WO2010009601A1 - 表达生长素合成相关基因的植物表达载体及其在棉花纤维性状改良的应用 - Google Patents

表达生长素合成相关基因的植物表达载体及其在棉花纤维性状改良的应用 Download PDF

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WO2010009601A1
WO2010009601A1 PCT/CN2009/000095 CN2009000095W WO2010009601A1 WO 2010009601 A1 WO2010009601 A1 WO 2010009601A1 CN 2009000095 W CN2009000095 W CN 2009000095W WO 2010009601 A1 WO2010009601 A1 WO 2010009601A1
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cotton
gene
plant
fiber
iaam
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PCT/CN2009/000095
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English (en)
French (fr)
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裴炎
侯磊
李德谋
宋水清
李先碧
罗明
肖月华
郑雪莲
曾其伟
张觅
邱坤
罗凤涛
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西南大学
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Priority to US13/055,586 priority Critical patent/US20110145947A1/en
Publication of WO2010009601A1 publication Critical patent/WO2010009601A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8291—Hormone-influenced development
    • C12N15/8294—Auxins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a plant expression vector and use thereof, and more particularly to a plant expression vector for expressing an auxin synthesis-related gene and an application thereof for improving cotton fiber properties.
  • Cotton is the world's most important natural fiber crop and the most important cash crop. China is the world's largest textile producer and consumer, and the cotton industry plays a pivotal role in the national economy. In recent years, with the improvement of people's living standards and the innovation of textile technology, the requirements for the quality of cotton fiber have also increased accordingly; especially in recent years, the technological revolution of replacing spinning with air-jet spinning requires longer and stronger. Finer and more neat fibers. However, at present, most cotton varieties are low in quality, single in length, low in fiber strength, relatively thick in fiber, and lacking more than 60 varieties of high-grade cotton yarns, which are far from meeting the needs of the market. This directly led to the lack of competitiveness of raw cotton in the international market.
  • the yield and quality traits of cotton are quantitative traits controlled by multiple genes, and there is a genetic negative correlation between yield and quality traits.
  • Cotton cultivars are mainly upland cotton, and the superior fiber quality genes are mainly derived from diploid Thurber cotton (fiber strength), abnormal cotton (fiber strength and fineness), and tetraploid sea island cotton (fiber strength and fineness). Degree) and so on.
  • the utilization of these excellent trait genes has been limited in conventional breeding. It is difficult to greatly increase cotton yield by relying on existing cotton genetic germplasm resources and conventional breeding methods, and it is difficult to meet the rapid development of textile process revolution on fiber quality. Claim.
  • Breeding with genetic engineering technology can break the genetic barrier between species, achieve the targeted transfer of good target genes, and have the advantages of easy to stabilize offspring and short breeding cycle, which provides a new way for the improvement of cotton fiber yield and quality.
  • genes that are directly related to cotton fiber formation, yield and quality have not yet been obtained, making the use of genetic engineering to improve cotton fibers lacking effective target genes. Little is known about the molecular mechanisms involved in the development, development and quality of cotton fibers. These have greatly hindered the process of yield and quality improvement of cotton fiber.
  • Cotton fiber is a single cell developed from the outer leaves of cotton ovule by epidermal cells through differentiation, elongation (primary wall synthesis), thickening (secondary wall synthesis) and mature dehydration.
  • Cotton fiber cells have a final length of 20-30 mm, a height of 35-40 mm, and a length to diameter ratio of 1,000-3,000. Such a high aspect ratio is the result of the intense elongation of the fiber cells, which must be promoted. Participation in plant growth and elongation of plant hormones. The initiation and elongation of fibroblasts are closely related to auxin (Auxin, such as indole-3-acetic acid, IAA).
  • auxin auxin, such as indole-3-acetic acid, IAA
  • auxin is applied to flowers or buds one by one, the workload is very large, the labor cost is high, and large-scale promotion is difficult to achieve. Moreover, the large use of auxin increases both the production cost and environmental pollution.
  • This strategy has at least the following advantages: 1) High efficiency and low cost, because once the auxin biosynthetic enzyme gene is introduced into the plant, no additional application of auxin or other treatment is required.
  • auxin is diffused into the cell, while endogenous hormones are produced from the cell. Therefore, the use of transgenic endogenous control of auxin is often better than exogenous application; 2) the negative impact on crops is small, because the concentration of auxin is very low, the concentration is too high or too low will bring about plant development Bad effects.
  • Endogenous expression of the auxin synthase gene in the case of appropriate expression levels and expression sites, can be achieved only for specific target organs (tissue) without affecting the normal development of other parts of the plant; 3) Endogenously regulated auxin synthase genes have less environmental pollution and less harm to human health than exogenously applied auxin and synthetically produced regulatory substances (Li Y et al., 2004, Transgenics of plant hormones and their potential application). In: horticultural crops. In: Genetically Modified Crops: their development, uses, and risks. New York: Food Products Press, 101-112).
  • auxin synthase genes to increase yield and improve quality has not been successful in cotton breeding.
  • John ME placed two enzyme genes, iaaM and iaaH, involved in auxin IAA biosynthesis under the fiber-specific promoter E6, and transferred to the upland cotton DP50 by Agrobacterium.
  • the results showed that IAA content was found in most transgenic plants. There was a 2-8 fold increase in the line, however the fiber length, fineness and strength were not significantly different from the control wild type (Basra AS et al., 1999, Cotton Fiber, New York: Food Products Press, 271-292). .
  • no endogenous expression of hormones has been observed.
  • hormone biosynthetic enzyme genes to improve the quality of cotton fibers is also common. Summary of the invention
  • the technical problem to be solved by the present invention is to provide a plant expression vector for expressing an auxin synthesis related gene, and the plant expression vector is applied to the improvement of cotton fiber traits to solve the existing endogenous expression auxin biosynthesis
  • the method of enzyme gene is difficult to improve the yield and quality of cotton fiber.
  • the invention further provides the use of the plant expression vector of the invention for the improvement of cotton fiber traits.
  • the invention also provides a method of preparing a transgenic plant based on the plant expression vector of the invention.
  • the plant expression vector of the present invention contains at least a nucleotide expressing an auxin synthesis-related gene consisting of an auxin synthase gene and a plant seed coat-specific promoter, which is encoded by The auxin synthase gene is operably linked to a gene encoding a plant seed coat-specific promoter.
  • the preferred auxin synthase gene is the soil tumour TMS (tumour morphology shooty) gene (often called the iaaM gene).
  • the preferred plant seed coat specific promoter is the FBP7 (Floral Binding Protein 7) gene promoter.
  • a preferred nucleotide encoding a gene for expressing a auxin synthesis is a nucleotide having the sequence of SEQ ID NO. After obtaining a nucleotide expressing a gene related to auxin synthesis, the nucleotide is inserted into an expression vector to construct a plant expression vector for expressing an auxin synthesis-related gene of the present invention, and the preferred plant expression vector has a vector structure as shown in FIG. . Since a plant expression vector is constructed, a portion of the variable non-coding sequence exists between the FBP7 promoter and the iaaM gene based on the convenience of gene manipulation.
  • a transformant which is obtained by transfecting a plant expression vector of the present invention into a host, and the transformant can be used for transforming a plant to obtain a transgenic plant.
  • the use of the plant expression vector of the present invention for the improvement of cotton fiber traits By expressing the auxin synthesis-related genes constructed in the present invention in plants, the expression of auxin synthase is regulated to achieve the purpose of improving cotton fiber traits.
  • a method for producing a transgenic plant wherein the transformant of the present invention is transformed into a plant (cotton) to obtain a transgenic plant.
  • the method for improving cotton fiber traits includes the following steps: 1) obtaining seed coat and fiber Dimensional expression of the promoter; 2) obtaining auxin synthesis related genes; 3) merging the specific promoter cloned in step 1) with the auxin synthesis related gene isolated and cloned in step 2) to construct specific expression growth a plant expression vector for synthesizing a gene; 4) integrating the plant expression vector for the specific expression of the auxin synthesis gene obtained in the step 3) into the cotton genome; 5) further cultivating the cotton obtained by the step 4) And obtain transgenic cotton plants.
  • the specific expression promoter described in the step 1 may be a natural promoter isolated from an animal, a plant or a microorganism, or may be a artificially engineered or designed synthetic promoter.
  • the auxin synthesis-related gene described in the step 2 may be a natural gene isolated and cloned from an animal, a plant or a microorganism, or may be an artificially engineered or designed gene.
  • the method in which the specific promoter described in the step 3) is fused with the auxin synthesis-related gene to construct an expression vector for specifically expressing the auxin synthesis-related gene is a conventional method in the art, and the vector used may be used in the field of plant transgenics. Conventional carrier.
  • the method for integrating the expression vector into the genome of cotton described in the step 4) is a commonly used plant transgenic method such as Agrobacterium tumefaciens-mediated or gene gun method.
  • the above specific promoter is an ovary-specific promoter or a seed coat-specific promoter or a seed coat and a fiber-specific promoter. More preferably, the seed coat and the fiber-specific promoter are the FBP7 (Floral Binding Protein 7) gene promoter, the ovary-specific promoter is the AGL5 (Agamous Like protein 5) gene promoter, and the fiber-specific promoter is the E6 gene. Promoter.
  • FBP7 Floral Binding Protein 7
  • the plant hormone synthesis-related gene is a gene related to auxin synthesis. More preferably, the phytohormone synthesis-related gene of the present invention is a soil tumor Agrobacterium tumefaciens tms (tumor morphology shooty) gene (commonly referred to as iaaM gene).
  • the "cotton fiber trait” referred to in the present invention means the quantity and quality traits of cotton fibers, including the number, length, fineness, strength, uniformity, and the like of the fibers.
  • transgenic cotton refers to cotton which has been genetically modified by biomimetic means to transfer the genes of other organisms into cotton, thereby transforming the genetic material of the modified cotton.
  • the genes used for engineering can be derived from plants, animals, and microorganisms or synthetically engineered.
  • clothing as used in the present invention means the ratio of the weight of the fiber on the seed cotton to the weight of the seed cotton, expressed as a percentage. That is, the weight of the fiber accounts for the total weight of the whole seed and fiber.
  • fiber strength refers to the maximum load that a bundle of fibers can withstand when it is about to break, expressed in centigrams per tex (cN/tex), and the tex is a gram of 1000 meters of fiber. Multiplicity.
  • the appropriate promoter can be selected to control the expression of genes related to plant hormone synthesis in a specific part of cotton, at a specific time of development, with appropriate intensity, and accurately regulate the concentration, duration and action site of hormones in plants. Effectively affect the growth and development of cotton fiber and achieve the desired results.
  • the variety of promoters is extremely large, and it is impossible to predict which promoter will be effective against cotton fibers after attaching auxin-related genes.
