CN111218472B

CN111218472B - Fusion gene for improving cotton yield, plant expression vector, transformant and application

Info

Publication number: CN111218472B
Application number: CN202010156464.7A
Authority: CN
Inventors: 曾健晏; 阎星颖; 白文钦; 张觅; 侯磊; 代永露; 吴怡萍; 肖月华; 裴炎
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-03-09
Filing date: 2020-03-09
Publication date: 2020-11-03
Anticipated expiration: 2040-03-09
Also published as: CN111218472A

Abstract

The invention relates to a fusion gene for improving cotton yield, a plant expression vector, a transformant and application, and belongs to the technical field of plant genetic engineering. The fusion gene comprises a cotton cytokinin oxidase gene GhCKX3bRNAi containing RNA interference and an anthogen specific promoter AGIP; the nucleotide sequence of the GhCKX3bRNAi is shown as SEQ ID NO. 1; the nucleotide sequence of the floral primordium specific promoter AGIP is shown in SEQ ID NO. 2. The cotton plant improved by the fusion gene of the invention has normal growth, obviously improved boll weight and fiber yield, and no obvious change of cotton fiber quality, can provide more protein and oil sources for food industry under the condition of not occupying limited cultivated land, and brings more fiber raw materials for textile industry, thereby generating great economic benefit.

Description

Fusion gene for improving cotton yield, plant expression vector, transformant and application

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a fusion gene for improving cotton yield, a plant expression vector, a transformant and application.

Background

Cotton is the most important natural fiber crop in the world, and is also an important oil crop (Chen ZJ et al, (2007) heated sequencing cotton (Gossypium) genes. plant Physiology 145: 1303. sup. 1310). The number of countries for planting commercial cotton is more than 80, the planting area is about 3300 ten thousand hectares, and the planting area occupies 5 percent of the cultivated land area in the world. Currently, over 150 countries participate in the import and export trade of cotton, and the economic value of the cotton-related industry exceeds $ 5000 billion per year (Zhang Aimin et al (2016) quality supports the crop industry and future development. Chinese agricultural science 49: 4265-. China is the largest textile production country and consumer country in the world, and the cotton industry plays a significant role in national economy in China. In addition, the oil content of the shelled Cottonseed can reach 30-38%, and the Cottonseed oil contains a large amount of unsaturated fatty acids, wherein the content of the essential amino acid-linoleic acid in the human body accounts for nearly 50%, and the Cottonseed oil is a rich-nutrition edible oil source, and is the sixth place in the ranking list of the use of the edible oil in the world (Sekhar SC, Rao VKB (2011) cottonsed oil as a nutritional oil. Pertanika Journal of nutritional Agricultural Science: 17-24; Xu C, Shanklinj (2016) triacylglycerols, Function, and culture in Plant specific tissues. Annu Rev Plant Biol 67: 179. 206). Meanwhile, the cottonseed is not only an important oil resource, but also an important protein resource. The cottonseed cake obtained after shelling and oil extraction has a protein content of 40-50%, the quality of the cottonseed cake is similar to that of bean protein, and the nutritional value of the cottonseed cake is higher than that of most cereal proteins (research progress of cottonseed protein in Zhao winter, Liu Xiao Yu (2009). 5:27-30 processing of agricultural products). Since ordinary cottonseed contains high concentration of the toxic chemical gossypol, it is not suitable for human and most animals, and is generally used as feed for animals with multiple stomachs, such as cattle and sheep. However, with the continuous progress of the breeding technology of Low-Gossypol cotton, a plurality of Low-Gossypol cotton materials have been cultivated at present, and the united states of 2019 approves the novel Low-Gossypol cotton transgenic cotton to be used as food raw materials for human and animals ("fertility for ultra-Low Gossypol cotton coded (ULGCS) TAM 66274"), which greatly increases the application range of the cotton seeds. With the economic progress and the increase of population, the available cultivated land area is gradually reduced, and meanwhile, the demand of pure cotton products, edible protein and oil is continuously increased. How to increase the yield of cotton fibers and cotton seeds on the premise of not occupying limited farmland resources is an important subject in front of the majority of cotton researchers. Whether the cotton yield of China can be rapidly improved or not is directly related to the growth and the decline of the cotton industry of China and the survival and the development of the textile production and processing industry.

The cotton genetic basis is narrow, and the cotton yield is difficult to be greatly improved only by the existing cotton genetic germplasm resources and conventional breeding means. The genetic disorder among species can be broken through by utilizing the genetic engineering technology for breeding, the directional transfer of the excellent target gene is realized, and meanwhile, the method has the advantages of easy and stable progeny, short breeding period and the like, and creates a new way for improving the yield of cotton fibers and seeds. Although researchers have increased cotton fiber or seed yield to some extent by increasing the starting number of cotton ovule epidermal fibers or increasing the accumulation of plant photosynthetic products, the fiber yield potential is limited due to the constant surface area of the individual ovules, while the increase in ovule surface fibers competes for seed nutrient supply and severely inhibits seed growth and development.

