同时具有后代易于稳定,育种周期短等优点。 The introduction of new biological macromolecules in the cotton fiber is an important strategy for the use of genetic engineering of cotton fiber quality improvement can break genetic barriers between species, to achieve excellent directional transfer of the gene of interest, while having easy descendants stable, short breeding cycle, etc. . 因此,分离与棉花纤维发育相关的基因及其特异启动子,进而利用基因工程的手段对棉花纤维品质实行定向改良,近年来一直是国内外棉花研究的热点。 Therefore, the separation associated with cotton fiber development and its gene-specific promoter, then the use of the means of implementation of targeted genetic engineering to improve the quality of cotton fiber, cotton in recent years has been a hot research at home and abroad. 利用纤维特异启动子,人们尝试通过生物工程技术改良棉花纤维的品质,如将聚羟基丁酸合成酶系在棉花纤维中特异表达改善了棉花纤维的保暖性能。 Using fiber-specific promoter, have been tried by improving the quality of cotton fiber biotechnology, as will polyhydroxybutyric acid biosynthesis enzymes are specifically expressed in cotton fiber improves the thermal properties of the cotton fiber. 1996年美国Agracetus公司首次报道基因工程改良棉纤维的探索性试验。 In 1996 the company was first reported US Agracetus exploratory test genetically engineered cotton fibers. 经遗传转化的棉纤维中含有一种天然的脂肪族聚酯化合物——多聚一D--(-) -3羟基丁菔(PHB)。 Genetically transformed cotton fibers contains a natural aliphatic polyester compound - a poly-D - (-) -3 turnip hydroxybutyrate (PHB). PHB聚酯是一种天然的可降解的热性塑料(thermoplastic),其理化性质与聚丙烯相似。 PHB polyester is a biodegradable natural thermal plastic (thermoplastic), their physical and chemical properties similar to polypropylene. PHB常以包涵体的形式存在于某些细菌的体内作为碳源。 PHB certain bacteria normally present in the body as a carbon source in the form of inclusion bodies. PHB的生物合成涉及3种酶:(3-酮硫解酶,乙酰CoA还原酶和PHB合成酶。棉纤维本身含有内源性的P-酮硫解酶。John等(John ME, Keller G. Metabolic Proc Natl Acad Sci, USA 1996,93:12768-12773.)将乙酰CoA还原酶基因(-W)和PHB合成酶基因(/7^C)分别与纤维特异性启动子五6和FWL2A连接,构建植物表达载体,运用基因枪技术,将/^aS和/^aC 基因共转化到陆地棉栽培种DP50中,成功获得同时表达p/w5和;?^C基因的转基因棉花。Gt/S基因表达、Northern杂交等分子检测结果均证明:p^5和p/wC在转基因棉株纤维中表达。荧光显微镜和透射电子显微镜观察一致显示:转基因棉纤维细胞中含有PHB颗粒。对转基因棉纤维提取物的GC—MC 分析表明,纤维中有与细菌PHB质谱相同的物质,证明转基因棉纤维中合成了PHB大分子物质。HPLC分析结果表明:每克纤维干重含约30-3440吗的PHB, PHB的合成始于开花后10d,纤维 PHB biosynthesis involves three enzymes: (3-ketothiolase, acetyl-CoA reductase and PHB synthase cotton fiber itself contains endogenous P- ketothiolase .John the like (John ME, Keller G. Metabolic Proc Natl Acad Sci, USA 1996,93:. 12768-12773) the acetyl-CoA reductase gene (-W) and PHB synthase gene (/ 7 ^ C) are fiber specific promoter with five sub connector 6 and FWL2A, Construction of plant expression vectors, using gene gun technique, / ^ aS and / ^ aC genes cotransformed into Gossypium hirsutum cultivar DP50, the successful simultaneous expression of p / w5 and;? ^ transgenic cotton gene .Gt C / S gene expression, Northern hybridization molecular detection results demonstrate: p ^ 5 and p wC expression / in transgenic cotton fibers fluorescence microscopy and transmission electron microscopy observations that show: transgenic cotton fiber cells containing PHB granules fiber extraction transgenic cotton GC-MC analysis thereof showed that the fibers have the same mass material PHB bacteria, demonstrated in transgenic cotton fibers of PHB synthesized macromolecules .HPLC analysis results show that: the fibers per gram dry weight of the PHB containing about 30-3440 do, after PHB synthesis begins flowering 10d, fiber 熟后PHB含量没有降低。禾ij用植物体内自身的乙酰CoA合成大分子PHB,并没有对棉花纤维品质如长度、强度和马克隆值带来任何影响,然而PHB的存在的确提高了纤维的热绝缘性能。 但是因纤维中PHB含量仍较低,绝缘性提高的幅度尚有限。在基因工程改良棉纤维方面颇为人们关注的另一个焦点是美国Auburn大学Daniell的研究工作。Daniell (Henry Daniell, Beftwide Coftow Cow/erenc幼, 1998, 595〜598)试图将编码含Val-Pro-Gly-Val-Gly重复单元的蛋白聚体(protein-based polymers,PBPs)基因遗传转化棉花,使棉花细胞能特异表达出PBPs蛋白。PBPs蛋白广泛存在于自然界中,如哺乳动物结缔组织中的弹性蛋白,分子内含有由多个氨基酸组成的重复顺序,PBPs分子常表现出较强的弹性(弹性系数10.6〜10.9Pa)。 Danidl (Henry Daniell, O?加w Co"/e^"cey, 1998, 595〜598)认为将PHBs引进棉纤维, 一方面有望 Cooked PHB content is not reduced. Ij Wo plants with acetyl CoA synthetase itself PHB molecules, and does not have any effect on cotton fiber quality such as length, micronaire, and strength, however, the presence of PHB fibers improves the heat does insulating properties. However, due to the fiber content of PHB is still low, insulation of increase is still limited. another focus of attention in quite the fiber aspect of genetically engineered cotton in the United States Auburn University Daniell's research .Daniell (Henry Daniell, Beftwide Coftow Cow / erenc Young, 1998, 595~598) attempts to contain coding (protein-based polymers Val-Pro-Gly-Val-Gly repeating mer unit protein, PBPs) genetic transformation of cotton, cotton cells can enable specific .PBPs proteins PBPs protein expressed widely in nature, such as mammalian elastin in connective tissue, the molecule containing a plurality of repeating sequences of amino acids, the molecular PBPs often show strong elasticity (elastic coefficient 10.6~10.9 Pa). Danidl (Henry Daniell, O? plus w Co "/ e ^" cey, 1998, 595~598) believe that the introduction of PHBs cotton fibers, on the one hand is expected to 高纤维的弹性和强度,另一方面纤维中蛋白质含量的提高,纤维吸水能力增强,与染料的亲和力亦相应增强,这是目前常规育种方法所做不到的。 High elasticity and strength of the fiber, on the other hand increase the protein content of the fibers, fiber-reinforced water absorption capacity, and also a corresponding increase in the affinity of the dye, which is impossible by conventional breeding methods. 我国中科院植生所科学家利用花粉管转化途径,将兔角蛋白基因置于棉花纤维特异启动子E6控制下,获得了转兔毛角蛋白基因的棉花,从而使棉纤维的伸长率明显增加,纤维比强度有所提高(张震林,棉花学报2004, 16(2): 72〜76)。 Academy of Sciences of the scientists using green plant pollen tube pathway transformation, placed Tujiao cotton fiber protein gene under control of E6-specific promoter obtained cotton rabbit keratin gene transfer, so that the elongation of the cotton fibers increases significantly, fiber specific strength has increased (Zhangzhen Lin, cotton Science 2004, 16 (2): 72~76). 启动子是RNA聚合酶能够特异识别的一段DNA分子,也就是使转录开始的部位。 RNA polymerase promoter is capable of specifically identifiable piece of DNA molecules, i.e. that the transcription start site. 在基因表达的调控中,转录是基因表达的第一步,也是表达调控的关键一步(Lewi, 2005基因VIII669-705,北京:科学出版社)。 In the regulation of gene expression, the transcription is the first step of gene expression, but also a key step in the regulation of the expression (Lewi, 2005 gene VIII669-705, Beijing: Science Press). 随着基因工程的发展,常常需要构建一种能高水平表达异源蛋白质的表达载体,启动子对外源基因在生物中的表达部位与表达水平影响很大,是基因工程表达载体的重要元件。 With the development of genetic engineering, often need to construct an expression vector capable of high levels of heterologous protein expression, the promoter and exogenous gene expression level in the biological part of a great influence, it is an important component of genetic engineering expression vector. 在棉花纤维品质的基因工程改良中,往往需要外源基因特异地在纤维细胞中表达,从而降低外源基因对棉花正常生长的影响。 In the genetic engineering of cotton fiber quality improvement, often we need Jiyin Te exogenous fibroblasts expressed in different places, thus reducing the effects of exogenous genes in normal growth on cotton. 在有关棉花纤维发育的基因调控机理研究中,通常采用转基因的手段,将目的基因超量表达或表达沉默,从而推测其功能。 For the study of gene regulation mechanism in cotton fiber development, usually means transgenic overexpression or the expression of the gene silencing, presumably so that its function. 因此,在通过转基因手段验证其功能时,会干扰转基因棉的正常生长,产生无法预料的结果,影响实验结果的准确性和实验进度。 Therefore, when verifying its function by transgenic means, turn interfere with the normal growth of transgenic cotton, produced unexpected results, affect the accuracy of results and progress of the experiment. 如果采用棉花纤维特异启动子,则可以使目的基因的表达控制在纤维发育过程中。 If cotton fiber-specific promoters, it is possible to control the expression of the gene during fiber development. 因此,棉花纤维或种皮特异启动子的克隆及其表达分析,对棉花纤维发育的基因功能研究和纤维品质的基因工程改良,都具有十分重要的价值。 Therefore, clones of cotton fiber or testa start and expression analysis, gene function studies and fiber quality of genetically engineered cotton fiber development, are of great value. 发明内容本发明的一个目的在于提供一种棉花纤维特异启动子序列,其至少含有SEQ ID NO. 3所示的核苷酸序列,更优选的是具有如SEQID NO.2所示的序列,该序列为根据棉花GhSCFP基因的cDNA序列设计引物,采用YADE(肖月华,遗传学报2002, 29 (1): 22~26)方法,得到的长度为1037bp 的GhSCFP基因的5'上游序列。 It is an object of the present invention is to provide a cotton fiber-specific promoter sequence comprising at least SEQ ID NO. 3 shown in the nucleotide sequence, and more preferably having the sequence as shown in SEQID NO.2, the the cDNA sequence primers were designed cotton GhSCFP gene using YADE (Xiao Yuehua, genetics 2002, 29 (1): 22-26) method, to obtain a length of 5 'upstream sequence of 1037bp GhSCFP gene. 该序列中包含种子特异的RY-dement(CATGCATC)、 AT富集区(TAAAATAATTATT)、多个CAAT-box、八-box、 G-box、 AAGAA-motif等大量顺式调控元件及与TFH结合的核心序列TATAA。 The sequence contains a seed-specific RY-dement (CATGCATC), AT rich regions (TAAAATAATTATT), a plurality of CAAT-box, eight -box, G-box, AAGAA-motif large number of cis-regulatory elements and a combination of the TFH core sequence TATAA. TATA-box下游25bp处为GhSCFP基因的转录起始位点。 25bp downstream of the TATA-box transcription initiation site GhSCFP gene. 通过在转基因烟草和转基因棉花中GUS组织化学染色、GUS酶活测定以及GFP荧光检测,证实该启动子驱动GUS基因和GFP基因在转基因烟草种子中,进一步地在种皮中特异性优势表达,在转基因棉花中驱动GUS基因在陆地棉纤维中特异地表达。 By transgenic tobacco and transgenic cotton GUS histochemical staining, GUS enzyme assays and GFP fluorescence detection confirmed that the promoter driving the GUS gene and the GFP gene in transgenic tobacco seeds, further in the seed coat-specific predominant expression in transgenic cotton driving the GUS gene expressed specifically in Gossypium hirsutum fibers. 另外,为了鉴定GhSCFP基因启动子的核心区,分析了GhSCFP基因5'序列的系列缺失片段分别与GUS基因融合的转基因烟草的GUS酶活性。 Further, in order to identify the core area GhSCFP gene promoter, the GUS enzyme activity analyzes of transgenic tobacco series GhSCFP gene deletion fragment 5 'sequence fused to the GUS gene, respectively. 结果显示当删除启动子的-634至-543区域(即SEQ ID NO. 3 序列),开花后5天的转基因烟草种子中GUS酶活性降低约30倍,表明该区域含有一个增强子序列,当删除启动子的-634至-543区域,茎和叶中的GUS酶活性高于种子和花冠,表明该区域含有控制启动子在种子中特异表达的元件。 The results show that when the delete start -634 to -543 promoter region (i.e., SEQ ID NO. 3 sequences), transgenic tobacco seeds 5 days after blossom GUS activity decreased about 30-fold, indicating that the region contains an enhancer sequence, when remove -634 to -543 promoter region and the GUS activity of the leaf stems in the seed and above the corolla, indicating that the control element comprises a promoter region-specific expression in seeds.