  • the seed-specific promoter FBP7 Floral Binding Protein 7 gene promoter (derived from petunia)
  • ovary-specific promoter was creatively used.
  • AGL5 (Agamous Like protein 5) gene promoter (derived from Arabidopsis thaliana) and fiber-specific promoter E6 gene promoter (derived from upland cotton) to construct a new gene expression vector for major elements, and established a complete set A suitable method for improving cotton fiber traits.
  • the manner in which the promoter and the target gene are fused into a new gene can be carried out in a manner commonly used in the art, and the fused new gene can be transferred into the field.
  • the vector is constructed into an expression vector and then transferred into cotton.
  • the above expression vector can be constructed as a monovalent vector containing a single gene, or a vector of a bivalent or trivalent type containing a plurality of genes can be constructed.
  • a single gene can be expressed in only one site, or multiple genes can be expressed in multiple sites and at multiple developmental stages, and the method of the present invention has provided a technical solution.
  • the method for improving cotton fiber traits of the present invention is to regulate the expression of auxin synthase by specifically expressing auxin synthesis related genes in the seed coat and fiber of cotton, and to regulate the content of corresponding hormones in specific tissues and organs of cotton by endogenous regulation.
  • the test results show that the number of cotton fibers improved by the method for improving cotton fiber traits of the present invention is obviously increased, the yield is obviously increased, the quality of cotton fibers is obviously improved, and the number of seeds is increased, and the number of clothes is significantly increased.
  • the method of the invention is simple and convenient, and has remarkable effects, and can bring high-yield, high-quality fiber raw materials to the textile industry, and generate huge economic benefits.
  • Figure 1 Flow chart of construction of auxin synthesis gene expression vector under the control of specific promoters (including FBP7, AGL5 and E6)
  • Km kanamycin resistance gene
  • Amp ampicillin resistance gene
  • ⁇ neomycin phosphotransferase gene
  • GUS ⁇ -glucosidase gene
  • 35S plant constitutively derived from cauliflower mosaic virus Promoter
  • Pnos opine synthase gene promoter
  • nos opine synthase gene terminator
  • LB T-DNA left border
  • RB T-DNA right border.
  • the backbone vector used to construct the plant expression vector is a p5 vector engineered on the basis of pBI121, which is under the control of the CaMV 35S promoter.
  • GUS gene which facilitates GUS staining of transformants during plant genetic transformation.
  • Figure 2 Structure of plant expression vector containing the specific promoter FBP7 of the present invention
  • P 5-FBP7 The genomic DNA of 11 strains of iaaM transgenic cotton was digested with Xbal, and then subjected to southern hybridization with the iaaM gene fragment. Different sizes of hybrid fragments were obtained in different strains. There was no hybridization signal in the wild type control. 1, 2, 6, ... 20 are different transgenic lines of p5-FBP7daaM; WT is a wild type control.
  • P 5-E6 Genomic DNA of 6 strains of iaaM transgenic cotton was digested with Xbal, and then subjected to southern hybridization with the iaaM gene fragment. Hybrid fragments of different sizes were obtained in different strains. There was no hybridization signal in the wild type control. 1, 2, 5, 8, 10, and 11 are different transgenic lines of p5-E6-iaaM (IE1- ⁇ 1-2, ⁇ 1-5, IEl-8, IEl-10, and IEl-11); WT is wild Type control.
  • the genomic DNA of 8 strains of C, P 5-AGL5-iaaM transgenic cotton was digested with Xbal, and then subjected to southern hybridization with the iaaM gene fragment.
  • 3, 4, 6, 7, 10, 11, 12, and 14 are different transgenic lines of p5-AGL5-iaaM (IG1-3, IG1-4, IG1-6, IG1-7, IG1-10, IG1- 11. IG1-12 and IG1-14); WT is a wild type control, and there is no hybridization signal in the wild type control.
  • FIG. 4 RT-PCR analysis of the specifically expressed auxin synthase gene iaaM in transgenic FBP7:iaaM cotton
  • FBP7 Expression analysis of 11 strains of iaaM transgenic cotton and wild type, and the results were detected in 9 # , 14 # , 20 # three strains. Among them, 9# has the strongest expression, 14 # centered, 20 # weakest; 1, 2, 6, 7, 9, 10, 11, 14, 15, 18, 20: different strain numbers.
  • ⁇ In the different developmental stages of FBP7:iaaM transgenic cotton 9 # ovule and fiber, the expression gradually decreased with time, and the expression of M was not detected after 15 days; -2: -2dpa, two days before flowering Materials; 0, 1, 2, 3, 5, 10, 15, 20, 30: Odap, ldpa ... 30dpa, different days of material after flowering.
  • FIG. 5 RT-PCR analysis of the specifically expressed auxin synthase gene iaaM in transgenic E6:iaaM cotton
  • E6 11 different transgenic lines of iaaM transgenic cotton were analyzed for expression of iaaM with wild type. The results showed that there were higher expression levels of 1 M, 1 #, 2 # , 8 # , 10 # , 14 in 11 # lines. The expression of the ⁇ ⁇ ⁇ gene was also detected in the # , 17# strain. 1, 2, 5, 8, 10, 11, 13, 14, 17, 19, 21: Different transgenic lines number. Top: Results of gene RT-PCR, amplification products of iaaM gene-specific primers (sequences 9 and 10) were amplified for 35 cycles.
  • FIG. 6 RT-PCR analysis of the specifically expressed auxin synthase gene iaaM in transgenic Agl5:iaaM cotton
  • Agl5 11 lines of iaaM transgenic cotton and wild type were analyzed for expression of iaaM. The results showed that higher levels of M gene expression were detected in 6 # , 7 # , 10 # three lines. Followinged by 15 # and 23 # transgenic lines, 2 # , 3 # , 4 # , 16 # , 17 # and 21# also have the expression of M gene. 2, 3, 4, 6, 7, 10, 15, 16, 17, 21, 23: Different strain numbers. Top: The results of the ⁇ gene RT-PCR, amplification products of the iaaM gene-specific primers (sequences 9 and 10), were amplified for 35 cycles.
  • FIG. 8 Comparison of FBP7/E6/AGL5: IAAM transgenic cotton with control ovule and free IAA in fiber
  • the test sample is a mixed extract of ovule and fiber. Control, isolated negative plants served as controls. The samples were repeatedly measured at all time points and averaged for mapping analysis.
  • FIG. 9 Scanning electron microscopy of FBP7: iaaM transgenic cotton and wild-type cotton ovules
  • A the surface of the wild-type ovule on the day of flowering, showing the initial fiber, magnified 70-fold;
  • B the specific promoter FBP7-controlled iaaM transgenic cotton ovule surface, showing the starting fiber, magnified 70 times;
  • C Figure A further Magnification, showing the shape and number of starting fibers, magnified 500 times;
  • D further enlargement of Figure B, showing the shape and number of starting fibers, magnified 500 times; the initial fiber distribution in D is significantly denser than C, and the number is more many.
  • C D
  • Figure 10 Scanning electron microscopy of E6: iaaM transgenic cotton and wild-type cotton ovules A, the surface of the wild-type ovule on the day of flowering, showing the initial fiber, magnified 80 times; B, further enlargement of Figure A, showing the shape and number of the starting fiber, magnified 500 times; (, specific promoter E6 controlled transgene The surface of the cotton ovule shows the starting fiber, magnified 80 times; D, Figure C is further enlarged, showing the initial fiber shape and number, magnified 500 times.
  • Figure 11 Scanning electron microscopy of AGL5:iaaM transgenic cotton versus wild-type cotton ovules.
  • Figure A the surface of the wild-type ovule on the day of flowering, showing the starting fiber, magnified 80 times;
  • B further enlargement of Figure A, showing the starting fiber The shape and number, magnified 500 times;
  • C specific promoter AGL5 controlled ⁇ ⁇ transgenic cotton ovule surface, showing the starting fiber, magnified 80 times;
  • D Figure C is further enlarged, showing the starting fiber shape and number, Zoom in 500 times.
  • Figure 12 Microscopic observation of ovule and fibrous tissue sections of FBP7:iaaM transgenic cotton
  • FBP7 iaaM transgenic cotton was evident in the protoplasts of the ovule surface of the ovule at 0 dpa; the growth of the fiber primordia on the surface of the ovule of the transgenic cotton at 1 dpa was also significantly higher than that of the control; the fiber had significantly increased on the surface of the transgenic ovule at 2 dpa. And more in number than the control.
  • the number of fibers on the ovules of the transgenic plants after two days of flowering increased significantly compared to the control.
  • the number of ovule surface fibers was about 6000
  • the number of controls was about 5940
  • the two lines of ⁇ and 14 # were significantly increased compared with wild type. Among them, 9 # increased by 11.3%, and 14 # increased by 15.1%.
  • A the seed of the transgenic cotton is significantly smaller than the control; the short pile is less.
  • B Desulfurization analysis of sulfuric acid The content of short velvet showed that the linters of transgenic cotton were reduced by about 10% compared with the control.
  • Control isolated negative plants served as controls; FBP7: iaaM, FBP7: iaaM transgenic cotton. detailed description
  • reagents in the examples of the present invention are not commercially available, and the materials are not specifically described in the "Molecular Cloning Experiment Guide” (Sambrook and Russell, 2001).
  • chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) were extracted once (10,000 r/min, centrifuged at 4 ° C for 5 min), and the supernatant was taken and ethanol precipitated. The precipitate was rinsed with 75% ethanol, air dried, dissolved in 200 LTE, and stored at -20 °C.
  • the amplification procedure is: 94 ° C, 5 min ; 94 ° C, 30 sec, 56 °
  • Fragments having a length of 2.0 kb or less were recovered by centrifugation. Under a UV lamp, cut the agarose gel block containing the desired fragment with a clean blade. Use a No. 5 needle to drill a small hole in the bottom of a 0.5 mL centrifuge tube and fill in a suitable size of glass wool.
  • the agarose block containing the target fragment was placed in a 0.5 mL centrifuge tube filled with glass wool, and the liquid N 2 was frozen.
  • the frozen 0.5 mL centrifuge tube was placed in a 1.5 mL centrifuge tube and centrifuged at 13,000 r/min for 3 min. .
  • the recovered fragments were linked to the pUCm-T (Shanghai Shenggong) vector as follows:
  • the molar ratio of the vector DNA fragment to the exogenous ligation product DNA fragment was 1:3, and the ligation was carried out at 16 ° C for 12 h.
  • the ligation product was then transformed into E. coli DH5a.
  • an E6-specific primer (SEQ ID N0.4, 5) was designed, and a 1.4 kb fragment was amplified from the upland cotton genome. After cloning the amplified DNA fragment into pUCm-T (product of Shanghai Shenggong Bioengineering Co., Ltd.), the sequencing analysis showed that it was an E6 fiber-specific promoter of Upland cotton, see SEQ ID N0.6, and the cloning vector was pUC-E6.