Disclosure of Invention

The invention aims to provide a fusion gene for improving cotton yield, a plant expression vector, a transformant and application. The cotton plant improved by the fusion gene of the invention has normal growth, obviously improved boll weight and fiber yield, and no obvious change of cotton fiber quality, can provide more protein and oil sources for food industry under the condition of not occupying limited cultivated land, and brings more fiber raw materials for textile industry, thereby generating great economic benefit.

The invention provides a fusion gene for improving cotton yield, which comprises a cotton cytokinin oxidase gene GhCKX3bRNAi containing RNA interference and a floral primordium specific promoter AGIP; the nucleotide sequence of the GhCKX3bRNAi is shown as SEQ ID NO. 1; the nucleotide sequence of the floral primordium specific promoter AGIP is shown in SEQ ID NO. 2.

The invention also provides a plant expression vector for improving the yield of cotton, which comprises a cotton cytokinin oxidase gene GhCKX3bRNAi containing RNA interference and a floral primordium specific promoter AGIP; the nucleotide sequence of the GhCKX3bRNAi is shown as SEQ ID NO. 1; the nucleotide sequence of the floral primordium specific promoter AGIP is shown in SEQ ID NO. 2.

Preferably, the backbone vector used to construct the plant expression vector comprises the p5 vector.

The invention also provides a transformant of the plant expression vector in the technical scheme, wherein the transformant is obtained by transforming a host with the plant expression vector.

The invention also provides a preparation method of the transgenic cotton based on the fusion gene in the technical scheme, which comprises the following steps:

1) constructing a plant expression vector containing a fusion gene of the cotton cytokinin oxidase gene GhCKX3bRNAi containing RNA interference and an floral primordium specific promoter AGIP according to the technical scheme;

2) transforming a host with the plant expression vector to obtain a transformant;

3) and transforming plants by using the transformant to obtain transgenic cotton.

The invention also provides application of the fusion gene in the technical scheme, the plant expression vector in the technical scheme or the transformant in the technical scheme in improving the content of cytokinin in cotton buds.

The invention also provides application of the fusion gene in the technical scheme, the plant expression vector in the technical scheme or the transformant in the technical scheme in increasing the number of cotton single-boll seeds.

The invention also provides application of the fusion gene in the technical scheme, the plant expression vector in the technical scheme or the transformant in the technical scheme in improving cotton yield.

The invention also provides application of the fusion gene in the technical scheme, the plant expression vector in the technical scheme or the transformant in the technical scheme in improving the yield of cotton seeds and fibers.

The invention also provides application of the fusion gene in the technical scheme, the plant expression vector in the technical scheme or the transformant in the technical scheme in breeding new plant varieties.

The invention provides a fusion gene for improving cotton yield. The fusion gene combines a cotton cytokinin oxidase gene GhCKX3bRNAi containing RNA interference with an flower primordium specific promoter AGIP, can specifically interfere genes related to cytokinin metabolic enzyme in cotton flower primordium, further regulates and controls the expression of the cytokinin oxidase gene, increases the number of ovules and seeds in cotton bolls in a mode of endogenously regulating and controlling the content of corresponding hormones in cotton, and achieves the purpose of improving the yield of cotton. Test results show that the cotton plants improved by the fusion gene of the invention grow normally, the boll weight and the fiber yield are obviously improved, and the quality of cotton fibers is not obviously changed. The invention improves the cotton by utilizing the fusion gene, is simple and easy to operate, has obvious effect, can provide more protein and oil sources for the food industry under the condition of not occupying limited cultivated land, brings more fiber raw materials for the textile industry and generates great economic benefit.

Drawings

FIG. 1 shows the in situ hybridization results of cytokinins in cotton flower buds at the ovule differentiation stage provided by the present invention;

FIG. 2 shows phylogenetic analysis results of cytokinin oxidase genes of upland cotton, Asian cotton, Raymond cotton and Arabidopsis provided by the present invention;

FIG. 3 is a schematic diagram of the structure of the expression vector of the cytokinin oxidase gene of cotton of the present invention;

FIG. 4 is a heat map of the expression patterns of the cotton cytokinin oxidase gene in different tissues of cotton, which is drawn according to the results of digital expression profiles of the cotton cytokinin oxidase gene in different tissues;

FIG. 5 is Real-time PCR analysis of the GhCKX3b gene in the flower bud of GhCKX3bRNAi transgenic cotton according to the AGIP provided by the invention;

FIG. 6 shows statistics of the number of single-boll ovules of the AGIP, GhCKX3bRNAi transgenic cotton on the day of flowering;

FIG. 7 is a graph showing statistics of the number of ovules per chamber in bolls on the day of flowering of AGIP, GhCKX3bRNAi and 35S, GhCKX3bRNAi transgenic cotton according to the present invention;

FIG. 8 shows the phenotype of the AGIP GhCKX3bRNAi transgenic cotton plant provided by the present invention;

FIG. 9 shows the growth of fiber on the ovule epidermis at the day of flowering (0DPA) and 1d (1DPA) after flowering of the GhCKX3bRNAi transgenic cotton provided by the present invention;

FIG. 10 shows the length statistics of mature fibers of AGIP, GhCKX3bRNAi transgenic cotton provided by the present invention.