本发明的又一个目的在于提供含有GhSCFP基因启动子的植物表达载体。 Yet another object of the present invention is to provide a plant expression vector containing the gene promoter GhSCFP. 采用基因工程方法,将GhSCFP基因启动子插入到适宜的表达载体中即可获得本发明的含有GhSCFP基因启动子的植物表达载体。 Genetic engineering method, the gene promoter GhSCFP inserted into a suitable expression vector to obtain a plant expression vector containing the gene promoter GhSCFP the present invention.

在本发明的一个具体实施方案中,构建了含有GUS基因的GhSCFP二GUS 植物表达载体,其具有如图4所示的结构。 In one particular embodiment of the present invention, constructed GhSCFP two plants containing the GUS gene GUS expression vector having the structure shown in FIG.

在本发明的另一个具体实施方案中,构建含有GFP基因的GhSCFP::GFP 植物表达载体,其具有如图5所示的结构。 In another embodiment of the present invention, plant expression vectors comprising GhSCFP :: GFP GFP gene, having the structure shown in FIG. 5.

发明的又一个目的在于提供含有GhSCFP基因启动子序列的转化体。 A further object of the invention to provide a transformant containing the gene promoter sequence GhSCFP. 将上述植物表达载体转化适宜的宿主即可获得本发明的转化体,在本发明的一个具体实施方案中,采用电击法将含有GhSCFP基因启动子的植物表达载体转化到根癌农杆菌LBA4404中获得转化体。 The above plant expression vector to transform an appropriate host to obtain a transformant of the present invention, in a particular embodiment of the present invention using electroporation GhSCFP containing plant gene promoter expression vector is transformed into Agrobacterium tumefaciens LBA4404 obtained transformants.