  • AGL5 specific primer SEQ ID N0.7, 8 was designed and a 2 kb fragment was amplified from the Arabidopsis genome. After cloning the amplified DNA fragment into pUCm-T (product of Shanghai Shenggong Bioengineering Co., Ltd.), the sequence analysis indicated that it was an AGL5-specific promoter of Arabidopsis thaliana, see SEQ ID N0.9, and the cloning vector was pUC-AGL5 o.
  • Agrobacterium tumefaciens iaaM gene sequence (GenBank accession number: K02554) (see SEQ ID NO. 10 and 11), and amplified by PCR from Agrobacterium tumefaciens Ti plasmid T-DNA.
  • the cloned vector was cloned into pUCm-T (product of Shanghai Shenggong Bioengineering Co., Ltd.), and sequenced and analyzed to obtain SEQ ID NO. 12, and the cloning vector was pUC-iaaM.
  • the carrier construction process is shown in Figure 1.
  • the starting plasmid vectors were derived from the above 1 and 2, respectively, and p5 was modified on the basis of pBI121 (Clontech) using the method of Molecular Cloning Assay (Sambrook and Russell, 2001). All restriction enzymes were purchased from Roche and operated according to the instructions for use.
  • the constructed plant expression vector containing the specific promoter FBP7 is shown in Fig. 2, which includes a nucleotide (SEQ ID NO. 13) expressing a gene related to auxin synthesis and various elements required for expression screening [Example 3] transformant And preparation of transgenic plants
  • the constructed plant expression vector plasmid was introduced into Agrobacterium LBA4404 by electroporation.
  • the above vector was introduced into Agrobacterium LBA4404 by electroporation using the Bio-RAD MicroPulser User's Manual.
  • a vector that specifically expresses genes related to plant hormone synthesis is integrated into the cotton genome
  • MSB MS inorganic salt + B5 organic
  • Seed germination medium l/2MSB+30g/L glucose +7.5 g/L agar, pH 6.5
  • Liquid suspension medium MSB+1.9 g/L KN0 3 +30g/L glucose, pH 5.8
  • Somatic embryo maturation medium MSB+1.9 g/L KN0 3 +30g/L glucose+2.0g/L Gelrite, pH6.5
  • Seedling culture medium SH medium +0.05% activated carbon +20g/L sucrose, pH6.5
  • the above expression vector is introduced into cotton by a method of Agrobacterium-mediated embryogenic callus.
  • the specific method is as follows:
  • Agrobacterium strain containing the above expression vector was picked, and YEB solid medium (0.5% sucrose (W/V) containing 50 mg/L kanamycin and 125 mg/L streptomycin , 0.1% bacterial extract (W/V), 1% bacterial tryptone (W/V), 0.05% MgSO 4 .7H 2 O
  • the germinated mature embryos were transferred to somatic embryo elongation induction medium and cultured under 28 ⁇ dark conditions for 2 weeks to induce somatic embryo elongation and germination.
  • Large germination embryos (>0.5 cm) were transferred to SH medium to form seedlings, and cultured at 28 ° C for 16 h. When the seedlings grow to a height of about 2 cm, the seedlings are harvested and grafted onto cotton seedlings with 3-4 true leaves.
  • One-strand cDNA of various RNAs was synthesized using a cDNA-strand synthesis kit (MBI), and the procedures were carried out according to the kit instructions.
  • the 1 - strand product was used as a template for PCR amplification.
  • the 25 L system included lxPCR buffer, 0.2 mmol/L dNTPs, 1.5 mmol/LMgCl 2 , and upstream and downstream primers of iaaM gene (SEQ ID N0.14 and SEQ ID N0. 15) Each 0.2 ⁇ /L, 1 U Taq DNA polymerase (Promega).
  • the temperature cycle parameters were pre-denaturation at 94 ° C for 5 min; 94 ° C, 30 sec, 56 ° C, 30 sec, 72 ° C, lmin, 30 cycles; 72 ° C extension for 5 min.
  • the histone Histone3 gene was used as an internal standard. Primer sequences 16 and 17 of histone Histone 3 are referenced (Zhu YQ et al., 2003, Plant Physiol., 133, 580-88). The results of RT-PCR are shown in Figures 4-6.
  • Example 5 Detection of the introduced gene in cotton fiber by real-time PCR method
  • the total RA of ovule and fiber of the control and transgenic cotton 10 days after flowering was extracted, and the cDNA-strand was synthesized by reverse transcription. This was used as a template for quantitative real-time PCR amplification.
  • the specific procedure is as follows: One-strand cDNA of various RNAs is synthesized by DNA-strand synthesis kit (MBI), and the operation is carried out according to the kit instructions.
  • PCR was performed on a quantitative real-time PCR machine, including 12.5 L MIX buffer (Bio-Rad, including PCR buffer, DNA polymerase, dNTPs, and MgCl 2 ) in a 25 L reaction system.
  • the upstream and downstream primers were 0.2 ⁇ . /L and 1 - chain products.
  • the temperature cycle parameter was pre-denaturation at 94 ° C for 3 mins; 94 ° C, 30 sec, 56 ° C, 30 sec, 72 ° C, 0.5 min, the preset number of cycles was 35.
  • the cotton Histone3 gene was used as an internal standard, and the upstream and downstream primers were SEQ ID NOS. 16 and 17.
  • the upstream and downstream primers for amplifying the ⁇ gene are SEQ ID N0.14 and sequence 15. Amplify once in the same temperature control program with the same primers and template before running the quantitative real-time PCR, and check and ensure that the amplified product is a single band by agarose electrophoresis. The result is shown in Figure 7.
  • Cotton bolls were selected two days after flowering. Each plant was randomly selected from 3 individual plants, and each plant took 5 bells. Two seeds per bell were used to count the fibers on the surface of the ovule. Each sample was counted 10 times, and the average was calculated as the number of early fibers per plant. The specific method is as follows:
  • the ovules at different stages of flowering were selected for paraffin sectioning to observe the effect of specific expression of genes in ovules on the growth and development of cotton fibers from the tissue level.
  • the apex was removed immediately after the plant was removed, and divided into small pieces or small pieces of about 0.5 cm, which were sliced by conventional tissue sectioning and sliced on an OLYMPUS BX41TF microscope. Observe and take pictures. As a result, as shown in Fig.
  • the protrusion of the fibrous original body was clearly observed on the surface of the ovule of the transgenic cotton Odpa; while the surface of the control was relatively smooth, the protrusion was not obvious, and the number was small.
  • On the surface of the ovule of ldpa prominent fibrous cells were observed in both the control and the transgenic cotton.
  • the number of fibroblasts on the surface of the transgenic cotton ovule was significantly higher than that of the control.
  • the 2dpa ovule surface observation also showed that the fiber cells increased significantly, and the fiber of the transgenic cotton was significantly longer than the control.
  • Hormone extraction and purification Take cotton ovule/fiber, freeze it in liquid nitrogen, and grind it into powder. Accurately weigh the sample powder (differentially weighed) about 0.5g, add 7ml of extract (80% pre-cooled methanol), and 10ngl3C6-IAA (dissolved in 100% methanol, final concentration 500ng ⁇ L) internal standard. Mix well, seal in a glass tube, and immerse in the dark at -20 °C overnight. The leached sample was transferred to a centrifuge tube, 10,000 g, 4 ° C, and centrifuged for 20 min.
  • the supernatant was aspirated into a distillation flask, and a drop of ammonia water was added to make the pH of the solution alkaline, and concentrated to dryness by rotary evaporation under reduced pressure in a 100 mL distillation flask at 40 °C.
  • the sample was reconstituted with 5 mL of 0.1 M HAC. After the dissolution, the pH was again checked and kept constant with the complex solution (0.1 M HAC).
  • the detection instrument used is Shimadzu Corporation (shimazu) high pressure liquid chromatography- Mass spectrometer (LCMS-2010A). The dried sample was reconstituted with 10% methanol, loaded through a micro-sampler, 10% methanol, 5 min, 85% methanol, 30 min, 100% methanol, 31 min, 100% methanol, 39 min; 10% methanol, 40 min; % methanol, 60 min.
  • the chromatographic peak retention time, peak area, and mass spectrum of the internal standard [ 13 C 6 ]IAA were recorded.
  • the IAA in each sample was identified based on retention time and mass spectrum, and the peak area of the internal standard and endogenous IAA was calculated. Repeat three times to take the average of the peak areas.
  • the IAA content in the sample was calculated by the internal standard method. The results are shown in Figure 8.
  • the transgenic cotton plants were planted in the genetically modified base of Southwest University, and were designed by randomized blocks. Each plot was set up with 3 plots and routine field management. The mature cotton is harvested in the sub-district, and the seed cotton yield is accurately weighed. In the seed cotton harvested in each plot, 100 seeds were selected, and the weight of the 100-seed cotton was accurately weighed. The total amount of the fibers and the total amount of the seeds were weighed by hand, and the size of the clothing was calculated. Finally, the average of the three plots was taken as the final result (Table 2).
  • the seed cotton yield and the clothing score of the non-transgenic control were 3.37Kg and 42.19%, respectively, while the clothing scores of the FBP7-iaaM transgenic lines were Above 43%, the highest reached 49.67% was significantly greater than the control, while the E6-iaaM and AGL5-iaaM transgenic plants had lower coat scores.
  • the lint yield of the FBP7-iaaM transgenic lines was higher than that of the control, indicating that the cotton fiber yield could be increased after transferring to the FBP7-iaaM gene.
  • the lint yield of E6::iaaM and AGL5::iaaM transgenic plants was lower than that of the control, and the 100-grain weight of all transgenic lines was not significantly different from the control.
  • FBP7 seed linters reduction in iaaM transgenic cotton
  • the cotton seeds are de-velted by a stripping machine, and placed under an environment condition of 65% relative humidity of 65% for more than two days, and the weight of the cottonseed after de-velveting is weighed.
  • the seeds are then de-velted by concentrated sulfuric acid de-veling. After the de-velveted seeds are dried, they are placed in an environment of 20 ° C and a relative humidity of 65% for 24 hr or more and weighed again, and the difference is calculated as the weight of the short piles.
  • the content of short velvet is expressed as a percentage. The results are shown in Fig. 14.
  • the weight of the control linters accounted for about 34% of the total weight of the seeds, and the transgenic 9 # and 14 # were 24%-27%, which was significantly reduced compared with the control.
  • the average length of the upper part of the transgenic cotton fiber was not significantly different from that of the control.
  • the uniformity index of the transgenic fiber was 85.70%-86.95%, and the t test showed no significant difference from the control.
  • the results of the two ratios of fiber strength and elongation at break indicate that the fiber strength of the transgenic lines was not significantly different from that of the control.