Detailed Description

The invention provides a fusion gene for improving cotton yield, which comprises a cotton cytokinin oxidase gene GhCKX3bRNAi containing RNA interference and a floral primordium specific promoter AGIP; the nucleotide sequence of the GhCKX3bRNAi is shown as SEQ ID NO.1 (GGATCCAGATCTCCAGTCTGCTACTCTAGTTCAGTTCCTTTTGGTATAGCAGCCAAAGGGCACGGTCATTCTGCCAGGGGTCAAGCGATGGCAGAAAACGGGGTCGTGGTGGACATGAGATCGATGGCGAACAATCGTCGGAACGGAACCGGAATCCGGGTCTCGATCGACAGGCTTTACGCCGATGTCGGCGGCGAACAGCTTTGGATCGACGTGTTGAATGCGACATTGGAATACGGACTTGCACCCGTTTCTTGGACCGATTATTTGTACTTAACCGTCGGCGGAACGCTCTCCAATGCTGGAATCAGTGGACAAACTTTTCGTTATGGTCCTCAGATCAGTAATGTTCTTGAAATGGATGTTATAACAGGTATATGATTTCGTATTCTTTTTCATTTTTAATCACGAGTTCGGTTCAATATTCTTTTGTCCTGTTCACAAAGATTCCAACAACAAACTTATTTTCCAGCATTTCTTTGGGTAAAATTACCATAAACATCTTACTGCATTTTACTCATTAATCTATGTACGTTAGATAAAAAAATAAATTTATCTTTTTGTCAAAAATCTCTTTCATTTTTATGGTTAAAAAATCTATTCCTATACATCAAAATGAGGTACATATAACATGTCAATTGTTTGATTATCCTTAAATCACGTAAATTTTTTAACAATATAATTAAATGAAATTTTTAATAAAAACAATCAATTTACTTTAACTTAAATTTATAAAAATTTTAAATAAAAAAGATAAAAATACTATTTAACTATTGATATAAATATTTCTATGTATTTTTTATCCATTTCTTAGCTGGTGTCTGATGGTTCATCATATGTATATACATTTAATTTTAAATGGCAGTTCATTTCATGCCAACCTATTAAAATATTTTTCTATAAATGTATTCGTTCTAGGGTCCCCACTAGGAAACCCCATAGGACAAAAGTTCAAACGGTGAACATCATCACCATCATCATCATCATTTATATAATATTATATATATGTTGGTTGCATCATGCATGCATGCAGTTATTTACGAGCAAATTTTATTTTTAAATTCAGGGAAAGCCGATTTCAAGAACATTCTGATCTGAGGACCATAACGAAAAGTTTGTCCACTGATTCCAGCATTGGAGAGCGTTCCGCCGACGGTTAAGTACAAATAATCGGTCCAAGAAACGGGTGCAAGTCCGTATTCCAATGTCGCATTCAACACGTCGATCCAAAGCTGTTCGCCGCCGACATCGGCGTAAAGCCTGTCGATCGAGACCCGGATTCCGGTTCCGTTCCGACGATTGTTCGCCATCGATCTCATGTCCACCACGACCCCGCTTTCTGCCATCGCTTGACCCCTGGCAGAATGACCGTGCCCTTTGGCTGCTATACCAAAAGGAACTGAACTGGAGACCTGGTCTGCAGGCGGCCGCCCATGGGATATCATCGATCATATGTCGCCCTATAGTGAGTCGTATTACGGTACC); the nucleotide sequence of the floral primordium specific promoter AGIP is shown in SEQ ID NO.2