本发明的又一个目的在于提供制备含有GhSCFP基因启动子的转基因植物的方法。 Still another object of the present invention is a method wherein a transgenic plant gene promoters provide GhSCFP prepared containing. 将本发明的GhSCFP基因启动子与报告基因构建植物表达载体,将所述植物表达载体转化宿主获得含GhSCFP基因启动子的转化体,以及用所述转化体转化植物获得转基因植物。 The present invention GhSCFP gene promoter and reporter construct plant expression vector, transforming a host vector to obtain a transformant containing the GhSCFP gene promoter, and the transformant transformed with a plant to obtain transgenic plant expressing the plant. 所述转基因植物优选为烟草或陆地棉。 The transgenic plant is preferably a tobacco or Gossypium hirsutum. 本发明分离获得了棉花纤维特异基因的启动子,并证实了该启动子序列在烟草中具有外种皮优势表达活性,在陆地棉中具有纤维表达特异性,为基因工程改良烟草、棉花品种提供了可能性和有效的途径。 The present invention is an isolated promoter of the cotton fiber-specific genes, and confirmed that the promoter sequence has activity testa predominant expression in tobacco, a fiber-specific expression in G. hirsutum, genetic engineering to provide an improved tobacco, cotton varieties the possibility and effective way. 附揮简l^说明图1表示GhSCFP基因启动子的核苷酸序列,其中阴影覆盖部分,为-634~-543的核心区域,在序列表中该序列的编号是从1至1037;而-634〜543 的编号是将转录起始点作为+1,转录起始点以上的序列从-1开始;转录起始点以下的序列从+l开始;没有O位点,序列的整个顺序是从5'至3';图2表示扩增DNA所用的引物,其中,P-upl.O和P-down用于扩增1037bp 的GhSCFP启动子,P-up947和P-down、P-up739和P-down、P-up530和P-down、 P-up465和P-down、 P-up258和P-down分别用于扩增946bp、 739 bp、 530 bp、 465 bp、 258bp的GhSCFP启动子片段。 Brief Description play attached l ^ GhSCFP FIG. 1 shows the nucleotide sequence of the gene promoter, wherein the shading cover portion, the core region -634 ~ -543 in the sequence table sequence No. from 1 to 1037; and - 634~543 numbered +1 is the transcription initiation point as, or more transcription start sequence starting at -1; the transcription start sequence begins + l; O no site, the entire sequence is the sequence from 5 'to 3 '; Figure 2 shows DNA amplification primer used, wherein, P-upl.O P-down and used to amplify a 1037bp GhSCFP promoter, P-up947 and P-down, P-up739 and P-down, P-up530 and P-down, P-up465 and P-down, P-up258 P-down, respectively, and used to amplify 946bp, 739 bp, 530 bp, 465 bp, GhSCFP promoter fragment of 258bp. Pgfi)-up和Pgf^-down用于扩增GFP 基因。 Pgfi) -up and Pgf ^ -down used to amplify the GFP gene. pgus-up和pgus-down用于扩增GUS基因;图3表示GhSCFP启动子克隆到pGEM-T载体上的质粒图谱;图4表示pGhSCFP1.0-GUS植物表达载体图谱;图5表示pGhSCFP1.0-GFP植物表达载体图谱;图6表示GhSCFP启动子5'端删除后的系列分析表达载体模式图;图7表示以32P标记的G/^C7T和18sRNA基因为探针,与陆地棉根、茎、 叶、花、胚珠、纤维(R、 S、 L、 FL、 O、 F)中的总RNA杂交得到的Northern 结果;图8表示以32P标记的GMCFP和18sRNA基因为探针,与陆地棉开花后(dpa)不同时间纤维中的总RNA杂交得到的Northern结果;图9表示以GhSCFP蛋白抗体为探针,与陆地棉开花后不同时期胚珠、纤维(0、 F)的总蛋白进行杂交,得到的Western杂交结果,M为标准分子量蛋白;图10表示GhSCFP启动子5'端删除系列,经PCR扩增后的电泳图,其中1、 2、 3、 4、 5分别为946bp、 739bp、 530bp、 465bp、 258bp的GhSCFP启动子DNA, 6为标准分 pgus-up pgus-down and used to amplify the GUS gene; Figure 3 shows GhSCFP promoter was cloned into the plasmid map of the vector pGEM-T; Figure 4 shows pGhSCFP1.0-GUS plant expression vector map; FIG. 5 shows pGhSCFP1.0 -GFP plant expression vector map; FIG. 6 shows a series of analysis GhSCFP promoter expression vector pattern view of the 5 'end deleted; FIG. 7 shows the 32P-labeled G / ^ C7T and 18sRNA genes as probes and upland cotton roots, stems , Northern result leaves, flowers, ovules, total RNA hybrid fiber (R, S, L, FL, O, F) is obtained; FIG. 8 shows GMCFP to 32P-labeled probes and the 18sRNA genes, and flowering Upland Northern results (DPA) total RNA hybrid fibers at different times obtained; FIG. 9 shows in GhSCFP antibody as probes hybridize to different periods ovules, fibers (0, F) Upland total protein after flowering, to give the result of Western blotting, M being the molecular weight protein standards; FIG. 10 shows GhSCFP promoter 5 'end of the series of deleted by electrophoresis after PCR amplification, wherein 1, 2, 3, 4, 5 respectively, 946bp, 739bp, 530bp, 465bp, GhSCFP promoter DNA 258bp, and 6 minutes for the standard 量DNA;图11表示GhscFPi.o: :gus融合基因表达载体构建流程图;图12表示GhSCFP1.0: :GFP融合基因表达载体构建流程图; 图13表示pGhSCFP1.0: :GUS载体的限制性酶切验证琼脂糖电泳图谱, 其中1为标准分子量DNA, 2为pGhSCFP1.0-GUS重组质粒经限制性内切酶Xbal和Smal消化后产物的电泳,表明1037bp的GhSCFP启动子己经与GUS 基因融合;图14表示pGhSCFP1.0: :GFP载体的限制性酶切验证琼脂糖电泳图谱,其中l、 2、 3为不同的p5-GhSCFP1.0-gft)-m重组质粒经限制性内切酶Xbal和BamHI消化后产物的电泳,表明1037bp的GhSCFP启动子己经与GFP基因融合,4为标准分子量DNA;图15表示转G/LSCFi^.O::GC/S基因烟草的PCR验证结果,其中l为标准分子量DNA, 2-7为不同的转基因株系,8为阳性对照,9为阴性对照。 The amount of the DNA; FIG. 11 shows GhscFPi.o:: gus fusion gene expression vector constructed flowchart; Figure 12 shows GhSCFP1.0:: GFP fusion gene expression vector constructed flowchart; FIG. 13 shows pGhSCFP1.0:: GUS vector limiting agarose gel electrophoresis to verify cleavage pattern, wherein 1 is a standard molecular weight DNA, 2 to the Smal and Xbal endonuclease digestion products within the electrophoretic pGhSCFP1.0-GUS plasmid by restriction indicating GhSCFP promoter and the GUS gene had the 1037bp fusion; FIG. 14 shows pGhSCFP1.0:: GFP vector restriction verified by agarose gel electrophoresis pattern, wherein l, 2, 3 different of p5-GhSCFP1.0-gft) -m recombinant plasmid by restriction endonuclease Xbal and BamHI digestion product of electrophoresis, indicating the GhSCFP 1037bp promoter had fused with the GFP gene, the DNA molecular weight standard 4; FIG. 15 shows PCR validation results revolutions G / LSCFi ^ .O :: GC / S gene of tobacco, wherein l is a standard molecular weight DNA, 2-7 for the different transgenic lines, 8 as a positive control, a negative control 9. 表明3、 4、 5、 6为转基因阳性株系,2、 7为转基因阴性株系;图16表示转GASCFP/.(K:GC/S基因棉花的PCR验证结果,其中1为标准分子量DNA, 2-6为不同的转基因株系,7为阳性对照,8为阴性对照。表明2、 3、 4、 5、 6均为转基因阳性株系;图n表示转G/iSCfP7.0::GaS基因烟草中的GUS染色结果,GUS在转基因烟草种子上优势表达。开花后5天的转G/^0^/力-GOS基因烟草种子GUS染色后,再按常规方法进行石蜡切片,最后得到如图的观察结果,表明GhSCFPl.O启动GUS在烟草种子外表皮上表达;图18表示野生型和转G/2SC/?A0,, GFP基因烟草开花后5天的种子, 在荧光显微镜下的观察结果:野生型的种子被激发出红光而转基因的则被激发出绿色荧光;图19表示在转基因烟草的种子、花冠、叶片、茎、萼片和子房壁中,GUS 酶活性的变化;其中在种子中酶活最高,其余组织中均很低;图20表示转 Showed 3, 4, 5, 6 is positive transgenic lines, 2, 7 is a transgene-negative strain; FIG. 16 shows a revolution GASCFP / (K: PCR verification result GC / S gene cotton, wherein 1 is a standard molecular weight DNA,. 2-6 different transgenic lines 7 as a positive control, a negative control showed 8 2, 3, 4, 5, 6 were positive transgenic lines; Figure revolutions n represents G / iSCfP7.0 :: GaS gene GUS staining of tobacco, the expression of GUS in transgenic tobacco seeds advantages 5 days after flowering revolutions G / ^ 0 ^ / -GOS force after GUS staining of transgenic tobacco seeds, paraffin sections in the usual manner, to give the final FIG. the observations indicate that GUS expression GhSCFPl.O start tobacco seed in the outer skin; FIG. 18 shows A0 ,, GFP transgenic tobacco seeds 5 days after flowering of wild-type and genetically G / 2SC /, observation under a fluorescence microscope? : wild-type seeds are excited red and transgene were excited green fluorescence; FIG. 19 shows the seed, corolla, leaves, stems, sepals and the ovary wall transgenic tobacco, the change in activity of the GUS; wherein seed the highest activity, and the remaining tissue was very low; FIG. 20 shows rotation G7lSCF尸7.0: :C?OS基因陆地棉的GUS染色结果;其中A: 开花当天的胚珠;B:当天胚珠表面的放大;C:开花后3天的胚珠;D: 3天胚珠表面的放大; 图21表示转GASCF尸/.(?::GWS基因陆地棉,胚珠GUS染色后的纵切观察结果;其中,A中未着色的胚珠为对照,B为转基因胚珠的纵切,C为B的放大图;图22表示转GASCF尸i.0: :(K/S基因陆地棉,开花当天胚珠GUS染色后的石蜡切片结果;其中,B和D分别为A和B的放大图,表明仅有胚珠表面突起的纤维被着色;图23表示GhSCFP启动子5'端缺失后,转基因烟草种子中GUS酶活性分析结果;当删除该启动子的一634至一543区域后,转基因烟草种子中的GUS 酶活性降低约30倍,表明该区域含有增强子序列。其余4个区域删除后,种子中GUS酶活性变化不大;图24表示GhSCFP基因启动子缺失一634到一543区域后,转基因烟草不同组织中GUS酶活性测定结果,U-7和U-14 ? G7lSCF corpse 7.0:: C GUS staining OS gene Upland; and wherein A: anthesis, ovules; B: day ovule enlarged surface; C: 3 days after flowering ovules; D: 3 days ovule enlarged surface; 21 shows gene transfer GASCF dead /.(?::GWS cotton, slitting observation after GUS staining ovules; wherein, a is not colored controls ovule, ovule B gene was transfected slitting, C is a B an enlarged view; FIG. 22 represents a dead turn GASCF i.0:: (K / S gene cotton, paraffin sections anthesis result after GUS staining ovules; wherein, B and D is an enlarged view of a and B, show that only fiber ovule surface of the projection is colored; FIG. 23 shows GhSCFP promoter 5 'after the end of the deletion, transgenic tobacco seeds GUS activity analysis; after a 634 to 1 543 region to remove the promoter, transgenic tobacco seeds GUS activity decreased about 30-fold, indicating that the region contains enhancer sequence and the remaining four regions deleted, seeds GUS little activity change;. Figure 24 shows GhSCFP gene promoter deletion of a 634 to 1 543 region, transgenic tobacco different tissue activity assay results of GUS, U-7 and U-14 不同的转基因株系,表明当删除GhSCFP基因启动子的一634至一543区域后,茎和叶片中的GUS酶活高于种子、花冠,表明在该区域中还有控制基因在种子中表达的元件。具体实滩方式【实施例1】棉花纤维RNA的提取在开花当天对棉花花朵进行挂牌标记,分别取开花当天到开花后9天的棉铃。棉花胚珠和纤维RNA的提取参考Chang Shujun等的方法(Shujun Chang, PlantMolecularBiologyReporter 1993 11: 113-116),但更改了部分步骤,具体操作程序如下:取样后将棉铃立刻置于冰盒中,室内分别剥取开花后当天的胚珠、纤维以及开花后6天、8天的纤维,于液氮下快速研磨成粉末并装入50ml 离心管中。 Different transgenic lines showed GhSCFP deleted when a gene promoter region after 543 634-1, the GUS activity in the stems and leaves than seeds, corolla, in this region also shows that control of gene expression in seeds [Example 1] element. specific Beach embodiment extracts of cotton fibers RNA in anthesis cotton flowers to supervise the marker, were taken the day of anthesis and 9 days after flowering bolls extraction ovule and fibers RNA reference Chang Shujun like immediately after the sampling boll placed in an ice box, each peeling chamber day after taking ovule flowering, flowering and fibers: method (Shujun Chang, PlantMolecularBiologyReporter 1993 11: 113-116), but some of the steps is changed, the specific procedure is as follows 6 days, 8 days fibers in liquid nitrogen and ground to a powder charged quickly 50ml centrifuge tube. 加入10ml65。 Join 10ml65. C预热的提取缓冲液(CTAB2%, PVP2%, Tris-HCl 100mmol/LpH8.0, EDTA25醒ol/L, NaC12mol/L, Spermidine 0.5mg/ml),混匀于65'C水浴3min,再加入等体积的氯仿:异戊醇(24:1),混匀后离心:20'C、 10000rpm、 5 min。 C preheated extraction buffer (CTAB2%, PVP2%, Tris-HCl 100mmol / LpH8.0, EDTA25 wake ol / L, NaC12mol / L, Spermidine 0.5mg / ml), mixed in a water bath at 65'C 3min, then an equal volume of chloroform: isoamyl alcohol (24: 1), centrifuged after mixing: 20'C, 10000rpm, 5 min. 氯仿:异戊醇(24:1)重复抽提一次,取水相加入l/4体积的10mol/L的氯化锂,4'C沉淀过夜。 Chloroform: isoamyl alcohol (24: 1) extraction was repeated once, the aqueous phase was added 10mol / L lithium l / 4 volume chloride, 4'c precipitate overnight. 于4°C 10000rpm离心20min弃上清液, 5(MHilSSTE溶解,加入等体积的酚(pH4.5)、氯仿:异戊醇抽提,最后于水相中加入2倍体积乙醇,-8(TC存放。 【实施例2】棉花纤维cDNA文库的构建及基因克隆棉花纤维cDNA文库的构建方法主要参照STRATAGENE公司产品说明书进行,简述为取10(Hig棉花开花后当天胚珠和纤维以及开花后6天、8天纤维的总RNA,通过GIBCO BRL的Super-scriptII逆转录酶合成cDNA的第1 链,然后用STRATAGENEcDNA文库构建试剂盒(XZAP),建立了棉花纤维的cDNA文库。经测定,得到的初级库滴度为9.8xl0S放大文库的滴度为1x10"。共随机选取了20个克隆并亚克隆到质粒pBSsk上,检测表明所克隆的外源片段长度在0.7-2.5kb之间,可以满足基因克隆的需要。【实施例3】棉花基因组DNA的分离取约1克棉花叶片材料,放入研钵中,液氮下快速研磨成粉末,装入离心管并加入3ml65'C预热的CTAB提取 At 4 ° C 10000rpm supernatant was centrifuged 20min, 5 (MHilSSTE dissolved, add an equal volume of phenol (pH 4.5), chloroform: isoamyl alcohol, and finally the aqueous phase was added 2 volumes of ethanol, 8 ( TC storage. [Example 2] Construction of cotton fiber cDNA library and cloning of cotton fiber cDNA library construction methods are mainly manufactured by STRATAGENE reference manual for the product, is briefly taken after 10 (Hig cotton fiber and ovule anthesis, anthesis and rear 6 days, total RNA 8 days fibers by chain 1 GIBCO BRL's Super-scriptII reverse transcriptase synthesis of cDNA library is then constructed using STRATAGENEcDNA kit (XZAP), the establishment of a cDNA library of cotton fibers was determined for the obtained amplifying the library a titer of the primary library 9.8xl0S titer of 1x10. "total of 20 randomly selected clones and subcloned into the plasmid pBSsk, analysis showed that the cloned exogenous fragment length between 0.7-2.5kb, meet Clone needs. cotton genomic DNA isolated [Example 3] take about 1 gram of cotton leaf material, placed in a mortar, ground to a powder under liquid nitrogen fast charged centrifuge tube and preheated CTAB added 3ml65'C extract 液,快速振荡混匀,65'C水浴30min。 然后在该离心管中,加入lml5mol/LKAc混匀冰浴30min,加入等体积(4ml) 的氯仿:异戊醇(24:l,v/v)颠倒混匀,8000rpm,离心10min,取上层水相加入2/3 倍体积的预冷异丙醇,混匀,-20'C放置2h。用封口毛细管挑取絮状DNA沉淀于新1.5ml离心管中,70%乙醇洗涤2次后风干沉淀,加入适量TE充分溶解, 酚:氯仿:异戊斷24:23:l,v/v/v滩提后,加入2倍体积乙醇-20。C保存备用。【实施例4】棉花纤维特异基因G/iSCF尸的克隆及序列特征分析从所构建的棉花纤维cDNA文库中,采用随机克隆法获得了80个基因。 其中一个基因的核苷酸序列与拟南芥等植物的Subtilisin-likeProtease基因有较高的同源性,命名为棉花种皮纤维特异基因(Seed Coat Fiber specific Proteinase, G/lSOT),该基因长2436bp含有一个2337bp的开放阅读框,5, 端非编码区31bp, 3'端非编码区68bp。推测编码的 The liquid, rapidly oscillating mixing, 65'C water bath for 30min and then the centrifuge tube was added lml5mol / LKAc mixing ice bath was 30min, an equal volume (4ml) chloroform: isoamyl alcohol (24: l, v / v ) mix by inversion, 8000 rpm for, centrifuged 10min, the upper aqueous phase was added 2/3 volume of isopropanol pre-cooled, mixed, -20'C placed 2h. picked with a sealing capillary flocculent precipitate the new DNA 1.5ml centrifuge tube, washed with 70% ethanol twice and air-dried precipitate was sufficiently dissolved was added an appropriate amount of TE, phenol: chloroform: isoamyl off 24: 23: after l, v / v / v mention Beach, -20 was added 2 volumes of ethanol. C for use. [Example 4] cotton fiber-specific genes G / cloning and sequence analysis of characteristics iSCF dead cotton fiber cDNA library constructed in a random cloning obtained 80 genes a gene in which the nucleotide Subtilisin-likeProtease gene sequence of Arabidopsis plants have a high homology, designated as cotton fiber-specific genes seed coat (seed Coat fiber specific Proteinase, G / lSOT), the gene contains an open reading a long 2436bp 2337bp of block, 5, UTR 31bp, 3 'UTR 68bp. estimation coding 白质由778个氨基酸残基组成,分子量83474道尔顿,pi等于8.244,核苷酸序列如SEQ ID NO. 1所示。 同源性分析表明:在蛋白质水平,与马铃薯、拟南芥、水稻的Subtilisin-like Protease基因的同源性分别为72%、 70%和68%。 White matter of 778 amino acid residues, a molecular weight of 83,474 daltons, pi is equal to 8.244, the nucleotide sequence shown in SEQ ID NO 1 homology analysis showed: at the protein level, potato, Arabidopsis, rice homology to Subtilisin-like Protease gene were 72%, 70% and 68%. Subtilisin属于丝氨酸蛋白酶的一种,它有一个底物结合位点, 一个由D、 H和S三个区构成的接触反应域。 Subtilisin is a serine protease that belongs to, which has a substrate binding site, a catalytic domain composed of three regions D, H and S. Subtilisin在原核和真核生物中均有发现, 但植物中的研究相对较少。 Subtilisin have been found in prokaryotes and eukaryotes, but relatively few studies in plants. 目前已经从植物中克隆了一些SubtiUsin-like基因并进行了初步研究。 Now some SubtiUsin-like cloned genes from plants and studied. Berger和Altmann ( Berger D, Altmann T C7ewes Dev 2000, 14:1119-1131)在研究拟南芥表皮气孔簇生的sddl-l单隐性突变体时,通过作图克隆出&DjW基因,结果显示,它与subtilisin基因有很高的同源性。 Berger and Altmann (Berger D, Altmann T C7ewes Dev 2000, 14: 1119-1131) sddl-l when a single recessive mutant Arabidopsis stomatal clustered by gene for Tuke Long & DjW out showed that it and subtilisin gene has a high homology. sddl 由于缺少S区可能丧失了枯草杆菌蛋白酶的催化活性,失去了对表皮细胞分化成气孔的控制,从而导致气孔的簇生现象。 sddl region S may be lost due to lack of the catalytic activity of subtilisin, loss of control of epidermal cells differentiate into the pores, resulting in the phenomenon of clustered stomata. 但通过对突变株转化SZ)Z)/基因, 气孔簇生现象由于S/)i)/基因的互补作用可以得到恢复,表明subtilisin基因在植物中可能具有控制表皮细胞分化的功能。 However, by conversion SZ) Z) / genes clustered stomata phenomenon can be restored since the mutant complementation of S /) i) / gene showed subtilisin gene may have the function of controlling epidermal cell differentiation in a plant. Tanaka (Tanaka H, Onouchi H, Kondo M, Hara-Nishimura I, Nishimura M, Machida C, Machida Y. Deve/o戸e/^. 2001 128(23):4681-9)等的研究结果也证实,在拟南芥alel (abnormal leaf shapel)突变体发育过程中,胚胎外表面与残存的胚乳表皮不分离,通过转座示踪克隆的況£/基因也与subtilisin有很高的同源性。 Tanaka (Tanaka H, ​​Onouchi H, Kondo M, Hara-Nishimura I, Nishimura M, Machida C, Machida Y. Deve / o Kobe e / ^ 2001 128 (23):. 4681-9) also confirmed the results of other studies, in Arabidopsis alel (abnormal leaf shapel) during the development of mutant embryos outer surface of the skin without remaining endosperm isolated clones by transposition tracer conditions £ / gene also has a high homology with subtilisin. 进一步研究得知基因只在幼嫩的胚胎和紧贴胚胎的胚乳表皮细胞中表达, 一旦胚胎萌发,其表达即停止,表明ALE1基因可能影响胚胎或幼嫩植物表皮的形成。 Further research that was expressed only in embryos and young endosperm against embryonic epidermal cells, once the embryo germination, the expression of which is stopped, suggesting that the gene could affect embryonic ALE1 or young plant cuticle. Paik-Ro (Paik-Ro OG Seib JC Smith R丄.7Tieor Gewd 2002 104:236-240)等应用差减杂交的方法从花生的cDNA文库中克隆了四个种子发育特异基因,其中基因在种皮中特异表达,而且也编码subtilisin-like蛋白。 Paik-Ro (Paik-Ro OG Seib JC Smith R Shang .7Tieor Gewd 2002 104: 236-240), etc. Methods of subtractive hybridization development of specific genes cloned from the cDNA library four peanut seeds in which the seed coat gene specifically expressed, but also encoding subtilisin-like protein. 这些研究结果表明,subtilisin基因可能参与了植物胚胎幼嫩组织表皮细胞形成及种皮发育等过程。 These findings suggest that, subtilisin genes may be involved in young plant embryo tissue formation and epidermal cells testa development and the like. 【实施例S】棉花纤维特异基因G/zSC/T的表达分析我们首先对GT^O^基因的表达进行了Northem杂交分析。 [Expression] Example S cotton fiber-specific genes G / zSC / T we first analyzed the expression of GT ^ O ^ genes were Northem blot analysis. 以其保守区域为探针,进行该基因组织特异性表达分析,发现该基因在根、茎、叶、花中不表达,仅在胚珠和纤维中表达,可见棉花G/^OT基因的表达具有纤维发育特异性,可能与棉花纤维的发育有关。 Conserved region thereof as a probe, a tissue-specific gene expression analysis, it was found that the gene is not expressed in roots, stems, leaves, flowers, only expressed in the ovules and fibers, cotton visible G / ^ OT expression of genes having fiber-specific development may be related to the development of cotton fibers. 进一步用相同的探针,分别与不同发育时期纤维的RNA杂交,结果表明:在棉花纤维发育的早期G/^O^P基因开始表达, 至开花后8-10天表达量达到最高,随后该基因的表达开始下降,到开花后14天该基因关闭,即该基因的表达与棉花纤维的形成和初生壁的形成有关。 Further with the same probes were hybridized with RNA fibers different developmental stages, results showed that: In the early development of the cotton fiber G / ^ O ^ P gene began to express, after 8-10 days to flowering highest expression level, subsequently gene expression began to decline to 14 days after flowering gene of the closed, i.e., primary cell wall formation and expression of the cotton fiber genes. 这些结果暗示:棉花G/zSCFP基因可能参与了胚珠表皮细胞的分化、纤维细胞的发育等过程。 These results suggest that: Cotton G / zSCFP genes may be involved in the differentiation ovule epidermal cells, fibroblasts and other developmental processes. 其次,通过Westem杂交分析该基因在蛋白质水平的表达,结果表明从 Secondly, by Westem blot analysis of the gene expression at the protein level, the results show that the