  • the results of the micronaire value index correlation with fiber fineness and maturity showed that the micronaire value of FBP7-iaaM transgenic cotton was significantly lower than that of the control.
  • cotton is divided into three grades A, B and C according to the difference of micronaire value, and B is the standard grade.
  • the micronaire value of the transgenic cotton fiber is in the range of B1 and B2 and belongs to the standard grade.
  • the control and E6-iaaM and AGL5-iaaM transgenic cotton fiber micronaire values were significantly higher, belonging to C grade, and the quality was poor.
  • the deviation from the normal range of the control fiber micronaire value was related to the climatic conditions of the cultivated area (Chongqing) in the field trial (2007).
  • FBP7:iaaM transgenic cotton had no significant difference in fiber length and strength from the control, and the fiber fineness was finer than the control, and the quality was improved.
  • the above examples show that the method for improving cotton fiber traits of the present invention can specifically express auxin synthesis related genes at specific developmental stages of specific parts of cotton, and thereby endogenously regulate the content of auxin in specific tissues and organs of cotton to achieve improvement.
  • the purpose of cotton fiber yield and quality (fineness and strength).
  • the test results show that the cotton bolls improved by the method of improving cotton fiber traits of the present invention increased, the number of seeds increased, the number of cotton fibers increased significantly, the clothing fraction increased significantly, the fiber yield increased significantly, and the cotton fiber strength remained unchanged, fineness. And maturity has been improved.
  • the method of the invention is simple and easy to perform, has remarkable effects, and has a good market prospect.

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Description

表达生长素合成相关基因的植物表达载体及其在棉花纤维性状改良的应用 技术领域
本发明涉及一种植物表达载体及其应用，尤其涉及表达生长素合成相关基 因的植物表达载体及其棉花纤维性状改良的应用。 背景技术
棉花是世界上最重要的天然纤维作物，也是最重要的经济作物。中国是世 界上最大的纺织品生产国和消费国，棉花产业在国民经济中具有举足轻重的地 位。近年来， 随着人民生活水平的提高和纺织技术的革新， 对棉花纤维品质的 要求也相应提高；尤其是近年来以气流纺纱取代环锭纺纱的技术革命，要求更 长、更强、更细和更整齐的纤维。但是， 目前棉花推广品种大都纤维品质偏低， 长度单一， 纤维强度偏低， 纤维较粗， 缺乏纺 60支以上的高档棉纱的品种， 远不能满足市场的需要。这直接导致了原棉在国际市场上缺乏竞争力。同时也 导致了棉花生产近年来一直处于一种结构性矛盾的困境： 一方面原棉产量逐 年下降， 而库存原棉仍不断增加， 造成大量资金积压； 另一方面进口棉花数量 却不断上升。能否迅速提高棉花品种的纤维产量和品质，直接关系到棉花产业 的兴衰和纺织品生产加工业的生存与发展。
棉花的产量和品质性状为多基因控制的数量性状，且产量与品质性状之间 存在遗传上的负相关。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， 却在常规育种中 受到诸多限制，单靠现有的棉花遗传种质资源和常规育种手段已经难以大幅度 提高棉花产量，难以满足快速发展的纺织工艺革命对纤维品质的要求。利用基 因工程技术育种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，这为棉花纤维产量和品质的改良 提供了新的途径。 但是目前人们还未得到与棉花纤维形成， 以及产量和品质 (强度、细度和长度等）直接相关的基因， 使得利用基因工程改良棉花纤维缺 乏有效的目的基因。人们对棉花纤维发生、发育和品质形成的分子机理也知之 甚少。 这些都极大的阻碍了对棉花纤维进行产量和品质改良的进程。
现有研究表明，棉花纤维是由棉花胚珠的外珠被表皮细胞经分化起始、伸 长（初生壁合成）、 增厚 （次生壁合成）和成熟脱水四个过程发育而成的单细 胞纤维. 棉花纤维细胞的最终长度可达 20-30mm，高的可达 35-40mm，其长径 比达 1，000-3,000。这样高的长径比是纤维细胞剧烈伸长的结果，其中必然有促 进细胞生长和伸长的植物激素参与。纤维细胞的起始与伸长都与生长素 (Auxin, 如吲哚乙酸 indole-3-acetic acid, IAA)密切相关。 利用离体培养的棉花胚珠，人 们发现未受精的胚珠培养后不能产生纤维，但在培养基中添加赤霉素 （GA) 和 IAA能诱导纤维的生长； 生长素拮抗剂处理表明生长素是纤维原始体伸长 的关键因素 (Beasley CA, 1973, Science, 179 :1003-1005； Beasley CA等， 1973, American J Bot, 60, 130-139)。 Giavalis等 2001年报道了 GA3 (赤霉素 A3 ) 和 IAA处理对培养的未受精胚珠纤维数量的影响，研究结果表明，在开花前或开 花后施用外源 GA和 IAA， 可以使培养的未受精胚珠纤维的数量显著增加 (Giavalis S等， 2001, J Cotton Sci, 5, 252-258). Seagull和 Giavalis 2004年进一 步发现，在自然生长状态下， GA3和 IAA处理棉花的蕾或铃， 可以明显增加纤 维细胞的数量。 而且 IAA处理开花前或开花后的棉铃可使单个棉籽上的纤维 细胞数量增加 58%( Seagull RW等， 2004, J Cotton Sci，， 8, 105-111)。 以上研究 都表明 IAA能促进棉花纤维的产生，与纤维发育生长的关系密切。
外源生长素施用虽然有很好的效果，但在生产上往往难以做到：将生长素 逐一涂抹在花或蕾铃上， 工作量甚大， 劳动力成本高， 大面积推广难以实现。 而且，生长素的大量使用，既增加了生产成本，又会带来环境污染。相比之下， 通过控制生长素生物合成酶基因，从内源的角度来调节特定器官中的生长素含 量， 促进收获器官的发育， 是个十分有效的策略。这一策略至少具有下述几方 面的优点： 1 )效率高、成本低， 因为一旦将生长素生物合成酶基因导入植物， 就无需外加施用生长素或其它处理。而且，外源施用生长素是通过扩散进入细 胞， 而内源激素则产生自细胞内。 因此， 用转基因内源控制生长素的效果往往 比外源施用更好； 2)对作物负面影响小， 由于生长素的作用浓度很低， 浓度 过高或过低均会对植物的发育带来不良影响。 而内源表达生长素合成酶基因， 在表达水平和表达部位合适的情况下， 就可以做到只对特定目标器官 （组织） 起作用， 而不影响植物的其它部分的正常发育； 3)与外源施用生长素和人工 合成生产调节物质相比， 内源调控生长素合成酶基因对环境污染小，对人类健 康造成的危害也小 （Li Y等， 2004， Transgenics of plant hormones and their potential application in horticultural crops. In: Genetically Modified Crops: their development, uses, and risks. New York: Food Products Press, 101-112)。
然而， 利用生长素合成酶基因提高产量和改善品质这一策略在棉花育种上 却未获成功。 1999年 John ME将涉及生长素 IAA生物合成的两个酶基因 iaaM和 iaaH置于纤维特异性启动子 E6下，通过农杆菌介导转入陆地棉 DP50中，结果发 现 IAA含量在大多数转基因株系里都有 2-8倍的增加，然而纤维长度，细度和强 度与对照野生型相比并没有显著差异 (Basra AS 等， 1999, Cotton Fiber, New York: Food Products Press, 271-292)。迄今为止还未见有通过内源表达激素生物 合成酶基因改良棉花纤维品质的成功的报道。人们对能否应用激素生物合成酶 基因改良棉花纤维品质也普遍持有疑义。 发明内容