(CTTAATCTACGCTTAAATCTGCATTTTGATAAGTAATAAAAAATAAAATTGATAGGGTCAAATCGACCACTTGCACAGTTAAGTGATTCTAATACGAAACCTTAAAAGCAAACATCGGTTCTTTTGAGTCAGAAGAAATGCAACTTAATGTGACACATGATGTGAAGAAAAAACAAAAGTAATATAAGAAAAGGGAACAATTAAATAGTTAATAAAATATTTCCTTAAAGTTGTAACAAATAAAGAATCATTTTATGAAACAATATGAACCCTAAATAAATTAAAATTCCTCTGAAACCTTAAATTTATCGAGCTAGTGATTGGCTGCCAACTGCCATGCTGGCAAAATTAGAGTGACATGATTGGTCTGAACATGTCTAGGGTTTCAGACATGTGACATGTGTCAACAACCCATTAACACATTGGGTATAAATCCAATAGACATTTGATAGTATTAAAATTGTAACCATTGGATTAAATTTAAACGTGATGGATGTAACTAAATGACTTGTCCGAGTAACATCACAACGTTCCATACTTTCCTTATTTGGAATATAATTAAATTTACCATTTATTCTTTTTTCTTGAGTTTCCTGTATATGTACTTGTACATAGATATATATACACAAATACGTATTACAATGACATATTATAGACTTTGATGTCTGAACTCTCAACCTTCTCGATGGAGAGATCATGACCGTAGATTTTTTTGGATCGTAGAAGGCAGACCAAACTCTTAAACTATTGGATCCGTACTAAAAATCTCACTTTCCTCTCAGTACCCATAATGAGAGAGAAAATGATAAAAATCCCTAACATTATTCTCTCTCTAGAAAAAAAAAAGATACTTCAAAAAGAAAGAGAAATTGCATAAATCTATCTACACCAAAGATGTTGAAGCAATTCCAATGCTATACTTCTATGCCAAATCTATTTATTCAGTGATCATTAATCTTTTTACTTCCAAGAAATATGAACAATTTAGTATCCTTATAATTTTTGTCTCTATATATGTAATATGAACATTGGGTATTGACCAAATGAGAAATCTAATATTAAATGGTCAAAAGTAGTAATATGATGACATTTTTGAATTTATAAATAGGTTACAAATTAATTCATTATGACATAAAACCTTCTTGTCAGAAGTCAAGAACTGAAACTAACAAAACTTTATAATAAATTAGTAAAAATACAAGTGAAAAATAAAAAGAAATAATATCTGAGTGATGACGTGATCAAAGATTCTTTAACAAAGACAACAAATCTTACAGACCCAAAACCTAATCTTGCGCTCAATTCCAACCTCTGAAAAAACCTCAAAAATCTTATAAAAGAAAATAAATAAAGAAACGAAACTCTGATTTCGTAGAGTACCCATCGGATATATAAAAAGAAATTAGTAGGTAAATGAAGACTAATTTTGATTGACTGATTTAATTTGAAGTCGTTGTTAGCTTTTCTTGTTTTGGACATGAGAATTATATATTTCAGGACATGAGAGTTGACAACTGTAAACGATTAAGAAAATTGATCTTTTAATTTTCAAACACCATTTAATCTTGACATGTTTTATGTTTTGGTGGAGAAGAAAGTAATCACGTGGGACTCTCTACTAATAAGTATTTGGAAATTGCGTGTCGAATTAGAGATTACTAGAGATCCGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAGCTAG). An RNA Interference cotton cytokinin oxidase (RNA Interference Gossypium hirsutum. L cytokinin hydrogenase) gene (GhCKX3bRNAi gene for short) is a gene for interfering the expression of a cotton cytokinin metabolic gene, and the gene can interfere the mRNA expression of a cytokinin oxidase gene and endogenously regulate and control the content of cytokinin. The selection of the flower primordium specific promoter AGIP can interfere the expression of GhCKX in cotton, can effectively avoid the adverse effect of high-concentration cytokinin on the growth and development of cotton plants, properly increase the cytokinin content in cotton carpels, increase the number of ovules and seeds and improve the yield of cotton seeds and fibers. The invention utilizes the flower primordium specific promoter AGIP to regulate and control the expression of the RNA interference sequence of GhCKX, can improve the cytokinin content in flower buds, and further increases the number of seeds.

The invention also provides a plant expression vector for improving the yield of cotton, which comprises a cotton cytokinin oxidase gene GhCKX3bRNAi containing RNA interference and a floral primordium specific promoter AGIP; the nucleotide sequence of the GhCKX3bRNAi is shown as SEQ ID NO. 1; the nucleotide sequence of the floral primordium specific promoter AGIP is shown in SEQ ID NO. 2. The plant expression vector can regulate and control the level of cytokinin oxidase by specifically interfering the expression of cytokinin metabolism related genes in cotton flower primordium, thereby improving the plant characters; the plant expression vector is applied to the improvement of the number of cotton seeds, can increase the yield of cotton, and solves the problems that the yield per unit area is difficult to improve and the available cultivated land is limited. In the present invention, the backbone vector used for constructing the plant expression vector includes a p5 vector. The structure of the plant expression vector of the present invention is preferably as shown in FIG. 3, NPTII: neomycin phosphotransferase gene; and (4) GUS: a beta-glucosidase gene; 35S: a plant constitutive promoter derived from cauliflower mosaic virus; nos Terminator: opine synthase gene terminators; 35S Terminator: a 35S gene terminator; LB: the T-DNA left border; RB: the T-DNA right border; the skeleton vector for constructing the plant expression vector is a p5 vector modified on the basis of pBI121, has a GUS gene under the regulation and control of a CaMV 35S promoter, and is convenient for screening GUS staining on a transformant in the process of plant genetic transformation.

The invention also provides a transformant of the plant expression vector in the technical scheme, wherein the transformant is obtained by transforming a host with the plant expression vector. The transformation method of the present invention is not particularly limited, and a conventional transformation method using a plant expression vector known to those skilled in the art may be used.

The invention also provides a preparation method of transgenic cotton (AGIP: GhCKX3bRNAi transgenic cotton) based on the fusion gene in the technical scheme, which comprises the following steps:

The method of the present invention for gene fusion, transformation of a host and transformation of a plant is not particularly limited, and any method known to those skilled in the art for experimental manipulation of gene transformation may be used.

The invention also provides application of the fusion gene in the technical scheme, the plant expression vector in the technical scheme or the transformant in the technical scheme in increasing the number of cotton single-boll seeds. The invention can reduce the expression of cytokinin oxidase gene in cotton through RNA interference, and can improve the content of endogenous cytokinin in the cotton carpel, thereby increasing the number of cotton single-boll ovules and seeds and improving the yield of cotton seeds and fibers.