4DPA就能够检测到目标蛋白的出现,在8DPA达到最高表达量;随着纤维细胞的发育,GhSCFP蛋白持续存在(超过30DPA)且含量并未有明显的下降。 4DPA can detect the presence of the target protein, the highest expression in 8DPA; with the development of fibroblasts, GhSCFP persistent protein (more than 30 DPA) and the content did not decrease significantly. 【实施例6】棉花纤维特异启动子的克隆及特征分析根据棉花G/iSCFP基因的cDNA序列设计DNA引物,采用YADE的方法, 在棉花基因组上向该基因的5'端进行了延伸,得到了长度为1037bp的GMO^ 基因的5'上游序列,如SEQ ID NO. 2所示。 [Example 6] Cloning and Characterization of cotton fiber specific promoter analysis The cDNA sequence of DNA primers were designed cotton G / iSCFP gene, the method YADE performs extending to the 5 'end of the gene in the cotton genome obtained length of 1037bp of GMO ^ 5 'upstream sequences of genes, such as SEQ ID NO. 2 in FIG. 利用分析软件(Plant CARE, http://intra.psb.ugent.be:8080/PlantCARE/),从植物启动子数据库中对该序列进行启动子调控元件分析,从中发现大量的顺式调控元件及其与TFu结合的核心序列TATAA ,在该TATA-box下游25bp处为GhSCFP基因的转录起始位点(initiator, Inr)。 Using analysis software (Plant CARE, http://intra.psb.ugent.be:8080/PlantCARE/), a promoter regulatory elements from the analysis of the plant promoter sequence database, and found a large number of cis-regulatory elements, and TFu which binds to the core sequence TATAA, 25bp downstream of the TATA-box of the transcription start site of the gene GhSCFP (initiator, Inr). 这些顺式调控元件包括种子特异的RY-element(CATGCATC)、 AT富集区(TAAAATAATTATT)、多个CAAT-box、 A-box、 G-box、 AAGAA-motif 等。 These cis-regulatory element comprises a seed-specific RY-element (CATGCATC), AT rich regions (TAAAATAATTATT), a plurality of CAAT-box, A-box, G-box, AAGAA-motif and the like. 【实施例7】棉花GASC/T基因启动子分析表达载体的构建通过设计的引物(P-upl.O, P-down)利用PCR扩增出这段DNA片段并克隆到TA载体pGEM-T中为pGEM-GhSCFPl.O,最后通过DNA测序对该DNA序列进行鉴定。 [Example 7] Cotton GASC / T gene promoter expression vectors analysis of this amplified DNA fragments by designing primers (P-upl.O, P-down) by PCR and cloned into the TA vector pGEM-T as pGEM-GhSCFPl.O, and finally the DNA sequence was identified by DNA sequencing. 根据该启动子序列中各种调控元件的分布,按照5'端缺失的原贝U,设计以下引物(图2)分别扩增出946bp、 739bp、 530bp、 464bp和257bp 的启动子片段。 According to the promoter sequence the various regulatory elements distributed, according to the 5 'end deletions of the original shell U, following primers were designed (FIG. 2) were amplified 946bp, 739bp, 530bp, 464bp and 257bp of the promoter fragment. 将扩增的DNA片段克隆到TA载体pUCm-T中,通过序列测定予以确认。 The amplified DNA fragment was cloned into the TA vector pUCm-T, to be confirmed by sequence determination. 由于在启动子5'端和3'端的引物后分别引入了XbaI和SmaI限制性内切酶位点,通过XbaI和SmaI将确认的启动子缺失片段定向克隆到pGPTV-KAN 载体中与wW^基因融合,构建GhSCFP::GUS植物表达载体(图4)。 Since the promoter 5 after 'and 3' ends of the primers introduced endonucleases XbaI and SmaI restriction sites, clonal deletion fragments directed by XbaI and SmaI to confirm the promoter to pGPTV-KAN vector with wW ^ Gene fusion, GhSCFP :: GUS construct plant expression vector (FIG. 4). PCR反应条件:反应总体积为25|^1,包括10xLA Taq buffer 2.5^1、每种dNTP 100nmol/L、 1.5 mmol/L MgCl2、模板DNA 10ng、上下游引物各400nmol/L、 1单位LA Taq DNA聚合酶(TaKaRa公司)。 PCR reaction conditions were: total reaction volume was 25 | ^ 1, comprising 10xLA Taq buffer 2.5 ^ 1, of each dNTP 100nmol / L, 1.5 mmol / L MgCl2, template DNA 10ng, upstream and downstream of each primer 400nmol / L, 1 unit of LA Taq DNA polymerase (TaKaRa company). 扩增条件:94'C变性4min,继以94。 Amplification conditions: 94'C denaturation 4min, followed by 94. C变性30s、 55。 C denaturation 30s, 55. C退火30s、 72。 C annealing 30s, 72. C延伸lmin,共35个循环,最后72'C延伸10min。 C extending Lmin, for 35 cycles, and finally extending 72'C 10min. GFP基因来源于pBINm-gfi)5-ER,但去除其中的碱性几丁酶信号肽。 GFP gene derived from pBINm-gfi) 5-ER, but to remove a basic chitinase signal peptide therein. 通过合成引物(Pgfj)-up, Pgf^down)扩增并克隆到pUCm-T中为pUCm-gfi)5, Amplified by synthesis primer (Pgfj) -up, Pgf ^ down) and cloned into pUCm-T for pUCm-gfi) 5,