本发明所要解决的技术问题是提供一种表达生长素合成相关基因的植物 表达载体，将所述的植物表达载体应用于棉花纤维性状的改良， 以解决现有的 内源表达植物生长素生物合成酶基因的方法难以改良棉花纤维产量和品质的 问题。
本发明进一步提供本发明所述的植物表达载体在棉花纤维性状改良上的 应用。
本发明还提供基于本发明植物表达载体的转基因植物的制备方法。
根据本发明的一方面，本发明的植物表达载体至少含有由植物生长素合成 酶基因和植物种皮特异启动子组成的表达生长素合成相关基因的核苷酸，所述 核苷酸通过将编码植物生长素合成酶的基因与编码植物种皮特异启动子的基 因可操作的连接而构建， 优选的植物生长素合成酶基因为土壤根癌农杆菌 tms(tumour morphology shooty)基因（常称 iaaM基因）；优选的植物种皮特异启 动子为 FBP7(Floral Binding Protein 7)基因启动子。因此，优选的编码表达生长 素合成相关基因的核苷酸为具有 SEQ ID NO. 13所示序列的核苷酸。获得表达 生长素合成相关基因的核苷酸后，将该核苷酸插入表达载体从而构建本发明表 达生长素合成相关基因的植物表达载体，优选的植物表达载体具有如图 2所示 的载体结构。 由于在植物表达载体构建过程中， 基于基因操作上的便利， 在 FBP7启动子和 iaaM基因之间， 存在一部分可变的非编码序列。 而且， 在将 FBP7启动子通过基因操作的手段， 置于 iaaM基因 5' 上游过程中， 采用不同 的克隆手段，会产生不同的非编码序列，但这些都不影响 FBP7启动子和 iaaM 基因的连接以及 FBP7启动子和 iaaM基因在一起时所起的生物学功能。 故， 只要 FBP7启动子置于 iaaM基因 5 ' 上游， 启动了 iaaM基因的表达， 不管它 们之间存在什么非编码序列， 均在本专利的保护范围之内。
根据本发明的另一方面，提供一种转化体，将本发明的植物表达载体转染 宿主而获得转化体， 该转化体可用于转化植物获得转基因植物。
根据本发明的再一方面，提供本发明的植物表达载体在棉花纤维性状改良 的应用。通过在植物体内表达本发明构建的生长素合成相关基因，而调控生长 素合成酶的表达， 达到改良棉花纤维性状的目的。
根据本发明的再一方面，提供转基因植物的制备方法，将上述本发明转化 体转化植物 （棉花)， 获得转基因植物。
具体地说，棉花纤维性状改良的方法包括以下几个步骤： 1)获得种皮和纤 维特异表达启动子； 2)获得生长素合成相关基因； 3)将步骤 1)中分离克隆 到的特异启动子与步骤 2)中分离克隆到的生长素合成相关基因进行融合，构 建特异表达生长素合成相关基因的植物表达载体； 4)将步骤 3)得到的特异表 达生长素合成相关基因的植物表达载体整合到棉花基因组中； 5)将通过步骤 4)获得的棉花进行进一步的培养，栽培，并获得转基因的棉花植株。
其中，步骤 1中所述的特异表达启动子可以是由动物、 植物或微生物中分 离克隆得到的天然启动子， 也可以是人工改造或设计合成的启动子。
其中，步骤 2中所述的植物生长素合成相关基因可以是由动物、 植物或微 生物中分离克隆得到的天然基因， 也可以是人工改造或设计的基因。
其中，步骤 3)中 所述的特异启动子与生长素合成相关基因融合以构建特 异表达生长素合成相关基因的表达载体的方法为本领域的常规方法，使用的载 体可以是植物转基因领域所使用的常规载体。
其中，步骤 4)中所述的将表达载体整合到棉花的基因组所使用的方法为常 用的植物转基因方法， 比如根癌农杆菌介导法或基因枪法。
优选的，上述特异启动子为子房特异启动子或种皮特异启动子或种皮和纤 维特异启动子。 更优选的， 上述种皮和纤维特异启动子为 FBP7(Floral Binding Protein 7)基因启动子， 上述子房特异启动子为 AGL5(Agamous Like protein 5) 基因启动子， 上述纤维特异启动子为 E6基因启动子。
优选的， 上述植物激素合成相关基因为生长素合成相关基因。 更优选的， 本发明植物激素合成相关基因为土壤根癌农杆菌 tms(tumour morphology shooty)基因 （常称 iaaM基因）。
本发明中所指的"棉花纤维性状"是指棉花纤维的数量和品质性状， 包括 纤维的数量多少、 长度、 细度、 强度、 整齐度等。
本发明中所指的"转基因棉花"是指通过分子生物学、生物技术手段， 将其 他生物的基因转移到棉花中， 从而使被改造棉花的遗传物质得到改造的棉花。 用于改造的基因可以来源于植物、 动物以及微生物或者人工合成改造。
本发明中所指 "衣分 "是指籽棉上纤维重量对籽棉重量之比， 以百分率表 示。 也就是纤维重量占整个种子及纤维总重量的比例。
本发明中所指 "纤维强度"是指拉伸一束纤维在即将断裂时所能承受的最 大负荷， 以厘牛 /特克斯 (cN/tex)表示， 特克斯是 1000米纤维的克重数。
本发明通过大量研究和分析以及前期实验，认为 John等未能成功并不意味 着利用植物激素生物合成酶基因来提高棉花产量和改善纤维品质的策略是不 可行的。 因为， 既然外源施用 IAA等植物激素对棉花纤维细胞数量的增加和纤 维品质的提高有明显的作用， 那么， 通过控制植物激素合成酶基因， 从内源角 度来调节激素含量， 促进棉花纤维发育， 应该是可行的。而现有研究的结果未 能取得预期效果的原因关键在于：未能找到合适的启动子。因此选择适当的启 动子， 在棉花的特定部位、发育的特定时间、 以适宜的强度控制与植物激素合 成相关基因的表达，精准的调控植物体内激素的作用浓度、作用时间与作用部 位， 才可以有效的影响棉花纤维的生长发育， 获得预期的效果。但是，启动子 种类极为繁多，不可能预料哪种启动子连接上生长素相关基因后就能对棉花纤 维有效。本发明根据棉花纤维发育的特点，在大量筛选启动子的基础上， 创造 性地采用了种皮特异启动子 FBP7(Floral Binding Protein 7)基因启动子（来源于 矮牵牛）， 子房特异启动子 AGL5(Agamous Like protein 5)基因启动子（来源于 拟南芥）和纤维特异启动子 E6基因启动子 （来源于陆地棉） 为主要元件构建 新的基因表达载体， 并建立起了一整套与之相适应的改良棉花纤维性状的方 法。
本发明方法在选定了启动子和目的基因的情况下，启动子和目的基因融合 成新的基因的方式可以采用本领域常用的方式，并可以将融合成的新的基因转 入本领域常用的载体中构建成表达载体再转入棉花中。显然，上述表达载体可 以构建成含单个基因的单价载体，也可以构建含多基因的双价或三价等类型的 载体。在使用本发明方法改良棉花的过程中， 既可以只在一个部位表达一个目 的基因，也可以在多个部位和多个发育阶段表达多个基因，本发明方法已经提 供了技术方案。
本发明改良棉花纤维性状的方法，是通过在棉花的种皮和纤维特异表达生 长素合成相关基因， 进而调控生长素合成酶的表达，通过内源调控棉花特定组 织器官中相应激素的含量的方式，控制棉花种子发育以及纤维发育的起始和伸 长，达到改良棉花纤维产量和品质 (长度，细度和强度)的目的。试验结果证明， 经本发明改良棉花纤维性状的方法所改良的棉花纤维的数量明显增多，产量明 显增加， 棉花纤维品质明显改良； 其种子数量增加， 衣分明显提高。本发明方 法简便易行， 效果显著， 能为纺织工业带来高产， 高品质的纤维原料， 产生巨 大的经济效益。 附图说明
图 1:特异启动子 （包括 FBP7、 AGL5和 E6) 调控下的生长素合成基因 表达载体的构建流程图
Km,卡那霉素抗性基因； Amp， 氨苄青霉素抗性基因； ΝΡΤΠ， 新霉素磷 酸转移酶基因； GUS, β-葡萄糖酸苷酶基因； 35S， 来源于花椰菜花叶病毒的 植物组成性启动子； Pnos, 冠瘿碱合成酶基因启动子； nos， 冠瘿碱合成酶基 因终止子； LB, T-DNA左边界； RB, T-DNA右边界。 用于构建植物表达载体 的骨架载体为 pBI121基础上改造的 p5载体，具有 CaMV 35S启动子调控下的 GUS基因，便于在植物遗传转化的过程中对转化子进行 GUS染色的筛选 图 2: 本发明含特异启动子 FBP7的植物表达载体结构图
图 3 : 生长素合成酶基因 iaaM在转基因棉花中的 southern分析
A, P5-FBP7:iaaM转基因棉花的 11个株系的基因组 DNA经 Xbal酶切后， 用 iaaM基因片段进行 southern杂交。 在不同的株系中得到了不同大小的杂交 片段。 野生型对照中没有杂交信号。 1、 2、 6......20为 p5-FBP7daaM的不同 转基因株系； WT为野生型对照。
B, P5-E6:iaaM转基因棉花的 6个株系的基因组 DNA经 Xbal酶切后， 用 iaaM基因片段进行 southern杂交。 在不同的株系中得到了大小不同的杂交片 段。 野生型对照中没有杂交信号。 1、 2、 5、 8、 10和 11分别为 p5-E6-iaaM的 不同转基因株系 (IE1- ΙΕ1-2、 ΙΕ1-5、 IEl-8、 IEl-10和 IEl-11 ) ; WT为野 生型对照。
C， P5-AGL5-iaaM转基因棉花的 8个株系的基因组 DNA经 Xbal酶切后， 用 iaaM基因片段进行 southern杂交。 3、 4、 6、 7、 10、 11、 12和 14分别为 p5-AGL5-iaaM的不同转基因株系（IG1-3、IG1-4、IG1-6、IG1-7、IG1-10、IG1-11、 IG1-12和 IG1-14) ; WT为野生型对照， 野生型对照中没有杂交信号。
图 4: 特异表达的植物生长素合成酶基因 iaaM在转 FBP7:iaaM基因棉花 中的 RT-PCR分析
A， FBP7:iaaM转基因棉花 11个株系与野生型进行 的表达分析， 结 果在 9#， 14#， 20#三个株系中检测到了 的表达。 其中 9#的表达最强， 14# 居中， 20#最弱； 1、 2、 6、 7、 9、 10、 11、 14、 15、 18、 20： 不同的株系编号。 Β， 在 FBP7:iaaM转基因棉花 9#胚珠和纤维的不同发育时期， 的表达随 着时间增长逐渐减弱， 在 15天之后基本检测不到 M的表达； -2: -2dpa, 开花前两天的材料； 0、 1、 2、 3、 5、 10、 15、 20、 30： Odap, ldpa ......30dpa, 开花后的不同天数的材料。 上： «Μ基因 RT-PCR的结果， iaaM基因特异引 物 (序列 9和 10)的扩增产物， 扩增进行 35个循环。 中： GhHis基因 RT-PCR 的结果； 组蛋白 Histone特异引物 (序列 7和序列 8)的扩增产物， 扩增 35个循 环。 下： 以 KNA为模板进行的^ M的 RT-PCR结果， 结果显示所用 RNA中 没有可被检测的 DNA 污染。 control : 分离的阴性植株作为对照； P : 以 pUC-iaaM质粒为模板的阳性对照。