The invention also provides application of the fusion gene in the technical scheme, the plant expression vector in the technical scheme or the transformant in the technical scheme in improving the yield of cotton seeds and fibers. The invention specifically interferes the cytokinin metabolic enzyme related gene in cotton flower buds to further regulate the expression of cytokinin oxidase genes, increases the number of cotton ovules and seeds on the premise of not influencing the normal growth and development of cotton, and achieves the aim of improving the yield of cotton seeds and fibers.

The fusion gene, the plant expression vector, the transformant and the application for improving cotton yield according to the present invention will be described in further detail with reference to the following embodiments, but the technical solutions of the present invention include, but are not limited to, the following embodiments.

Reagents and drugs in the examples of the present invention are not specifically described, and they are all generally available on the market, and materials and methods are not specifically described, which are referred to in the molecular cloning laboratory Manual (Sambrook and Russell, 2001). The experimental cotton material used was Ji Cotton 14, from Gossypium hirsutum L.

As used herein, "number of cotton single bolls" refers to the number of ovules that are present in each boll during the day of cotton flowering.

As used herein, "the number of cotton single boll seeds" refers to the number of mature seeds per boll after harvesting of cotton.

The "transgenic cotton" refers to cotton which is transformed from the genetic material of the transformed cotton by transferring the target gene into the cotton through molecular biology and biotechnology means. The genes for modification may be derived from plants, animals and microorganisms or artificially modified.

As used herein, the term "child finger" means the weight of 100 pieces of cotton expressed in grams (g).

The "clothing" as used herein means the weight of ginned cotton expressed in grams (g) after ginning 100 pieces of cotton.

The term "lint" as used herein refers to the percentage of the weight of lint cotton to the weight of the lint cotton, i.e., the weight of the fibers to the total weight of the seeds and fibers, after the cotton is ginned.

The boll weight in the invention refers to the average value of the dry weight of the cotton in the bolls of 100 bolls in which the cotton plant normally cracks and bolls are opened in the middle stage of boll opening, and the unit is gram (g), which is also called the single boll weight.

"cotton bolls" as used herein refers to the cotton that has been stripped from the bolls that opened the bolls.

The term "number of bolls per plant" as used herein refers to the average number of effective bolls of a single plant of cotton. Is the average of 30 cotton plants in the middle two rows of each plot during field trials.

The term "plot" as used herein refers to a single area of cotton planted in a random block design during field trials. Each cell area is about 18m²The seeds are planted in 4 rows, the row spacing is 1.0 meter, 60 plants are planted in 15 rows, and the spacing between adjacent plants is 0.3 m.

Example 1

Accumulation mode of cytokinins in cotton buds

1. Tissue section

(1) Placing the bolls in the required period in newly prepared 2% EDAC solution, vacuumizing at4 deg.C to remove air from the sample, and maintaining for 1 h.

(2) And (3) transferring the sample into FAA stationary liquid, vacuumizing at4 ℃ again to remove air in the sample, and fixing for 2-4 h.

(3) The fixed sample is transferred into dehydrating agents (I-VI) of each stage for dehydration treatment and then embedded by paraffin, and the formula of the dehydrating solution is shown in Table 1.

(4) The embedded samples were cut into 10 μm thin pieces and attached to

On a Plus Gold slide, the slide was baked at 42 ℃ until the water evaporation was complete.

TABLE 1 tissue slice dehydration solution formulation

2. In situ hybridization of cytokinins

The method comprises the following specific steps:

(1) the samples were sequentially dewaxed and rehydrated in 100% xylene (2 times treated), 75% xylene (diluted with ethanol), 25% xylene (diluted with ethanol), 100% ethanol, 95% ethanol (diluted with water, the same is applied), 80% ethanol, 60% ethanol, 30% ethanol, and water for 2min each time.

(2) The rehydrated sample was immersed in 1 × PBS solution for 5 min.

(3) The treated sample is transferred into blocking liquid for blocking for 45 min.

(4) The samples were immersed in RSR solution for 5 min.

(5) After the sample was briefly soaked in PBS-BSA solution for 2min, 100. mu.L of a cytokinin ZR polyclonal antibody diluted with PBS-BSA solution at a ratio of 1:100 was applied to the slide to which the sample was attached, and a Parafilm seal, which was large enough to fit the slide, was dark-treated in a wet box at4 ℃ overnight.

(6) The next day, 100mL of H-RSR solution was poured into the petri dish (90mm) and the slides were horizontally placed in the plate and rinsed off the Parafilm.

(7) Washed 2 times with H-RSR solution for 2min each time, and then soaked with RSR solution for 10 min.

(8) After the sample was soaked in PBS-BSA solution for 2min, 100. mu.L of a secondary antibody (Dylight 549 label) diluted 1:100 with PBS-BSA solution was applied to the slide on the side to which the sample was applied, and treated with Parafilm seal, such as a slide, in a wet box at 37 ℃ for 2h in the dark.

(9) After the secondary antibody incubation was completed, the slides were directly mounted (80% glycerol, pH 7.4) after washing twice with RSR (2min each) and 1 time with 1 XPBS solution (2 min).