利用限制性内切酶HindIII和EcoRI将该基因定向克隆到P5中为p5-g*-m。 Enzymes HindIII and EcoRI restriction use within the targeted gene cloned into P5 for p5-g * -m. 其次,利用EcoRI和Smal将GhSCFPl.O从pGEM-GhSCFPl.O中切出并克隆到pBS上为pBS-GhSCFPl.O;再通Xbal和Bamffl将GhSCFPl.O从pBS-GhSCFPl .0中切出并定向克隆到p5-g$-m载体中与GFP基因融合,构建成GhSCFP: :GFP植物表达载体(图5)。 Secondly, the EcoRI and Smal GhSCFPl.O excised from pGEM-GhSCFPl.O and cloned into pBS of pBS-GhSCFPl.O; recanalization Xbal and the GhSCFPl.O Bamffl cut out and pBS-GhSCFPl .0 cloned into the p5-g $ -m vector fused with the GFP gene, constructed GhSCFP:: GFP plant expression vector (FIG. 5). 最后,通过电击法将这些启动子分析表达载体转化到根癌农杆菌LBA4404中。 Finally, by electroporation promoter analysis of these expression vectors were transformed into Agrobacterium tumefaciens LBA4404. 【实施例8】烟草的遗传转化1、 烟草无菌幼苗的获得以及根癌农杆菌侵染液的制备将烟草种子用1%的次氯酸钠消毒15min,无菌水冲洗4-5次,然后置于固体MS培养基上萌发,于25'C、 16h光照/8h黑暗光周期条件下培养。 Example 8 Transformation of Tobacco] 1. Preparation of seedlings infected with Agrobacterium tumefaciens, and obtaining liquid sodium hypochlorite 15min aseptically tobacco Tobacco seeds 1%, sterile water 4-5 times, then placed germinated on MS solid medium at 25'C, cultured under 16h light / 8h dark photoperiod. 在含50mg/L Km和125mg/L Sm的YEB固体培养基上,活化含表达载体的农杆菌保存菌株。 YEB on solid medium containing 50mg / L Km and 125mg / L Sm, activation of the expression vector containing Agrobacterium strain preservation. 挑取一个含目的基因的农杆菌单菌落,接种于5ml附加相同抗生素的YEB液体培养基中,28'C、 200rpm培养过夜。 A single colony was picked Agrobacterium containing a desired gene, YEB liquid medium was inoculated in 5ml denoted by the same antibiotics, 28'C, 200rpm overnight culture. 取250pl该菌液接种于25mlYEB培养基中,28°C、 200rpm培养至006。 The inoculated broth taken 250pl 25mlYEB medium, 28 ° C, 200rpm grown to 006. . 约0.6~0.8,然后将菌液于6000rpm离心5min收集菌体,用液体MS培养基稀释至原菌液体积。 From about 0.6 to 0.8, and then the bacteria, diluted to the original volume of broth with liquid MS medium 5min to collect bacteria by centrifugation at 6000rpm. 2、 根癌农杆菌介导的烟草遗传转化取无菌幼苗健壮叶片,切成约0.5cm2大小叶盘,加入OD6oo为0.6^0.8的农杆菌菌液,轻轻振荡侵染15min后,立刻倾去菌液,将外植体转入表面铺有一层滤纸的共培养基上,24'C暗处共培养3d。 2, tobacco Agrobacterium tumefaciens-mediated genetic transformation to take robust sterile seedling leaves, leaf discs were cut to size from about 0.5cm2, was added 0.6 ^ 0.8 OD6oo Agrobacterium bacteria, after infection with gentle shaking 15min, poured immediately to bacteria, the explants were transferred on a medium surface covered with a layer of filter paper, 24'c, dark coculture 3d. 3、 转化植株的筛选与再生共培养后,将外植体转入筛选培养基中进行筛选分化培养,每4周继代一次。 3, after the screening and regeneration of transformed plants co-cultivation, the explants were transferred to selective differentiation medium for screening culture passaged once every 4 ZHOU Ji. 当愈伤出现绿芽时,将绿芽切下转入生根培养基中生根,直到幼苗长到高约5cm时,对转基因幼苗进行检测。 When callus appears green shoots, rooting the shoots transferred to rooting medium cut, until the seedlings grow to a height of about 5cm, transgenic seedlings were detected. 对融合GFP的启动子分析转基因烟草, 对根及叶片直接进行GUS组织化学染色,将GUS阳性幼苗移栽入土钵中;对融合GUS的转基因烟草,釆用PCR的方法进行鉴定,将PCR阳性的烟草移栽并管理。 Of the promoter fused to the GFP in transgenic tobacco root and leaf directly GUS histochemical staining, transplanted into the soil mortar positive seedlings GUS; transgenic tobacco fusion of GUS, Bian identified by PCR, the PCR positive tobacco transplanting and management. 【实施例9】棉花的遗传转化1、 棉花胚性愈伤组织诱导以及根癌农杆菌侵染液的制备棉花种子剥壳,用0.1%升汞(HgCl2)消毒10min,无菌水漂洗6次,接种于种子萌发培养基上,28°C、黑暗条件下萌发5〜7d,获得无菌的棉花幼苗。 [9] Example 1 Genetic transformation of cotton, cotton seed shelling prepared embryogenic cotton callus and the Agrobacterium was infected with 0.1% mercuric chloride (HgCI2) disinfection 10min, rinsed 6 times with sterile water inoculated in the seed germination medium, 28 ° C, the dark germination 5~7d, to obtain a sterile cotton seedlings. 选取生长健壮的无菌幼苗下胚轴,切成约0.4cm的小段,接种于棉花愈伤组织诱导培养基上诱导愈伤组织,每3-4周继代一次。 Select hypocotyl robust growth under sterile seedlings, cut into small pieces of about 0.4cm, the callus was inoculated to cotton callus induction medium every 3-4 ZHOU Ji passaged once. 选取松散的愈伤组织接种到胚性愈伤组织诱导培养基上,诱导胚性愈伤组织的产生。 Select loose callus was inoculated to the embryogenic callus induction medium to produce embryogenic callus. 在含50mg/LKm和125mg/L Sm的YEB固体培养基上,活化含表达载体的农杆菌保存菌株。 YEB on solid medium containing 50mg / LKm and 125mg / L Sm, activation of the expression vector containing Agrobacterium strain preservation. 挑农杆菌单菌落,接种于5mL含相同抗生素的YEB液体培养基中,28°C、 200r/min震荡培养过夜。 Pick a single colony of Agrobacterium was inoculated in YEB liquid medium containing the same antibiotics 5mL, 28 ° C, 200r / min shaking overnight. 培养后的农杆菌菌液按1: 20的比例转接到50mL含相同抗生素的YEB液体培养基中,继续培养3-5hr, 6000rpm 离心10min后,菌体用液体MSB (含10(Himol/L AS)培养基重悬,调整OD600 值为0.3-0.5备用。2、 浸染和共培养用无菌吸水纸吸去胚性愈伤组织表面的液体,放入调整好浓度的农杆菌菌液中,浸染20-30min。倾去菌液,将浸染过的胚性愈伤转移到共培养培养基(含100(amol/LAS)上,于24。C黑暗条件下共培养4d。3、 转化子的筛选共培养完成后把胚性愈伤组织转移到筛选培养基上进行脱菌和筛选培养, 每3周继代一次。l-2个月后大部分愈伤组织褐化死亡,少部分表现出卡那霉素抗性,生长出新鲜的胚性愈伤组织。愈伤组织块继代,每块组织增殖到2.0-3.0g时,接入液体悬浮培养基中,在摇床上120r/miii震荡悬浮培养以获得大量体胚。悬浮2周后用30目筛网过滤悬浮培养组织,网下沉 Agrobacterium culture broth by the ratio of 1: 20 to turn 50mL YEB liquid medium containing the same antibiotics, and cultured 3-5hr, 6000rpm centrifugal 10min, cells with a liquid MSB (containing 10 (Himol / L AS) medium was resuspended, adjusted OD600 value of 0.3-0.5 standby .2, dip and co-culture with sterile absorbent paper to suck the liquid surface of embryogenic callus tissue, into Agrobacterium bacteria concentration adjusted in disseminated 20-30min. was decanted broth, disseminated through the embryogenic callus was transferred to the co-cultivation medium (containing the 100 (amol / LAS), were cultured in 4d.3 24.C under dark conditions, transformant screening of co-culture after the completion of the transfer of embryonic callus on selective media were screened off bacteria and cultures, generations once every three ZHOU Ji-.l-2 most callus browning died months later, a small number of cards to show that when neomycin resistance were grown from fresh embryogenic callus. callus pieces subcultured, to 2.0 - 3.0 g of each tissue proliferation, the access liquid suspension medium, shaker 120r / miii shaking cultured in suspension to obtain a large number of somatic embryos. after 2 weeks, the suspension was filtered suspension with a 30 mesh screen tissue culture, sink network 物转接入体胚成熟培养基。萌发的成熟胚转入体胚伸长诱导培养基上,诱导体胚伸长、萌发。取长度大于0.5cm的萌发胚转接入SH培养基成苗。待幼苗长到约2cm高时,剪取幼苗嫁接到有3-4片真叶的棉花幼苗上。嫁接成活后移栽到温室生长。【实施例10】转基因植株的PCR验证采取DNA小量提取方法,提取转基因植株DNA用于PCR验证。具体步骤包括:分单株分别取少量TO代转基因烟草和棉花叶片,置1.5ml离心管中, 加入少许液氮并用玻棒研磨成粉末。加入0.5ml 65匸预热的DNA提取缓冲液, Body composition were transferred to embryo maturation media. Germinated mature embryos into the elongated somatic embryo induction medium elongated somatic embryo germination. Take a length greater than 0.5cm in germinating embryos were transferred to SH medium seedling. Be seedlings grown to approximately 2cm high, there is a clip to cotton seedlings grafted seedlings 3-4 true leaves. after grafting transplanted to a greenhouse growth. [Example 10] PCR of transgenic plants DNA miniprep method of verification to take , transgenic plants DNA extraction for PCR verification step specifically comprises: a small amount of points per plant were taken tO transgenic tobacco and cotton leaves, placed 1.5ml microcentrifuge tube, add a little liquid nitrogen and ground to a powder with a glass rod was added 0.5ml 65. Xi preheated DNA extraction buffer,