图 5 : 特异表达的植物生长素合成酶基因 iaaM在转 E6:iaaM基因棉花中 的 RT-PCR分析
E6:iaaM转基因棉花 11个不同转基因株系与野生型进行 iaaM的表达分 析， 结果在 11#株系中检测到^ M有较高的表达水平 1#， 2#， 8#， 10#， 14#， 17#株系中也检测到了 ζ· βΜ基因的表达。 1、 2、 5、 8、 10、 11、 13、 14、 17、 19、 21： 不同的转基因株系编号。 上： 基因 RT-PCR的结果， iaaM基因 特异引物 (序列 9和 10)的扩增产物，扩增进行 35个循环。中： GAMs基因 RT-PCR 的结果， 组蛋白 Histone特异引物 (序列 Ί和序列 8)的扩增产物， 扩增 35个循 环。 下： 以 RNA为模板进行的 的 RT-PCR结果， 结果显示所用 RNA中 没有可被检测的 DNA污染。 control: 分离的阴性植株作为对照。
图 6: 特异表达的植物生长素合成酶基因 iaaM在转 Agl5:iaaM基因棉花 中的 RT-PCR分析
Agl5:iaaM转基因棉花 11个株系与野生型进行 iaaM的表达分析， 结果在 6#， 7#， 10#三个株系中检测到了 M基因的较高水平的表达。 其次是 15#和 23#转基因株系， 2#、 3#、 4#、 16#、 17#和21#中也有^ M基因的表达。 2、 3、 4、 6、 7、 10、 15、 16、 17、 21、 23： 不同的株系编号。上： ^αΜ基因 RT-PCR 的结果， iaaM基因特异引物 (序列 9和 10)的扩增产物， 扩增进行 35个循环。 中： 基因 RT-PCR的结果， 组蛋白 Histone特异引物 (序列 7和序列 8)的 扩增产物，扩增 35个循环。下： 以 RNA为模板进行的 iaaM的 R -PCR结果， 结果显示所用 RNA中没有可被检测的 DNA污染。 control:分离的阴性植株作 为对照。
图 7: FBP7:iaaM转基因棉花胚珠中 «ο 基因的 Real-time PCR分析
基因在 11个转基因株系中表达程度不同， 9#、 14#的表达量高 (A); 基因在棉花胚珠和纤维中的表达水平从开花前两天到 lOdpa逐渐降低， Odap为峰值， 15天及之后检测不到 β«Μ基因的表达 (B)。 Control, 分离的阴 性植株作为对照。
图 8: FBP7/E6/AGL5:iaaM转基因棉花与对照胚珠和纤维中游离 IAA的 含量比较
通过测定开花前 1 d至开花后 5 d棉花胚珠中游离的 IAA含量， 结果发现 转 E6 aaM和 AGL5:iaaM基因棉花胚珠中游离 IAA，与对照相比无明显改变， 而转 FBP7:iaaM基因植株中的游离 IAA含量比对照明显增加，约是对照的 2-8 倍。其中检测样品为胚珠和纤维的混合提取物。 Control, 分离的阴性植株作为 对照。 所有时间点重复取材测定 3次， 取平均值进行作图分析。
图 9: FBP7:iaaM转基因棉花与野生型棉花胚珠表面扫描电镜比较图
A,开花当天的野生型胚珠表面，显示起始的纤维，放大 70倍； B,特异启 动子 FBP7控制的 iaaM转基因棉花胚珠表面，显示起始的纤维，放大 70倍； C, 图 A的进一步放大，示起始纤维的形状和数量，放大 500倍； D, 图 B的进一 步放大，示起始纤维形状和数量，放大 500倍； D中的起始纤维分布明显比 C 中密集，数量更多。 C,D中
Figure imgf000008_0001
图 10: E6:iaaM转基因棉花与野生型棉花胚珠表面扫描电镜比较图 A, 开花当天的野生型胚珠表面，显示起始的纤维，放大 80倍； B, 图 A 的进一步放大，示起始纤维的形状和数量，放大 500倍； （，特异启动子 E6控 制的 转基因棉花胚珠表面，显示起始的纤维，放大 80倍； D, 图 C的进 一步放大，示起始纤维形状和数量，放大 500倍。
图 11 : AGL5:iaaM转基因棉花与野生型棉花胚珠表面扫描电镜比较图 A,开花当天的野生型胚珠表面，显示起始的纤维，放大 80倍； B, 图 A 的进一步放大，示起始纤维的形状和数量，放大 500倍； C,特异启动子 AGL5 控制的 ^Μ转基因棉花胚珠表面，显示起始的纤维，放大 80倍； D, 图 C的 进一步放大， 示起始纤维形状和数量，放大 500倍。
图 12: FBP7:iaaM转基因棉花胚珠及纤维组织切片的显微观察
FBP7:iaaM转基因棉花在 0 dpa的胚珠表面纤维原始体的突起明显可见； 在 1 dpa的转基因棉花胚珠表面纤维原始体的增长也比对照明显； 到 2 dpa的 转基因胚珠表面， 纤维已经显著增长， 且数量上比对照多。 Control, 分离的阴 性植株作为对照； FBP7:iaaM， FBP7:iaaM转基因棉花； 0dpa， 开花当天的胚 珠； ldpa， 开花后一天的胚珠； 2dpa， 开花后两天的胚珠； A、 D、 E、 H、 I、 J， 放大倍率 10x， Bar = 5 μπΐ; B、 C、 F、 G， 放大倍率 40x， Bar = 2 m。
图 13: FBP7:iaaM转基因棉花早期纤维数量统计结果
转基因植株开花两天后的胚珠上纤维的数量明显比对照增长。 在 9#、 14#F.BP7:iaaM转基因棉花株系 2 dpa的胚珠表面纤维的数量在 6000左右， 对 照的数量为平均 5940左右， ≠、 14#两个株系都比野生型明显增多。 其中 9#大 约增加了 11.3%， 14#增加了 15.1%。
图 14: FBP7:iaaM转基因棉花与对照种子大小和短绒含量的比较
A， 转基因棉花的种子明显比对照的减小； 短绒变少。 B， 硫酸脱绒分析 短绒含量， 结果显示转基因棉花的短绒较对照减少了约 10%。 Control, 分离 的阴性植株作为对照； FBP7:iaaM， FBP7:iaaM转基因棉花。 具体实施方式
以下结合附图对本发明进行进一步的详细说明，但以下说明并不对本发 明进行限定，任何对本发明的变形和改变， 只要不脱离本发明的精神，均应属 于本发明所附权利要求所定义的范围。
本发明实例中的试剂药品未做具体说明的均为普通市售，材料方法未做 具体说明的均参考《分子克隆实验指南》（Sambrook和 Russell, 2001)。
【实施例一】棉花基因组的制备
1. DNA的提取
选取棉花幼叶 0.5-lg， 在液氮中迅速研磨成粉， 加入 3mL 65°C预热的 CTAB提取液 (100 mmol/L Tris-HCl (pH8.0), 20 mmol/L EDTA (pH8.0), 1.5 mol/L NaCl, 2% CTAB (WV), 4% PVP40 (W/V)和 2%巯基乙醇 (V/V)， PVP 和巯基乙醇使用前加入)， 快速振荡混匀。 65°(水浴3011 1, 然后加入 l mL5 mol/L KAc, 冰浴 20 min后， 用等体积的氯仿:异戊醇 (24:1)抽提 1次 (10，000 r/min, 4°C离心 5min)， 取上清， 加入 2/3倍体积的 -20 °C预冷异丙醇，混匀,静 置约 30 min, 用玻棒挑出絮状沉淀， 用 75%的乙醇反复漂洗数次， 再用无水 乙醇漂洗 1次， 吹干， 重悬于 500 μΙ^ΤΕ中。 加入 1 LRNaseA (10 mg/mL)， 37°C处理 1 h。 再用酚 (pH8.0):氯仿:异戊醇 (25:24:1)和氯仿:异戊醇 (24:1)各抽提 1次 (10,000 r/min， 4°C离心 5min)， 取上清， 乙醇沉淀。 沉淀用 75%的乙醇漂 洗， 风干， 溶于 200 LTE中， -20 °C保存备用。
2. RNA的提取
选取约 3 g新鲜棉花材料， 在液氮中迅速磨成精细粉末，装入 50 mL离心 管， 加入 15 mL 65°C预热的 RNA提取液 (2% CTAB (W/V), 2% PVP (W/V), 100 mmol/L Tris-HCl (pH8.0), 0.5g/L Spermidine, 2.0mol/LNaCl, 2%巯基乙醇 (V/V, 使用前加入: ))， 颠倒混勾。 65°C水浴 3~10min, 期间混匀 2~3次。氯仿: 异戊醇（24:1)抽提 2次（10,000 r/min， 室温， 5 min)。 取上清， 加入 1/4体 积 10 mol/L LiCl溶液， 4°C放置 6 h，以氯仿:异戊醇 (25:24:1 )各抽提 1次（ 10,000 r/min, 室温， 5min)。加 2倍体积的无水乙醇， 在 -70°C冰箱沉淀 30 min以上。 12,000 r/min, 4°C离心 20min， 弃上清。 沉淀用 200μ 的 DEPC处理水溶解。 酚 (ρΗ4.5)：氯仿:异戊醇（25:24:1)、氯仿:异戊醇 (24:1)各抽提 1次 (10,000 r/min,室温， 5 min)。加 1/10体积 3mol/LNaAc溶液和 2.5倍体积的无水乙醇， 在 -70°C冰箱沉淀 30min以上。 12,000 r/min， 4°C离心 20min， 弃上清。 沉淀用 70%的酒精漂洗一次， 风干。加 20(^L的 DEPC处理水溶解。用非变性琼脂糖 凝胶电泳和紫外分光光度计扫描检测 RNA的质量。
3.基因组序列的 PCR扩增
ΙΟχΕχ PCR buffer (Mg2+ free) 2.5 \L
2.5 mmol/L dNTPs 2 xL
25 mmol/L MgCl2 2 μΐ
引物 l(5 mol/L) 1
引物 2(5 mol/L) 1
ExTaqDNA聚合酶 1U
基因组 DNA 约 60ng
25 的扩增体系
扩增程序为： 94°C， 5min; 94°C， 30sec， 56°
35个循环； 72°C延伸 10min。 4. DNA片段回收， 连接， 转化大肠杆菌 DH5a
长度在 2.0kb以下的片段采用离心法回收。紫外灯下， 用洁净的刀片切下 含目的片段的琼脂糖凝胶块。 用 5号缝衣针在 0.5mL的离心管底部钻一小孔 并填入大小合适的玻璃棉。将含目的片段的琼脂糖块放入填有玻璃棉的 0.5mL 离心管中， 液 N2速冻， 将速冻的 0.5mL离心管套入到一 1.5mL的离心管中， 13,000r/min离心 3min。向流出液（含 DNA)中加入 1/10倍体积的 3mol/L NaAc (pH5.2), 3倍体积的无水乙醇，混匀后于 -70°C放置 30min。 13,000r/min、 4°C 离心 15min收集 DNA沉淀， 用预冷的 75%的乙醇洗涤沉淀。 室温干燥， 用适 量的 TE溶解沉淀即得到目的片段。 回收片段在琼脂糖凝胶上电泳定量。 长度 大于 2.0kb以上的片段用试剂盒 （Roche公司） 回收。