(10) Observations were performed with FV1000 confocal laser microscopy (Olympus).

The results observed by the above method are shown in FIG. 1 (in situ hybridization of cytokinins in cotton buds at the differentiation stage of ovule. A: observation of transmitted light; B: observation of fluorescence of Dylight 549 after hybridization with cytokinin (ZR + ZT) antibody; C: observation of fluorescence of Dylight 549 without hybridization with cytokinin (ZR + ZT) antibody; scale: 500 μm), and cytokinins accumulated in the heart pericarp in a large amount in cotton buds and were particularly evident at the primordial site at the initiation of ovule.

Example 2

Cotton cytokinin oxidase gene expression pattern

1. Cytokinin oxidase gene search

7 cytokinin oxidase genes, specifically AtCKX1(AT2G41510), AtCKX2(AT2G19500), AtCKX3(AT5G56970), AtCKX4(AT4G29740), AtCKX5(AT1G75450), AtCKX6(AT3G63440) and AtCKX7(AT5G21482) are searched in a dicotyledonous model plant Arabidopsis thaliana genome database website https:// www.arabidopsis.org/.

The amino acid sequences of the 7 Arabidopsis cytokinin oxidase genes searched in the cotton genome website http:// www.cottonfgd.org/respectively search homologous genes of 27 upland cotton, 14 Ramended cotton and 14 Asian cotton, and perform phylogenetic tree analysis by using MEGA5 by using an adjacency method to obtain the clustering result shown in FIG. 2 (the numbers on the branches are guide values obtained by 1000 times of repeated calculation).

2. Analysis of expression patterns of cytokinin oxidase in cotton

The published results of digital expression profiling of genes in different tissues of cotton (Zhang T et al, (2015) Sequencing of allelic cottons (Gossypium hirsutum L. acc. TM. 1) provides a resource for fiber improvement. Nature Biotechnology 33:531 537) were used to explore the expression patterns of the gene for cytokinin oxidase in Gossypium hirsutum and to map heatmaps. The results are shown in fig. 4 (heat map drawn by the results of digital expression profiles of the cotton cytokinin oxidase genes in different tissues according to the expression patterns of the cotton cytokinin oxidase genes provided by the present invention in different tissues), and from the heat map results, it can be seen that GhCKX3b is predominantly expressed in flower organs compared to other cytokinin oxidase genes, and GhCKX3b is selected as a target gene for cytokinin oxidase expression interference.

Example 3

Preparation of Arabidopsis and Cotton genomes

1. Extraction of DNA from Arabidopsis and cotton

Selecting 0.5-1 g of young leaves of arabidopsis thaliana or cotton, quickly grinding the young leaves in liquid nitrogen to fine powder, and extracting genome DNA by using an EASY spin plant DNA quick extraction kit of the Idela corporation, wherein the extraction method is strictly carried out according to the steps in the specification. The quality of the DNA was checked by non-denaturing agarose gel electrophoresis.

2. Extraction of RNA

Selecting about 3g of fresh cotton material, quickly grinding the fresh cotton material into fine powder in liquid nitrogen, and extracting total RNA by using an EASYspin plant RNA quick extraction kit of the Edley company, wherein the extraction method is carried out strictly according to the steps in the specification. The quality of RNA was checked by non-denaturing agarose gel electrophoresis.

3. Synthesis of cDNA

The DNA in the total RNA is removed, and then the RNA is reversely transcribed into cDNA. Synthesis of cDNA Using TaKaRa

The RT reagent Kit with gDNA Eraser Kit is strictly operated according to the product instructions.

4. PCR amplification of genomic sequences

The amplification procedure was: at 98 ℃ for 5 min; 98 ℃, 10sec, 56 ℃, 15sec, 72 ℃, 1kb/min, 35 cycles.

The example provides a general method for extracting cotton genome, amplifying genome sequence PCR, recovering DNA fragment, connecting and transforming. In a particular embodiment of the invention, the methods described above may be used, as well as methods described elsewhere in the invention.

Example 4

Preparation of nucleotide for expressing cytokinin oxidase gene and construction of expression vector

1. Acquisition of GhCKX3bRNAi nucleotide sequence

By adopting the method of directly amplifying from genome to obtain RNA hairpin structure with intron,

primers

1 and 2 are designed, which are respectively SEQ ID NO.3(GhCKX3bRNAi amplification upstream primer 1 containing SalI enzyme cutting site, 5-GTCGACGCTACTCCAGTTCAGTTCCT-3') and SEQ ID NO.4(GhCKX3bRNAi amplification downstream primer 2, EcoRI cleavage site containing, 5-GAATTCCAGATCAGTAATGTTCTTGAAATCGGCTTTCCCTGA-3'), and the nucleotide sequence of GhCKX3bRNAi is directly amplified and cloned from the genome of upland cotton, and is shown in SEQ ID NO. 1.