混匀,65X:水浴30min。 Mix, 65X: water bath 30min. 等体积氯仿:异戊醇抽提两次,吸取上清液于新离心管中,加入2倍体积无水乙醇-20'C沉淀2h。 An equal volume of chloroform: isoamyl alcohol extracted twice, the supernatant to a fresh centrifuge tube, adding 2 volumes of absolute ethanol precipitated -20'C 2h. 室温下12000rpm离心10min,弃取上清液风干沉淀,最后200nlTE缓冲液溶解。 Centrifuged 12000rpm at room temperature for 10min, the supernatant was discarded precipitate was air-dried, and finally 200nlTE buffer solution. 对提取的转基因植株DNA进行PCR体外扩增验证,反应总体积为25^1, 包括10xLATaq buffer 2.5pl、每种dNTP 100nmol/L、 1.5 mmol/LMgCl2、模板DNA10ng、上下游引物各400nmol/L、 1单位LA Taq DNA聚合酶(TaKaRa 公司)。 Transgenic plants DNA extraction for PCR verification vitro amplification in a total reaction volume of 25 ^ 1, comprising 10xLATaq buffer 2.5pl, each dNTP 100nmol / L, 1.5 mmol / LMgCl2, template DNA10ng, upstream and downstream of each primer 400nmol / L, 1 unit of LA Taq DNA polymerase (TaKaRa Co.). 扩增条件:94t:变性4min,继以94-C变性30s、 55'C退火30s、 72。 Amplification conditions: 94t: denaturation 4min, followed by 94-C denaturation 30s, 55'C annealing 30s, 72. C延伸lmin,共35个循环,最后72'C延伸10min。 C extending Lmin, for 35 cycles, and finally extending 72'C 10min. 扩增产物在含溴乙锭的ln/。 Amplification product containing ethidium bromide ln /. 琼脂糖凝胶上以5V/cm的电压电泳后,紫外灯下观察照像。 After the agarose gel to 5V / cm voltage electrophoresis, observed under ultraviolet light photography. 【实施例11】GUS的组织化学染色及观察取新鲜的转基因烟草、陆地棉材料,切成小块后置1.51111离心管中,加入GUS染色液(1Ommol/LEDTA, 10Ommol/L磷酸钠缓冲液pH7.0, 0.1mol/L K3[Fe(CN)6], O.lmol/L K4[Fe(CN)6],0.1%(V/V)Triton X-100,5mg/ml X陽Glue)。 [Example 11] GUS tissue staining and observation Fresh transgenic tobacco, Gossypium hirsutum material, cut into small pieces rear 1.51111 centrifuge tube, was added GUS staining solution (1Ommol / LEDTA, 10Ommol / L sodium phosphate buffer pH7 .0, 0.1mol / L K3 [Fe (CN) 6], O.lmol / L K4 [Fe (CN) 6], 0.1% (V / V) Triton X-100,5mg / ml X male Glue). 将含有植物材料和GUS染液的离心管,放入37.(TC恒温温箱中,染色3h。最后倾去染色液,通过70%乙醇脱色后,观察照相。如果对GUS染色后烟草种子和棉花胚珠进行石蜡切片观察,则倾去染色液后,用FAA固定液固定3至24h。洗去固定液,常规乙醇一—叔丁醇法6级脱水,浸蜡、包埋。石蜡包埋好的植物组织,于切片机上切片,切片厚度控制在10—15nm。【实施例12】转基因材料GFP的显微观察将切取的新鲜组织立即投入含4%多聚甲醛、2.5%戊二醛、20mmol/L磷酸缓沖液(pBS, pH7.0)的固定液中,经lh真空处理后,于(C下继续固定过夜。固定的样品经磷酸缓冲液浸洗2〜3次后,直接用Tissue-TekO.CT,包埋剂包裹在样品上,再用液氮迅速冷冻。随后尽快将样品移至-80。C超低温冰箱中保存备用。于切片前lh左右,将样品从-8(TC超低温冰箱中取出,置冰冻切片机腔内,经回温后再用 The dye-containing plant material and GUS centrifuge tube, placed 37. (TC thermostat incubator, 3H staining. Finally staining solution was decanted, after decolorization by 70% ethanol, photographic observation. If tobacco seeds and GUS staining after ovule paraffin sections were observed, the staining solution was decanted, with FAA fixative solution washed fixative 3 to 24h, a conventional ethanol - 6 tert-butanol dehydration method, dipping wax, paraffin embedded well. plant tissue, on a microtome, the slice thickness control 10-15nm. [Example 12] microscopic observation of GFP gene the material cut fresh tissue immediately into 4% paraformaldehyde, 2.5% glutaraldehyde, 20mmol after / fixing solution L phosphate buffer (pBS, pH7.0), the vacuum treatment after lh at (C continues under stationary overnight fixed samples were 2 or 3 times a phosphate buffer solution immersion, directly Tissue -TekO.CT, embedding medium wrapped on the sample, then quickly frozen in liquid nitrogen. the samples were then moves -80.C Ultralow freezer stored for use as soon as possible to the left and right front sections LH, the sample from -8 (TC cryogenic remove the refrigerator, home freezing microtome intracavity, then warmed with 埋剂粘在样品头上进行冰冻切片。切片厚度为,10网,切片机腔温度为-2(TC。切片自然晾干后,在荧光显微镜下观察并照相。 Sample head sticking agent buried frozen sections. Slice thickness, net 10, the chamber temperature is -2 slicer (after the TC. Sliced ​​natural drying, it was observed and photographed under a fluorescence microscope.

【实施例13】转基因植物组织GUS酶活性测定Gi[/S基因编码j3-葡萄糖苷酶,该酶是一种水解酶,表现活性时不需要辅酶, 能催化许多(3-葡萄糖苷酯类物质的水解。催化作用的最适pH为5.2~8.0,能适应较宽的离子强度范围。以4-甲基伞形酮酰-pD葡萄糖醛酸苷(4-MUG)为底物,GUS催化其水解为4-甲基伞形酮(4-MU)及PD葡萄糖醛酸。4-MU分子中的羟基解离后被365nm的光激发,产生455nm的荧光,可用荧光分光光度计定量,由于4-MU羟基的pKa在8〜9之间,欲使羟基大量离子化,溶液的pH应大于其pKa, 一般使用碳酸钠终止反应并创造碱性条件。1、 GUS粗酶液的提取选择己开花的转基因烟草,于开花当天挂牌标记开花时间。取开花后5d 的果实(其中每个单株3〜5个果实),室温下分别剥取种子、子房壁。另外, 常规方法对叶片、萼片、幼茎和开花当天的花冠取样。将待测的植物材料 [Example 13] Gene transfer activity in plant tissues GUS assay Gi [/ S j3- glucosidase gene encoding the enzyme is a hydrolytic enzyme, the coenzyme does not need to exhibit activity and can catalyze a number of (3-glucoside esters hydrolysis. catalysis optimum pH of 5.2 to 8.0, can adapt to a wide range of ionic strength. in -pD 4- methylumbelliferyl-glucuronide (4-MUG) as substrate, catalyze the GUS hydrolyzed to 4-methylumbelliferone (4-MU) and PD-hydroxy-glucuronic acid solution .4-MU molecule after light from the 365nm excitation, 455nm fluorescence, can be used fluorescent spectrophotometer, since 4 -MU pKa between 8-9 hydroxyl groups, a large number of hydroxyl purports ionized, pH of the solution should having a pKa greater than, sodium carbonate is generally used to terminate the reaction and to create basic conditions .1, GUS extraction select the crude enzyme solution had flowering the transgenic tobacco, to mark the day of anthesis listed flowering time. fruit (each fruit plant 3 ~ 5 months) taken after the flowering 5d, respectively, at room temperature for stripping seeds, ovary wall. Further, the conventional method leaves, sepals , young stems and flowering corolla sampling day. the plant material tested 置液氮中研磨成粉,加入2倍体积的GUS酶提取缓冲液(100mmol/L磷酸钠缓冲液pH8.0, 1%PVPW/V, 10mmol/l^-巯基乙醇)制成匀浆,冰上放置lh。于13000rpm/min离心10min,收集上清夜,立即测定GUS酶活,如需保存加入灭菌甘油(50%v/v) -20'(:保存。2、 Bradford法(汪家政,2000蛋白质技术手册42~46,北京:科学出版社)测定GUS粗酶液中总蛋白的含量。3、 4-MU标准曲线的制作进行启动子分析时,基于启动子表达的时空特异性,在转基因植物的不同组织中,会得到不同的GUS酶活。因此,为保证实验的准确性,制作了高、 低两种4-MU浓度梯度的标准曲线。首先,用少量的N,N-二甲基甲酰胺溶解4-MU固体,然后用双蒸水稀释成lmmoVL的母液。其次,用反应终止液将4-MU母液稀释成10Onmol/L、 80Mmol/L、 60nmoi/L、 40nmol/L和20^imol/L 的高浓度梯度样品,以及10Mmol/L、 8^mol/L、 6nmol/L、 4pmol/L和2[imol/L Pulverized in liquid nitrogen, was added 2 volumes of GUS enzyme extraction buffer (100mmol / L sodium phosphate buffer pH8.0, 1% PVPW / V, 10mmol / l ^ - mercaptoethanol) and homogenized, ice lh placed at 13000rpm / min centrifugal 10min, the supernatant was collected, GUS activity was measured immediately, sterile glycerol was added to save (50% v / v) -20 '(:. save .2, Bradford method (Wang Jiazheng, technical Manual 2000 protein 42 to 46, Beijing: Science Press, the content of .3) GUS assay of total protein in the crude enzyme solution, production of the 4-MU standard curve of a promoter analysis, based on the temporal and spatial specific expression promoter, in different tissues of transgenic plants, GUS activity will be different. Therefore, to ensure accuracy of the experiment, the production of high, low two kinds of standard curves 4-MU concentration gradient. first, a small amount of N, N- two dimethylformamide was dissolved 4-MU solid was then diluted with distilled water into a mother liquor lmmoVL Secondly, the mother liquor was diluted to 4-MU 10Onmol / L with the reaction-terminated liquid, 80Mmol / L, 60nmoi / L, 40nmol / L and 20 ^ imol / L sample of a high concentration gradient, and 10Mmol / L, 8 ^ mol / L, 6nmol / L, 4 pmol/L and 2 [imol / L 的低浓度梯度样品。于酶标仪上在激发光360nm,发射光460nm,狭缝25nm 的条件下测定各样品的荧光强度,以反应终止液为空白对照,每种浓度样品各设置3个重复,通过计算平均值绘制标准曲线。4、 GUS酶与底物反应于37'C水浴中预热反应缓冲液(100mlGUS酶提取缓冲液中加入35.23mg Low sample concentration gradient. In light of 360 nm excitation, 460nm emission light, the fluorescence intensity of each sample was measured under conditions of 25nm slit to blank control reaction-terminated liquid, the concentration of each sample of each set is repeated on a microplate reader 3 , by calculating the average standard curve .4, GUS enzyme reacts with the substrate reaction buffer preheated to 37'C in a water bath (100mlGUS enzyme extraction buffer was added 35.23mg

的4-MUG),同时在2个96微孔酶联板中,以180fil/孔加入反应终止液(200mmol/L碳酸钠)。 Of 4-MUG), while two 96 microwell ELISA plate in order 180fil / well of Stop Solution (200mmol / L sodium carbonate). 在样品离心管中加入195^1预热的反应缓冲液和5plGUS粗酶液,混匀,立即取出20m!加入到1号板中,此为反应o时的样品(荧光测定时以此为空白),并严格记时。 When added to the sample tube 195 ^ 1 of reaction buffer and preheat 5plGUS a crude enzyme solution, mixed, immediately remove 20m! No. 1 was added to the plate, this sample (fluorescent when measured as a blank reaction o ), and when strictly to remember. 将反应板放入37'C水浴中进行酶反应,于20min时各取20nl反应液加入到2号板中,混匀,为酶反应20min时的样品。 The reaction plate into a water bath 37'C enzyme reaction, when depicting 20min at 20 NL No. 2 was added to the reaction plate, mix, samples for 20min, the enzymatic reaction. 5、 GUS酶活的荧光测定样品荧光测定以1号板为空白,在酶标仪上以激发光360nm,发射光460nm,狭缝25nm的条件下测定2号板各样品的荧光强度。 5, the fluorescent GUS activity measured in the samples No. 1 fluorescence plate blank on a microplate reader at excitation of 360 nm, emission 460nm light, the fluorescence intensity of each sample was measured under the conditions No. 2 plate slit 25nm measured. 根据得到的荧光强度值选择相应的标准曲线,由回归方程计算出2号板中4-MU含量。 The fluorescence intensity value obtained to select the appropriate calibration curve, calculate the number plate 2 by the content of 4-MU regression equation. 酶活力单位定义为:每分钟水解4-MUG生成lnmo1 4-MU的酶量为一个单位。 Enzyme activity unit is defined as: hydrolyzed per minute an amount of 4-MUG generating enzyme lnmo1 4-MU is a unit. Gt/S 基因的表达活性以每毫克总蛋白的酶活力表示,即4-MUmnol/mg/min。 Expression activity Gt / S gene is expressed in enzyme activity per mg of total protein, i.e., 4-MUmnol / mg / min. 【实施例14】GhSCFP启动子的功能分析经过对转GMCF尸/力.vGW基因烟草不同组织的GUS染色,结果发现在叶片、茎、花冠、雄蕊、柱头中均未检测到蓝色出现,而在烟草幼嫩种子及部分胎座表面观察到蓝色,特别在种子表面有很深的蓝色出现。 [Example 14] Analysis of promoter function GhSCFP after GUS staining of dead GMCF revolutions / force .vGW different tissues of transgenic tobacco found in leaves, stems, corolla, stamens, stigma not detected appear blue, whereas blue was observed in the tobacco seeds and young bead seat surface portion, particularly a deep blue color appears in the surface of the seed. 同时在开花前以及开花后12天种子表面观察到蓝色,12天后蓝色消失。 Also observed blue before flowering and 12 days after flowering seed surface, 12 days after the blue color disappeared. 结果表明:GhSCFPl.O 启动子在种子发育早期有活性,且只在种子中表达。 The results showed that: GhSCFPl.O promoter active in early seed development, and expressed only in seeds. 进一步对GUS染色的种子切片观察发现,在种皮的五层细胞中,只有最外层细胞被着色,即该启动子具有种皮表达特异性。 GUS staining is further observed seed chip, seed coat of five cells, the cells were colored only the outermost, i.e. the promoter having a seed coat-specific expression. 对转G/^CF尸M.vG^P基因烟草进行荧光检测,在叶片、茎、花冠、雄蕊、柱头同样未观察到GFP的绿色荧光,而在幼嫩的种子上可以观察到较强的绿色荧光,非转基因的野生烟草种子只被激发出红色荧光。 Counter-rotating G / ^ CF gene dead M.vG ^ P tobacco fluorescence detection in leaves, stems, corolla, stamens, stigma same green GFP fluorescence was not observed, and can be observed on a strong young seeds green fluorescence, non-genetically modified wild tobacco seeds of red fluorescence is only excited. 上述结果表明G/^C/V::Gt/S和G/lSCF尸::G^P融合基因,可以特异地在幼嫩烟草的种皮中表达。 The above results show that the C / V :: Gt / S and G / lSCF dead :: G ^ P fusion gene can be expressed specifically G / ^ in the tobacco seed coat young. 因此,在烟草中,该启动子具有严格的种皮特异性和发育时间特异性。 Therefore, in tobacco, the promoter has a strict seed coat-specific and time-specific development. 进一步检测转G/^CfP/.a':GWS基因烟草不同组织的GUS酶活性,结果可见转基因烟草的花冠、叶片、茎、萼片和子房壁中,GUS酶活性最高只有88.5nmol/mg/min,但是开花后5天的种子中GUS酶活性在500nmol/mg/min 以上。 Further detects revolutions G / ^ CfP / .a ': GUS activity in different tissues of transgenic tobacco GWS, the results can be seen transgenic tobacco corolla, leaves, stems, sepals and the ovary wall, only the GUS activity in the highest 88.5nmol / mg / min , but 5 days after flowering seeds GUS activity in 500nmol / mg / min or more. 结果同样说明该启动子具有种子优势表达特异性。 Results also shows that the promoter has the advantage of seed-specific expression.