回收的片段与 pUCm-T (上海生工)载体建立如下连接体系：
10xT4 DNA连接缓冲液 l L
载体 DNA片段 1
外源连接产物 DNA片段 1
T4 DNA连接酶 1
用双蒸水补足体积至 10 的连接体系
载体 DNA片段与外源连接产物 DNA片段摩尔比为 1:3， 16°C连接 12h。 之后将连接产物转化大肠杆菌 DH5a。
【实施例二】表达生长素合成相关基因的核苷酸和植物表达载体的制备
1.特异启动子的获得
根据矮牵牛种皮特异启动子 FBP7(GenBank登录号： U90137), 设计引物 (SEQ ID NO.l 和 SEQ ID N0.2),从矮牵牛基因组中 PCR扩增获得一个 500bp 左右的片段。 将扩增 DNA片段克隆到 pUCm-T (上海生工生物工程公司产品) 后， 测序分析表明为矮牵牛的 FBP7特异启动子，见 SEQ ID N0.3, 克隆载体 为 pUC-FBP7。
根据棉花纤维特异启动子 E6,设计 E6特异引物 (SEQ ID N0.4, 5),从陆地 棉基因组中扩增获得一个 1.4kb 左右的片段。 将扩增 DNA 片段克隆到 pUCm-T (上海生工生物工程公司产品)后，测序分析表明为陆地棉 E6纤维特异 启动子， 见 SEQ ID N0.6, 克隆载体为 pUC-E6。
根据拟南芥种子特异启动子 AGL5(GenBank登录号： AC006931.6),设计 AGL5特异引物 (SEQ ID N0.7, 8),从拟南芥基因组中扩增获得一个 2kb左右的 片段。 将扩增 DNA片段克隆到 pUCm-T (上海生工生物工程公司产品)后， 测 序分析表明为拟南芥的 AGL5 特异启动子， 见 SEQ ID N0.9, 克隆载体为 pUC-AGL5 o
2. 土壤根癌农杆菌 iaaM基因的获得 根据土壤根癌农杆菌 Ti质粒 tms(iaaM)基因序列 (GenBank登录号： K02554) 设计引物 (见 SEQ ID NO.10和 11)， 从土壤根癌农杆菌 Ti质粒 T-DNA中通过 PCR扩增并克隆到 pUCm-T (上海生工生物工程公司产品)、 测序分析后得到 SEQ ID NO.12, 克隆载体为 pUC-iaaM。
3.特异表达植物激素合成相关基因的载体的构建
载体构建流程见图 1。 起始的质粒载体分别来自上述的 1和 2， p5为在 pBI121(Clontech公司）的基础上， 采用 《分子克隆实验指南》 （Sambrook和 Russell, 2001)中的方法改造而来。所有限制性内切酶购自 Roche公司， 按照使 用说明书操作。
构建的包含特异启动子 FBP7的植物表达载体结构见图 2， 其包括表达生 长素合成相关基因的核苷酸（SEQ ID NO.13) 以及表达筛选所需的各元件 【实施例三】转化体和转基因植物的制备
1. 用电激法将构建的植物表达载体质粒导入农杆菌 LBA4404。
参考 Bio-RAD MicroPulser用户说明书， 将上述载体通过电激转化法导入 农杆菌 LBA4404。
2. 特异表达植物激素合成相关基因的载体整合到棉花基因组
通过根癌农杆菌介导的方法进行棉花的遗传转化
表 1 根癌农杆菌介导的棉花遗传转化用培养基
培养基名称 成分
基本培养基 MSB (MS无机盐 +B5有机）
种子萌发培养基 l/2MSB+30g/L葡萄糖 +7.5 g/L琼脂， pH6.5
愈伤诱导培养基 MSB+0.5mg/L IAA +0.1mg/L Kt +30g/L葡萄糖 +2.0g/L Gelrite, pH5.8
胚性愈伤诱导培 MSB+1.9 g/L KN03 +30g/L葡萄糖 +2.0g/L Gelrite, pH5.8
养基
共培养培养基 MSB+1.9 g/L KN03 +30g/L葡萄糖 +2.0g/L Gelrite+100 μπιοΙ/L乙
酰丁香酮 , pH5.2
筛选培养基 MSB+1.9g/L KNOs+75mg/L卡那霉素 +500mg/L头孢青霉素
+30g/L葡萄糖 +2.0g/L Gelrite, pH5.8
液体悬浮培养基 MSB+1.9 g/L KN03 +30g/L葡萄糖， pH5.8
体胚成熟培养基 MSB+1.9 g/L KN03 +30g/L葡萄糖 +2.0g/L Gelrite, pH6.5
体胚伸长培养基 1/2 MSB+15g/L葡萄糖 +4.0g/L Gelrite, pH6.5
成苗培养基 SH培养基 +0.05%活性炭 +20g/L蔗糖， pH6.5
MS: Murashige & Skoog, 1962
B5: Gamborg, 1986 Gelrite: Sigma,货号: G1910
SH: Schenk & Hildebrandt, 1972
上述的表达载体通过农杆菌介导胚性愈伤的方法导入棉花。 具体方法如 下：
(1) 棉花胚性愈伤组织的诱导： 棉花种子 (冀棉 14)剥壳， 用 0.1%升汞 (HgCl2) 消毒 lOmiri, 无菌水漂洗 6次， 播种于种子萌发培养基 (培养基成分 见表 1)上。 28°C、 黑暗条件下萌发 5-7天， 以获得无菌棉花幼苗。选取生长健 壮的无菌幼苗下胚轴，切成长约 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， 28 C, 16h光照下培养， 每 3-4周继代一次。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， 28°C, 16h光照下培养。
(2)转化农杆菌的培养：挑取含上述表达载体的农杆菌菌株，在含 50mg/L 卡那霉素和 125mg/L链霉素的 YEB固体培养基 (0.5%蔗糖（W/V)， 0.1%细菌 用酵母提取物 （W/V)， 1%细菌用胰化蛋白胨 （W/V)， 0.05%MgSO4.7H2O
(W/V), 1.5%的琼脂粉 （W/V) pH7.0)上划线培养。 挑农杆菌单菌落， 接种 于 5 mL含相同抗生素的 YEB液体培养基中， 28°C、 200 r/min振荡培养过夜。 培养后的农杆菌菌液按 1:20的比例转接到 50 mL含相同抗生素的 YEB液体培 养基中， 继续在 28°C、 200 r/min振荡培养 3-5 h。 6,000 r/min离心 10 min后， 菌体用液体 MSB (含 ΙΟΟ μπιοΙ/L乙酰丁香酮）培养基重悬， 调整 OD6。Q值为 0.3-0.5备用。
(3)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 20-30 min。 倾去菌液， 将侵染过的胚性愈伤 转移到共培养培养基上， 于 24°C黑暗条件下共培养 4天。
(4)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 行脱菌和选择培养， 28°C, 16h光照下培养，每 3周继代一次。 1-2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。将愈伤组织块进行继代， 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 120 r/min振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， 网下沉淀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， 于 28Ό黑暗条件下培养 2周以诱导体胚伸长、 萌发。 取较大的萌发胚（>0.5 cm) 转接入 SH培养基成苗， 28°C, 16h光照下培养。 待 幼苗长到约 2 cm高时， 剪取幼苗嫁接到有 3-4片真叶的棉花幼苗上。
3.获得纤维性状改良的棉花
嫁接的棉花培养在温室中常规管理。 成熟后收集种子和纤维进行产量和 品质性状分析。获得的转基因棉花在表型和生长发育上与野生型的对照没有明 显区别。 【实施例四】用 RT-PCR的方法检测导入的生长素合成酶基因 iaaM在棉花中 的表达
用 cDNA—链合成试剂盒（MBI公司）合成各种 RNA的一链 cDNA， 操 作均按试剂盒说明书进行。 取 1 —链产物为模板进行 PCR扩增， 25 L体 系中包括 lxPCR缓冲液， 0.2 mmol/L dNTPs， 1.5 mmol/LMgCl2， iaaM基因的 上下游引物 (SEQ ID N0.14和 SEQ ID N0.15)各 0.2 μιηοΙ/L, 1U Taq DNA聚合 酶（Promega)。温度循环参数为 94°C预变性 5 min; 94 °C , 30sec， 56°C , 30sec, 72°C， lmin, 30循环； 72°C延伸 5min。 用组蛋白 Histone3基因作内标。 组蛋 白 Histone3的引物序列 16和 17参考 (Zhu YQ等, 2003,植物生理， 133, 580-88)。 RT-PCR的结果见图 4-6。
【实施例五】用 real-time PCR方法检测导入的 基因在棉花纤维中的表达 提取对照和转基因棉花开花后 10天的胚珠和纤维的总 R A, 通过逆转录 合成 cDNA—链。 以此为模板进行 quantitative real-time PCR扩增。 具体操作 步骤为： 用 DNA—链合成试剂盒（MBI公司）合成各种 RNA的一链 cDNA, 操作均按试剂盒说明书进行。 PCR在 quantitative real-time PCR仪上进行， 在 25 L的反应体系中包括 12.5 L MIX buffer (Bio-Rad公司，包括 PCR缓冲液、 DNA聚合酶、 dNTPs和 MgCl2)， 上下游引物各 0.2 μπιοΙ/L和 1 —链产物。 温度循环参数为 94°C预变性 3mins; 94 °C , 30 sec, 56°C， 30 sec, 72°C , 0.5min, 预设循环数为 35。用棉花 Histone3基因作内标，上、下游引物是 SEQ ID NO.16 和 17。 扩增 ^αΜ基因的上、 下游引物为 SEQ ID N0.14和序列 15。 在运行 quantitative real-time PCR前，用相同引物和模板在相同的温度调控程序中扩增 一次， 通过琼脂糖电泳， 检査并确保扩增产物是单带。 结果如图 7.