2. Obtaining of AGIP nucleotide sequences

By adopting the method of directly amplifying and obtaining the chimeric promoter AGIP with the second intron of the Arabidopsis AGI gene from the genome,

primers

3 and 4 are designed, and are respectively SEQ ID NO.5(AGIP amplification upstream primer 3, containing HindIII enzyme cutting site, 5' -AAGCTTCTTAATCTACGCTTAAATCTGCAT-3') and SEQ ID NO.6(AGIP amplification downstream primer 4, containing SalI cleavage site, 5-GTCGACGTCGACTAGCTTCAGCGTGTCCTCTCCAAATGAAATGAACT TCCTTATATAGAGGAAGGGTCGGATCTCTAGTAATCTCTAA-3'), wherein the downstream primer 4 contains 35S mini promoter sequence, and the nucleotide sequence of AGIP cloned from Arabidopsis genome is directly amplified, as shown in SEQ ID NO. 2.

3. Construction of vector interfering with cytokinin oxidase gene in cotton

After GhCKX3bRNAi and AGIP nucleotide sequences are obtained through respective amplification, the sequences are connected with a pUCm-T vector to transform escherichia coli, plasmid digestion and sequencing verification are extracted, and the plasmid with correct sequencing is digested by corresponding restriction enzymes. Meanwhile, the plasmid of the p5 vector was digested with restriction enzymes HindIII and EcoRI. The restriction enzymes were obtained from Thermo Scientific company and were digested as described in the specification. After the agarose gel electrophoresis separation of the digestion product, the gel is cut and recovered, and T is utilized₄And (5) DNA ligase connection. T4DNA ligase was a product of MBI and ligated as described in the specification. All restriction enzymes were purchased from Roche, following the instructions for use. The structure of the constructed plant expression vector is shown in FIG. 3, which includes expression interfering cell fractionThe nucleotides of the lyase gene (SEQ ID NO.1 and SEQ ID NO.2) and the elements required for expression screening.

Example 5

Preparation of transformants and transgenic plants

1. The constructed plant expression vector plasmid was introduced into Agrobacterium LBA4404 by electric stimulation.

The above vector was introduced into Agrobacterium LBA4404 by electroporation, referred to Bio-RAD MicroPulser user instructions.

The method comprises the following specific steps:

(1) the electric shock cup is washed by sterile water for 4-6 times in advance, dried on a super clean bench and then placed on ice for precooling.

(2) Taking Agrobacterium-infected cells stored at-80 deg.C, thawing on ice (2min), adding 2 μ L of Escherichia coli plasmid to be transformed, gently blowing, sucking, mixing, and standing on ice for 10 min.

(3) The electric shock conversion apparatus (Bio-Rad) was turned on, adjusted to the Agr mode, and 800. mu.L of liquid YEB medium was taken for use.

(4) Transferring the bacterial liquid into an electric shock cup, quickly adding a prepared YEB culture medium after electric shock, blowing, uniformly mixing, and carrying out shaking culture at 28 ℃ and 200rpm for 4 h.

(5) The bacterial liquid was centrifuged at 10000rpm for 1min at room temperature.

(6) The supernatant was discarded, and the cells were resuspended in 100. mu.L of liquid YEB medium, spread evenly on YEB plates containing 125mg/L Sm and 50mg/LKm, and subjected to inverted culture at 28 ℃ for 36 hours.

2. Integration of vectors interfering with the cotton cytokinin oxidase gene into the cotton genome

The expression vector is introduced into cotton by an agrobacterium-mediated hypocotyl method.

The specific method comprises the following steps:

(1) the kernel of Ji Cotton 14 is sterilized with 75% ethanol for 2min, and washed with sterile water for at least 2 times.

(2) Further 0.1% (w/v) of HgCl was added₂Sterilizing for 10min, and washing with sterile water for at least 5 times.

(3) Shaking and culturing with sterile water at normal temperature on a shaking table at 120rpm for 12 h.

(4) Inoculating the seeds into a germination culture medium, and culturing for 2-3 d at 30 ℃.

(5) Simultaneously preparing agrobacterium for transformation:

picking a single colony, inoculating the single colony in 15mL of liquid YEB culture medium (containing 125mg/L Sm and 50mg/LKm), and carrying out shaking culture at 28 ℃ and 200rpm for overnight;

secondly, putting 1mL of the bacterial liquid into 20-25 mL of YEB liquid culture medium, and carrying out shaking culture at 28 ℃ and 200rpm until OD600 is about 0.8;

③ centrifuging at 10000rpm for 1min at room temperature, collecting the thalli, and resuspending the thalli by using an equal volume of liquid coculture medium (containing AS).

(6) Cotton sterile hypocotyls were cut into small sections of about 1cm, and stained with resuspended agrobacterium solution: dip-dyeing is carried out at 28 ℃ and 100rpm for 45 min.

(7) The hypocotyls were inoculated onto solid co-medium (containing AS) and cultured in the dark at 28 ℃ for 2 d.

(8) Subcultures were carried out once at 2-week intervals and different tissue culture media (tables 2 and 3) were replaced according to the auxin status of the material, and kanamycin was used for resistance selection throughout the process until seedlings were formed.

(9) Transplanting the seedlings to a greenhouse for propagation after the seedlings take roots.