上述是转基因烟草的检测结果,经过对转G/^CF尸/力.vGI7S基因棉花不同组织的GUS染色,结果发现在叶片、幼茎、花冠、雄蕊、柱头和开花前l 一2天的胚珠中均未观察到蓝色信号,而在开花后胚珠表面突起的纤维上观察到很深的蓝色,特别在开花当天的胚珠表面,观察到有蓝色的点,而这些蓝色的点就是突起的纤维,直到开花后8天,仍然显示蓝色。 The above-described transgenic detection result of tobacco, after the counter-rotating G CF dead / force GUS staining of different tissues / ^ .vGI7S transgenic cotton, and found that a two-day ovules in leaves, young stems, corolla, stamens, stigma and before flowering l It was not observed in the blue color signal, and the deep blue color was observed on the fiber surface of the protrusion after flowering ovule, ovule surface of the particular day of flowering, observed blue dots, which is a blue dot fiber projections until 8 days after anthesis, still blue. 对开花后3天且经过GUS染色的胚珠进行纵切,观察纵切面发现,被GUS染成蓝色的部分,就是棉花的纤维部分。 For 3 days after flowering and after GUS staining of slitting ovules observed found longitudinal section, part of the GUS stained blue, is cotton fiber portion. 进一步通过石蜡切片观察,在开花当天的胚珠表面,突起的纤维细胞被染成蓝色,而未突起的表皮细胞着色很浅。 By further observation paraffin sections, the ovule surface of anthesis, projections fibroblasts were stained blue, whereas the projections epidermal cells slightly stained. 上述结果表明: GhSCFPl.O启动子具有陆地棉纤维发育特异性。 These results indicate that: GhSCFPl.O promoter has specific upland cotton fiber development. 【实施例15】棉花GhSCFP基因启动子核心区域鉴定按照5'端缺失原则,对GhSCFPl.O启动子进行删除。 [Example 15] Cotton GhSCFP gene promoter region identified in accordance with the core 5 'deletion principle, GhSCFPl.O promoter deletion. 删除了该启动子的-634至-543、 -634至-336、 -634至-127、 -634至-61和-634至146共5个区域后,分别与GUS基因融合构建植物表达载体并转入烟草中。 Remove the promoter -634 to -543, -634 to -336, -634 to -127, -634 to -634 and -61 to 146 after a total of five regions, each with a fusion construct plant expression vectors and GUS gene into tobacco. 对开花后5天的转基因烟草种子进行GUS酶活性分析,检测结果表明当删除该启动子的-634 至-543区域(SEQIDN0.3 )后,转基因烟草种子中的GUS酶活性降低了约30倍,表明该区域含有一个增强子序列。 After transgenic tobacco seeds for 5 days after flowering GUS activity assays, test results show that when the -634 to -543 region (SEQIDN0.3) deleting the promoter, gene transfer GUS activity in tobacco seeds is reduced about 30-fold , indicating that the region contains an enhancer sequence. 其余4个区域分别删除后,种子中GUS酶活性变化不大。 After the remaining four regions were deleted, the GUS activity in seed not change. 转基因烟草不同组织的GUS酶活性测定发现:当删除GhSCFPl.O启动子-635至U-543区域后,茎和叶片中的GUS酶活高于种子、花冠,表明在该区域中还有控制启动子在种子中特异表达的元件。 GUS activity in different tissues of transgenic tobacco was measured found: After deleting GhSCFPl.O promoter region -635 to U-543, GUS activity in the stems and leaves than seeds, corolla, signifies that the control of a promoter region in expression of the specific element in seeds. 序列表.ST25SEQUENCE LISTING<110>西南大学<120>棉花纤维特异启动子及其应用<130> 06P103129<160> 3<170> Patent In version 3.1<210> 1<211〉 2436<212> DNA <213>陆地棉<220><221> misc—feature<222> (1)..(2436)<223>棉花种皮纤维特异基因<400> 1ctagcacccc ctaaa幼gct tgtgcttgga tatggctgaa幼cccagtaa aatggctgU 60tctcattcta gcaagctgcc tgtgtttcgc tttcgtcctc tcggaaagca acttattgat 120a幼ga柳cc tacatcgtcc a幼tgcacaa gtctgcaatg ccagcatcat tctcaagtcc 180tctggaatgg tactcatcga aattaaagtc ggt幼tgtcc gataccc卿 gtg幼ggtga 240agg卿tggt ga朋at柳a tcatctatag ctacc幼aat gcttttcatg gtgttgccgc 300tcagttaact g幼gaagaag ctg卿ggct aaaac幼g幼gatggtgtag ttgctatact 360tccagagacc aagtatg幼t tacacaccac幼ggagtcct atgttccttg g3tt3g助CC 420agaagaaagt actagcatct ggtctcaaaa acttgcagat catgatgtca tsgUggggt 柳acttgacact ggaatttggc cggaaagtgc t8gcttt肪t gatac鄉ga tgacaccggt 540tcctgctcac tggaaaggaa catgtgaaac tggacgaggc tttcaa幼gt atcactgcaa 600caggaaaatt gtgggtgcaa gagtgttcta SEQUENCE LISTING .ST25SEQUENCE LISTING <110> University of Southwest <120> cotton fiber-specific promoter and its application <130> 06P103129 <160> 3 <170> Patent In version 3.1 <210> 1 <211> 2436 <212> DNA <213 > Gossypium hirsutum <220> <221> misc-feature <222> (1) .. (2436) <223> fiber cotton seed coat-specific gene <400> 1ctagcacccc ctaaa immature juvenile gct tgtgcttgga tatggctgaa cccagtaa aatggctgU 60tctcattcta gcaagctgcc tgtgtttcgc tttcgtcctc tcggaaagca acttattgat 120a Young ga Liu cc tacatcgtcc a child tgcacaa gtctgcaatg ccagcatcat tctcaagtcc 180tctggaatgg tactcatcga aattaaagtc ggt Young tgtcc gataccc State gtg Young ggtga 240agg State tggt ga friends at willow a tcatctatag ctacc Young aat gcttttcatg gtgttgccgc 300tcagttaact g Young gaagaag ctg State ggct aaaac baby g baby gatggtgtag ttgctatact immature immature t tacacaccac 360tccagagacc aagtatg ggagtcct atgttccttg g3tt3g co CC 420agaagaaagt actagcatct ggtctcaaaa acttgcagat catgatgtca tsgUggggt Liu acttgacact ggaatttggc cggaaagtgc t8gcttt fatty t gatac Township ga tgacaccggt 540tcctgctcac tggaaaggaa catgtgaaac tggacgaggc tttcaa immature gt atcactgcaa 600caggaaaatt gtgggtgcaa gagtgttcta ccgg鹏tat gaagctgcca ctggga幼at 660ca&tgaga犯aacgaatata aatcacctcg agatc幼gat ggacatggta ctcacacagc 720agccactgtt gctggctctc cggtacgagg tgcaaatctt cttggttatg cctatggaac 780agct卿gga atggctcctg gtgc幼g幼t tgcagcttac卿gtttgct ggactggtgg 840gtgcttcagc tcggatattc tgtcggcggt tgatagagcg gtgggtgatg 柳tg幼tgt 卿 ccgg Peng tat gaagctgcca ctggga Young at 660ca & tgaga guilty aacgaatata aatcacctcg agatc David gat ggacatggta ctcacacagc 720agccactgtt gctggctctc cggtacgagg tgcaaatctt cttggttatg cctatggaac 780agct State gga atggctcctg gtgc baby g baby t tgcagcttac State gtttgct ggactggtgg 840gtgcttcagc tcggatattc tgtcggcggt tgatagagcg gtgggtgatg Liu Qing tg Young tgt