【实施例六】特异表达植物激素合成酶基因 iaaM的棉花纤维数量性状的考察
1、 扫描电镜的观察
参照常规电镜制样方法（《电子显微镜生物标本制备技术》， 黄立， 南京： 江苏科学技术出版社， 1982) ， 选取开花当天的胚珠经戊二醛， 锇酸， 单宁酸 系列固定， 乙醇系列梯度脱水，置换，干燥，离子镀膜后做扫描电镜表面观察。 制备好的样品在 HITACHI 日立 S-3000N SEM扫描电镜下观察胚珠表面的纤 维起始情况。结果如图 9所示，转基因棉花胚珠表面的纤维原始体较野生型来 说， 分布更为密集， 数量更多。
2、 早期纤维计数
选取开花后两天的棉铃， 每个株系随机选择 3个单株， 每株取 5铃， 每铃 取 2个种子对胚珠表面的纤维进行计数。 每个样品分别计数 10次， 计算平均 数作为单株的早期纤维数量。 具体方法如下：
取开花后 2天的饱满胚珠两颗， 置于 1.5ml离心管中， 加适量 FAA固定 液固定一小时以上。用去离子水漂洗两次， 力 I] 20(^L 5mol/L HCl， 室温解离一 小时以上。 弃 HC1， 用去离子水漂洗两次， 加 lOO L Schiff试剂处理一小时以 上。弃 Schiff试剂，用去离子水漂洗两次，加 100μΙ^ 45%乙酸并用玻璃棒研磨， 使纤维从胚珠表面脱落分散。将研磨好的纤维液用移液枪混匀，置血球计数板 上在显微镜下观察、 计数。 结果见图 13， 转基因棉花的早期纤维数量明显增 加。
3、 早期胚珠的显微切片观察
选取开花不同时期的胚珠进行石蜡切片， 以从组织水平观察 基因在 胚珠中特异表达对棉花纤维生长和发育的影响。 采取开花后不同时期的子房 (含胚珠)从植株上取下后立即切去顶端， 分割成约 0.5cm的小块或小段， 采用 常规的组织切片方法进行切片，切片在 OLYMPUS BX41TF型显微镜上观察并 照相。结果如图 12所示，在转基因棉花 Odpa的胚珠表面可以明显的观察到纤 维原始体的突起； 而对照的表面相对光滑， 突起不明显， 数量很少。 在 ldpa 的胚珠表面， 无论是对照还是转基因棉花都可以明显的观察到突起的纤维细 胞， 转基因棉花胚珠表面的纤维细胞数量明显多于对照。 2dpa的胚珠表面观 察结果也表明， 纤维细胞都明显的增长， 转基因棉花的纤维明显长于对照。
【实施例七】特异表达植物激素合成酶基因 iaaM 的棉花胚珠和纤维中 IAA含量的考察
以 [13C6]IAA为内标， 采用高压液相色谱 -质谱联用分析法，
1. 内标的配制： [13C6]IAA内标溶解于适量体积的 100%甲醇， 配制终浓 度为 500ng^L的母液。
2. 激素提取和纯化： 取棉花胚珠 /纤维， 液氮速冻， 研磨成粉。 准确称取 样品粉末 （差减法称取） 约 0.5g， 加入 7ml提取液 （80%预冷甲醇）， 和 10ngl3C6-IAA (溶解于 100%甲醇， 终浓度 500ng^L)内标。混匀后于玻璃管 中密闭， 避光 -20°C浸提过夜。 浸提样品转入离心管中， 10,000g， 4°C , 离心 20min。 吸取上清至蒸馏瓶中， 并加入一滴氨水， 使溶液 pH显碱性， 40°C， 于 lOOmL蒸馏瓶中减压旋转蒸发浓縮至干。 样品以 5mL 0.1M HAC复溶。 溶 解后再次检测 pH值，保持与复溶液（0.1M HAC)—致。过柱纯化：以 5mL100% 甲醇过柱， 活化提取柱（Sep-Pak® Cartridges, Waters); 10mL 0.1M HAC分 2 次过柱， 洗去甲醇， 平衡提取柱； 上样， 缓慢过柱样品； 4mL 17%甲醇（0.1M HAC稀释） 缓慢过柱进行漂洗； ，6mL 40%甲醇 （0.1M HAC稀释）洗脱样品， 并收集滤过液。 40°C， 于 50mL蒸馏瓶中减压旋转蒸发浓缩至干。 1.5mL 80% 甲醇复溶样品， 转入 1.5mL离心管中， 真空干燥浓缩。 干燥样品密闭、 低温、 避光保存。
3. 激素的检测： 使用的检测仪器为岛津公司 （shimazu) 高压液相色谱- 质谱联用仪 （LCMS-2010A)。 将干燥的样品以 10%甲醇复溶， 通过微量上样 器上样， 10%甲醇， 5min， 85%甲醇， 30min， 100%甲醇， 31min， 100%甲醇， 39min; 10%甲醇， 40min; 10%甲醇， 60min。
记录内标 [13C6]IAA的色谱峰保留时间、 峰面积和质谱图。 根据保留时间 和质谱图鉴定各样品中 IAA， 并计算内标和内源 IAA的色谱峰面积。 重复三 次， 取峰面积的平均值。 通过内标法计算样品中的 IAA含量。 结果见图 8。
【实施例八】特异表达植物激素合成酶基因 iaaM的棉花纤维衣分和品质的 考察
1. 转基因棉花和对照的衣分含量及纤维产量对比分析
转基因 ^代棉花植株种植于西南大学转基因基地， 随机区组设计， 每个 株系设置 3个小区， 常规田间管理。分小区收获成熟的棉花， 准确称取籽棉产 量。于每小区收获的籽棉中，随即选取 100粒种子，准确称取百粒籽棉的重量, 手工脱纤维再分别称量纤维的总量和种子的总量， 计算衣分的大小。 最后取 3 个小区的平均值作为最终结果 （表 2)， 从实验结果可见： 非转基因对照的小 区籽棉产量、衣分分别为 3.37Kg和 42.19%，而 FBP7-iaaM转基因株系的衣分 均在 43%之上， 最高达到 49.67%显著大于对照， 而 E6-iaaM和 AGL5-iaaM转 基因植株的衣分均较低。 FBP7-iaaM转基因株系的皮棉产量均大于对照，表明 转入 FBP7-iaaM基因后， 可以提高棉花纤维产量。 E6::iaaM和 AGL5::iaaM转 基因植株的皮棉产量低于对照，所有转基因株系的百粒重，和对照相比差异不 明显。
表 2转基因棉花与对照的纤维产量比较
基因名称 百粒重 g 衣分％ 小区籽棉产量 Kg 小区皮棉产量 Kg 对照 10.34 42.19 3.37 1. 42
FBP7-iaaM-9 10.44 48.62 3.39 1. 65
FBP7-iaaM-14 10.30 49.67 3.14 1. 56
FBP7-iaaM-20 10.84 43.91 3.47 1. 52
AGL5-iaaM-6 10.92 41.53 2.74 1. 14
AGL5-iaaM-10 11.07 42.82 2.84 1. 22
E6-iaaM-4 10.31 37.70 2.81 1. 06
E6-iaaM-7 10.23 41.70 2.78 1. 16
E6-iaaM-19 10.52 37.50 2.84 1. 07 对照：以子代中分离出的 GUS染色阴性植株为对照
2. FBP7:iaaM转基因棉花的种子短绒减少 将棉花种子经脱绒机脱绒， 在 20°C相对湿度 65%的环境条件下放置两天 以上， 称量经脱绒后的棉籽重量。再将种子用浓硫酸脱绒法脱绒， 脱绒后的种 子燥后在 20°C相对湿度 65%的环境条件下放置 24 hr以上再次称量，计算差值 为短绒的重量。 短绒的含量以百分数表示。 结果如图 14所示， 经过对种子脱 绒前后的重量比较， 对照短绒重量占种子总重量的 34%左右， 转基因 9#、 14# 为 24%-27%， 比对照明显减少。
3. 转基因棉花纤维的品质检测
将各棉纤维样品送农业部棉花品质监督检测测试中心 (安阳)， 依据 ASTM D5867-95 《HVI900大容量纤维测试仪试验方法》，采用 HFT9000在温度 20°C 相对湿度 65%的环境条件下， 对上半部平均长度、 整齐度指标、 断裂比强度、 伸长率、马克隆值 5指标进行测试。对照材料选用 代分离的 GUS阴性植株， 这些植株中不含有转基因成分。 结果见表 3。
表 3 转基因棉花和对照的纤维品质比较
基因名称 上半部平均长度 cm 整齐度％ 断裂比强度 cN/tex 伸长率％ 马克隆值 对照 30.41 85.16 30.08 5.76 5.25
FBP7-iaaM-9 30.21 86.13 29.97 6.09 4.62
FBP7-iaaM-14 29.92 85.89 30.74 5.88 4.51
FBP7-iaaM-20 30.39 86.39 29.99 5.98 5.16
AGL5-iaaM-6 30.5 86.95 31.32 5.75 5.03
AGL5-iaaM-10 30.31 86.45 30.38 5.95 5.11
E6-iaaM-4 30.13 85.45 30.75 6.00 5.17
E6-iaaM-7 29.83 85.70 29.50 6.25 5.28
E6-iaaM-19 29.86 85.10 30.07 6.00 5.08
转基因棉花纤维的上半部平均长度与对照没有明显差异，转基因纤维的整 齐度指数在 85.70%-86.95%， t检测结果表明与对照差异不显著。 与纤维强度 相关的两个指标断裂比强度和断裂伸长率的结果都表明，转基因株系的纤维强 度与对照相比没有明显差异。与纤维细度和成熟度相关的马克隆值指标检测的 结果显示 FBP7-iaaM转基因棉花的马克隆值比对照显著下降。 根据国家棉花 分级标准， 按照马克隆值差异将棉花分为 A、 B、 C三级， B为标准级。 A级 取值范围在 3.7-4.2， 品质最好； B级取值范围为 3.5-3.6和 4.3-4.9； C级取值 范围为 3.4及以下和 5.0及以上， 品质最差。 转基因棉纤维的马克隆值在 B1 和 B2级范围内，属于标准级。而对照和 E6-iaaM以及 AGL5-iaaM转基因棉纤 维马克隆值明显偏高， 属于 C级， 品质较差。 对照纤维马克隆值偏离正常范 围与进行田间试验的受试地区 (重庆)栽培年份 (2007年)的气候条件有关。 高温 少雨， 日照时间长， 日照强度大， 昼夜温差小的气候条件影响了棉纤维的成熟 度和细度。但是在相同栽培条件下， FBP7-iaaM转基因植株的纤维细度和成熟 度指标马克隆值仍然显著优于对照， 说明转基因植株纤维的品质得到了提高。 而 E6::iaaM和 AGL5::iaaM转基因植株的马克隆值与非转基因的对照相比，无 明显的差异。
总之， 根据纤维品质测定的结果， FBP7:iaaM转基因棉花在纤维长度和强 度上与对照没有明显的差异， 而纤维细度比对照细， 品质得到了提高。 工业应用性
上述实例表明，本发明改良棉花纤维性状的方法， 能够实现在棉花的特定 部位的特定发育阶段特异性地表达生长素合成相关基因，进而内源调控棉花特 定组织器官中生长素的含量，达到改良棉花纤维产量和品质 (细度和强度）的目 的。试验结果证明，经本发明改良棉花纤维性状的方法所改良的棉花棉铃数量 增加， 种子数量增加， 棉花纤维数量明显增多， 衣分明显提高， 纤维产量显著 增加， 同时棉花纤维强度不变， 细度和成熟度得到了改良。本发明方法简便易 行，效果显著， 具有很好的市场前景。

Claims

权利要求书
1、 一种表达生长素合成相关基因的植物表达载体， 至少含有由植物生长 素合成酶基因和植物种皮特异启动子组成的表达生长素合成相关基因的核苷 酸。
2、 权利要求 1所述的植物表达载体， 其特征在于所述核苷酸至少包含植 物生长素合成酶基因 iaaM和植物种皮特异启动子 FBP7基因启动子， 且所述 FBP7基因启动子位于植物生长素合成酶基因 iaaM的 5 ' 上游。
3、 权利要求 1所述的植物表达载体，其特征在于所述核苷酸具有如 SEQ ID No. 13所示的序列。
4、 权利要求 1所述的植物表达载体， 其特征在于所述植物表达载体具有 如图 2所示的结构。
5、 一种转化体， 以权利要求 1所述的植物表达载体转化宿主获得。
6、 权利要求 1~4任一所述的植物表达载体在棉花纤维性状改良的应用。
7、 一种含有权利要求 1所述的植物表达载体的转基因植物的制备方法， 包含下述步骤：
1 )将植物生长素合成酶基因与植物种皮特异启动子可操作地连接；
2)构建含有植物生长素合成酶基因与植物种皮特异启动子的植物表达载 体；
3 )用所述植物表达载体转化宿主， 获得转化体；
4)用所述转化体转化植物， 获得转基因植物。
8、一种编码表达生长素合成相关基因的核苷酸，其特征在于其具有如 SEQ ID NO. 13所示的核苷酸序列。
PCT/CN2009/000095 2008-07-25 2009-01-22 表达生长素合成相关基因的植物表达载体及其在棉花纤维性状改良的应用 WO2010009601A1 (zh)

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