序列表.ST25gttgtcgata tctttgggtg gtggagcttc atcttactct cacgacagtt tggcgatagc 960aacttUgga gcaatggaga tgggtgtttt tgtctcttgc tcagctggca atggaggacc 1020agatcctgtc agc"cacta atgtaccacc atggatcact acagtcggtg ctagcaccat 1080ggatagagat tttccaggta atgttaagct agggagtggg agaaccatac ctggagtttc 1140actctacaaa gggcgaaggt tactgcaggc幼acaagcaa taccctcttg tttatatggg 1200tagtaactca agcagcccta atccaagttc attatgctta gagggaactt tggatccaca 1260tgttgtttct gggaaaattg tgatatgtga tcgaggaata agtcctagag tgcaaaaggg 1320tc幼gtagtg aaagatgctg g鄉agtagg aatgattttg acaaacactg cagc助atgg 1380gg柳柳tt gttgcagatt gtcacctact tccagcagtt gcagtgg柳agatggaagg 1440gaaagcaatx aaacattatg ccttaac幼a tggga幼cca accgcaactc tagccttttt 1500aggtaccaga ttgggtgtta ggccatcacc agtggtggca gcattttcat ctagaggacc 1560aaatttcctc acacttga犯ttctcaaacc tgatgtggtt gcgccagggg tg幼catcct 1620tgcagcctgg actggag幼t tgggtccgtx幼gtcttcca ac卿tcata ggagagtgag 1680attcaacata ttatcaggga cttcaatgtc atgccctxat gttagtggaa "ttgctgcctt 1740gat SEQUENCE LISTING .ST25gttgtcgata tctttgggtg gtggagcttc atcttactct cacgacagtt tggcgatagc 960aacttUgga gcaatggaga tgggtgtttt tgtctcttgc tcagctggca atggaggacc 1020agatcctgtc agc "cacta atgtaccacc atggatcact acagtcggtg ctagcaccat 1080ggatagagat tttccaggta atgttaagct agggagtggg agaaccatac ctggagtttc 1140actctacaaa gggcgaaggt tactgcaggc Immature acaagcaa taccctcttg tttatatggg 1200tagtaactca agcagcccta atccaagttc attatgctta gagggaactt tggatccaca 1260tgttgtttct gggaaaattg tgatatgtga tcgaggaata agtcctagag tgcaaaaggg 1320tc Immature gtagtg aaagatgctg g Township agtagg aatgattttg acaaacactg cagc co atgg 1380gg Liu Liu tt gttgcagatt gtcacctact ttctcaaacc tgatgtggtt gcgccagggg tg immature catcct 1620tgcagcctgg actggag immature t tgggtccgtx tccagcagtt gcagtgg Liu agatggaagg 1440gaaagcaatx aaacattatg ccttaac immature a tggga immature cca accgcaactc tagccttttt 1500aggtaccaga ttgggtgtta ggccatcacc agtggtggca gcattttcat ctagaggacc 1560aaatttcctc acacttga guilty Young gtcttcca ac State tcata ggagagtgag 1680attcaacata ttatcaggga cttcaatgtc atgccctxat gttagtggaa "ttgctgcctt 1740gat caaggcc agacacccag attggagtgc cgcagcagtt aaatctgctc taatgacaac 1800cgcttatgtt catgat肪ca tccataatcc actcc幼gac tcttccactg cagcagcttc 1860cactccctat gatcatggag ctggtcatat caaccctttg aaagctctag acccaggttt 1920gatctatgac atttcagccc aggattactt tgtattcctc tgcacac柳aattgactgc 1980aatgcagcta aaagctttta gcaagcattc caatatgtct tgccatcaca acacccttgc 2040cactccagga gatugaact acccagcaat ctcagttgtc ttcccag肪g atacagccat 2100ttcaactttg actctccata gaacagtcac aaatgttggt cctcctgctt cacattacca 2160tgttgtagtt tcaccatua aaggcgtcac catt幼agtt gagcccaaaa ctctaaattt 2220cactag卿a aaccagaaac tgtcttacaa gatcagtttc ac卿aaaat ccccacaaac 2280aatgcctgag tttggagggc tggtgtggaa鹏cggagtg cacaaagtaa g幼gccccat 2340tgctatcaca tggttaccac cattttaaac tagattttca gtcacctatg attccaaata 2400aacattatgt ttttatat站aaaa肪aaaa aaaaaa 2436<210> 2 <211> 1037 <212> DNA <213〉人工序列<220><22i> promoter <222> (1037) <223><220〉 caaggcc agacacccag attggagtgc cgcagcagtt aaatctgctc taatgacaac 1800cgcttatgtt catgat fat ca tccataatcc actcc Young gac tcttccactg cagcagcttc 1860cactccctat gatcatggag ctggtcatat caaccctttg aaagctctag acccaggttt 1920gatctatgac atttcagccc aggattactt tgtattcctc tgcacac Liu aattgactgc 1980aatgcagcta aaagctttta gcaagcattc caatatgtct tgccatcaca acacccttgc 2040cactccagga gatugaact acccagcaat ctcagttgtc ttcccag fat cacattacca 2160tgttgtagtt g atacagccat 2100ttcaactttg actctccata gaacagtcac aaatgttggt cctcctgctt tcaccatua aaggcgtcac catt immature agtt gagcccaaaa ctctaaattt 2220cactag Qing a aaccagaaac tgtcttacaa gatcagtttc ac State aaaat ccccacaaac 2280aatgcctgag tttggagggc tggtgtggaa Peng cggagtg cacaaagtaa g immature gccccat 2340tgctatcaca tggttaccac cattttaaac tagattttca gtcacctatg attccaaata 2400aacattatgt ttttatat station aaaa fatty aaaa aaaaaa 2436 <210> 2 <211> 1037 <212 > DNA <213> artificial sequence <220> <22i> promoter <222> (1037) <223> <220>

序列表.ST25<221><222>(1)..(91)<223><400> 2 tUsttacaacttttctcta ccaatcaaat ttaaaaaata gaaaaatgaaaatcgatgaattggatcacc acaatttagc ccaaag幼aa acacagtcaa cccctctcac agggt鄉幼120tgatUcgag gtatagatag acatagtaac gggcaacttt幼cteLttgct gcctcgattt 180gaggaaaata tcaaatccaa gacaaaaatt tcaattatac actatgccat accattettaa 240atatccccgt tcgcaatatc atcaccatta tttgaatttg cattgcaaca Ucgtxaccg 300ttagttatac catcaccatc acttaattac taaaat幼tt attggtttct caatatgaaa 360aagctcgagt gcattuctt ttgaatatca accgaaaaga aagaa幼aac taaagattU 420ggaagatgac ggggaaacca aaaaggaaat tttgggcatt tttaaaatga gaaagacgaa 480tgtaataacc cattutcu tcttactctg acaacgccac agatgcttta catgcatcat 540gtgatcgtgg gggaccccga aacttggcat acgg幼agca ccaacggcac agcattaaaa 600gaaattgtgt ataatgttaa aagaccatta attcagtctc atccaaccag gcttaaaagt 660cUcatgcct tttctcacct ctgatttcat ctaatgaaaa gcggacaagt tgaaggatca 720ctcgttgctt gtgtgagctt tcattattta ttattatgtt ttaggt幼cc ataggaagaa 780gccattaaca acagcatgaa aaacagctag tttctccgca aacaagataa acttttaaa SEQUENCE LISTING .ST25 <221> <222> (1) .. (91) <223> <400> 2 tUsttacaacttttctcta ccaatcaaat ttaaaaaata gaaaaatgaaaatcgatgaattggatcacc acaatttagc ccaaag Immature aa acacagtcaa cccctctcac agggt Township immature juvenile 120tgatUcgag gtatagatag acatagtaac gggcaacttt cteLttgct gcctcgattt 180gaggaaaata tcaaatccaa gacaaaaatt tcaattatac actatgccat accattettaa 240atatccccgt tcgcaatatc atcaccatta tttgaatttg cattgcaaca Ucgtxaccg 300ttagttatac catcaccatc acttaattac taaaat Young tt attggtttct caatatgaaa 360aagctcgagt gcattuctt ttgaatatca accgaaaaga aagaa Young aac taaagattU 420ggaagatgac ggggaaacca aaaaggaaat tttgggcatt tttaaaatga gaaagacgaa 480tgtaataacc cattutcu tcttactctg acaacgccac agatgcttta catgcatcat 540gtgatcgtgg gggaccccga aacttggcat acgg Young agca ccaacggcac agcattaaaa 600gaaattgtgt ataatgttaa aagaccatta attcagtctc atccaaccag gcttaaaagt 660cUcatgcct tttctcacct ctgatttcat ctaatgaaaa gcggacaagt tgaaggatca 720ctcgttgctt gtgtgagctt tcattattta ttattatgtt ttaggt child cc ataggaagaa 780gccattaaca acagcatgaa aaacagctag tttctccgca aacaagataa acttttaaa c 840tttttaccac tgcacccccc ccaaagacca gttttt幼ct ccacctacca agcattcaag 900aagcaccaac caacttaatt accagcttaa caagacagta caggtttctg ggatatttgt 960agtctctcaa ggacatcacc acctccactc accttcccat ttttctctag caccccctaa 1020aaagcttgtg cttggat 1037<210〉 3 <211> 91 <212> DNA <213>人工序列<220><221> enhancer <222> (91) <223><400> 3tttattacaa cttttctcta cc幼tcaaat ttaaaaaata g幼幼atg幼aatcgatgaa 60ttggatcacc acaatttagc cc幼agaa肪a 91 c 840tttttaccac tgcacccccc ccaaagacca gttttt Immature ct ccacctacca agcattcaag 900aagcaccaac caacttaatt accagcttaa caagacagta caggtttctg ggatatttgt 960agtctctcaa ggacatcacc acctccactc accttcccat ttttctctag caccccctaa 1020aaagcttgtg cttggat 1037 <210> 3 <211> 91 <212> DNA <213> Artificial Sequence <220> <221> enhancer <222 > (91) <223> <400> 3tttattacaa cttttctcta cc tcaaat ttaaaaaata g immature young young immature aatcgatgaa 60ttggatcacc acaatttagc cc atg immature fatty agaa a 91

Claims (10)

  1. 1. 一种增强子,其特征在于其为如SEQ ID NO.3所示的核苷酸序列。 An enhancer, a nucleotide sequence characterized in that it is as shown in SEQ ID NO.3.
  2. 2. —种棉花纤维特异启动子,其特征在于其为如SEQIDN().2所示的核苷酸序列。 2. - cotton fiber specific promoter, characterized in that it is such SEQIDN () the nucleotide sequence shown in Figure 2.
  3. 3. —种含有权利要求2所述的棉花纤维特异启动子的植物表达载体。 3. - cotton fiber-specific promoter of plant species according to claim 2 comprising the expression vector.
  4. 4. 根据权利要求3所述的植物表达载体,其特征在于其具有如图4所示的结构。 4. The plant expression vector according to claim 3, characterized in having a structure as shown in FIG. 4 thereof.
  5. 5. 根据权利要求3所述的植物表达载体,其特征在于其具有如图5所示的结构。 5. The plant expression vector according to claim 3, characterized in having a structure as shown in FIG. 5 thereof.
  6. 6. —种转化体,包含权利要求2所述的棉花纤维特异启动子及宿主。 6. - transformant, comprising the 2 cotton fiber-specific promoter and the host claims.
  7. 7. 根据权利要求6所述的转化体,其特征在于,所述宿主为根癌农杆菌。 7. The transformant according to claim 6, characterized in that the host is Agrobacterium tumefaciens.
  8. 8. 权利要求2所述的棉花纤维特异启动子在制备转基因植物中的应用。 Application of cotton fiber specific promoter according to claim 2 in the preparation of transgenic plants.
  9. 9. 根据权利要求8所述的应用,其特征在于所述转基因植物为烟草或陆地棉。 9. The use according to claim 8, wherein said transgenic tobacco plant or a Gossypium hirsutum.
  10. 10. —种含有权利要求2所述的棉花纤维特异启动子的转基因植物的制备方法,包括下述步骤:(1 )将棉花纤维特异的启动子序列可操作地插入表达载体中,构建植物表达载体;(2) 用所述植物表达载体转化宿主,获得转化体;(3) 将所述转化体转化植物,获得转基因植物。 10. - Preparation of cotton fiber specific promoter comprising a transgenic plant and seed as claimed in claim 2, comprising the steps of: (1) A cotton fiber-specific promoter sequence operatively inserted into an expression vector, a plant expression a carrier; (2) a host transformed with a vector, transformants were obtained with the plant expression; (3) the transformant transformed plant to obtain transgenic plants.
CN 200610156823 2006-11-10 2006-11-10 Cotton fiber specific promoter and its use CN100471954C (en)

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CN101633934B (en) 2008-07-25 2012-01-04 西南大学 Plant expression vector for expressing auxin synthetic related genes and application in improvement of cotton fiber traits
CN102485893B (en) * 2010-12-06 2014-01-22 华中农业大学 Two preferentially expressed strong promoters during cotton fiber development initiation and their application
WO2012093032A1 (en) * 2011-01-04 2012-07-12 Bayer Cropscience N.V. Fiber selective promoters
CN102367439B (en) * 2011-10-31 2013-06-26 中国农业大学 Plant stomata specific promoter and application thereof
CN102620966B (en) * 2012-03-31 2014-05-07 华中科技大学 Biological sample preparation method applicable to ultra-thin section and fluorescence imaging
CN103981217B (en) * 2014-06-05 2016-04-27 西南大学 Expression vectors iaaL auxin metabolism genes and their use
CN104651367B (en) * 2015-02-11 2017-09-08 上海交通大学 A variety of skin and fibrous tissue-specific expression promoter sfs its application

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