TABLE 2 Agrobacterium tumefaciens-mediated culture medium MSB mother liquor formulation for cotton genetic transformation

TABLE 3 Agrobacterium tumefaciens-mediated culture media for genetic transformation of cotton

Gelrite: sigma, cat No.: g1910; SH: schenk & Hildebrandt, 1972.

Example 6

Detection of expression of cytokinin oxidase gene GhCKX3b in cotton flower bud by Real-time PCR method

Total RNA was extracted from control and transgenic cotton flower buds and synthesized into one strand of cDNA by reverse transcription. This was used as a template for quantitative real-time PCR amplification.

The specific operation steps are as follows:

(1) the quality of the synthesized cDNA is firstly detected: taking 1 mu L of cDNA as a template, adding primers (SEQ ID NO.7 (5'-GAAGCCTCATCGATACCGTC-3') and SEQ ID NO.8 (5'-CTACCACTACCATCATGGC-3')) of a cotton internal reference gene Histone3, amplifying by using a real-time quantitative PCR instrument, detecting Ct values, and stabilizing the Ct values of all the templates between 16-20.

(2) The cDNA was diluted 5-fold with sterile distilled water.

(3) The analytical system and procedure were:

the RT-qPCR reaction system is (20 mu L): 10 μ L of 2 XSybr Green qPCR Mix (Aidlab) +3 μ L H₂O + 5. mu.L of cDNA + 5. mu. mol/L of each of the upstream and downstream specific primers, respectively.

The amplification procedure is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 20sec, annealing at 56 ℃ for 20sec, and extension at 72 ℃ for 30sec for 40 cycles. The specificity of the primers was judged by melting curve.

(4) Data were analyzed by Bio-Rad CFX Manager. Each experiment was performed in 3 replicates, from which one plot was taken.

The cotton Histone3 gene is used as an internal standard, and the upstream primer and the downstream primer are respectively SEQ ID NO.7 and SEQ ID NO. 8. The upstream and downstream primers for amplifying the GhCKX3b gene are respectively SEQ ID NO.9 (5'-GGATGATAGGATGTCAGCTGTA-3') and SEQ ID NO.10 (5'-CTCTGGCCTGGTGACAACAACA-3').

The expression level of the GhCKX3b gene in the flower buds of the transgenic and wild plants was determined by the above method, and the results are shown in FIG. 5(AGIP: Real-time PCR analysis of the GhCKX3b gene in the flower buds of GhCKX3bRNAi transgenic cotton; wild type: untransformed Ji Cotton No. 14 cotton as control). The lowest expression level of the GhCKX3b gene in the transgenic strain is ACR1, and then ACR5 and ACR7, the expression level is moderately reduced to ACR6, and the expression levels are ACR2, ACR3 and ACR4 without obvious change.

Example 7

AGIP GhCKX3bRNAi transgenic Cotton boll number survey

1. Comparative analysis of number of ovules per boll for transgenic Cotton and controls

The cotton planted in the test field was managed conventionally, and the number of ovules per boll was counted for 15 bolls on the day of flowering at the full-bloom stage, and the results are shown in FIG. 6(AGIP: statistics of the number of ovules per boll for the day of flowering for GhCKX3bRNAi transgenic cotton; the number of ovules per boll was different for 7 transgenic lines, the number of strains ACR1, ACR5, ACR6, and ACR7 was significantly increased as compared with the wild type control, and the number of strains ACR2, ACR3, and ACR4 was not significantly different as compared with the wild type) and FIG. 7(AGIP: statistics of the number of ovules per chamber for bolls on the day of flowering for GhCKX3bRNAi transgenic cotton and 35S: GhCKX3bRNAi transgenic cotton; scale: 1 mm. The number of ovules per bell of transgenic lines ACR1, ACR5, ACR6, ACR7 was significantly higher than the wild-type material. Analysis of the expression level of GhCKX3b in the flower buds combined with the transgenic material shows that the expression level of GhCKX3b interferes with more obvious strains, and the number of ovules is increased more. In the ACR1 strain in which GhCKX3b interference was most pronounced, the proportion of 10 ovules per chamber of bolls was significantly increased and a small number of 11 cases occurred, whereas the proportion of 9 ovules per chamber was greater in the wild type and 35S: CKX3bRNAi transgenic (35SCR) material, and 11 cases were not observed.

Example 8

AGIP (agricultural chemical marker: GhCKX3 bRNAi) transgenic cotton plant type, boll and fiber quality character investigation

1. Transgenic material plant type and fiber initiation observation

Even the line in which expression of GhCKX3b was most significantly disturbed in the flower buds grew normally with no significant difference compared to the wild type (FIG. 8, AGIP: GhCKX3bRNAi transgenic cotton plant phenotype; wild type: cotton Ji No. 14 cotton from a non-transgenic isolate as a control; scale 10 cm). The scanning electron microscope was used to observe the initiation of 2d fiber at the day of flowering and after flowering, which was not significantly different from the wild type (FIG. 9, growth of fiber on the ovule epidermis at the day of flowering (0DPA) and 1d (1DPA) after flowering of AGIP: GhCKX3bRNAi transgenic cotton; scale: 100 μm).

2. Seed and fiber yield statistics

After approval of the transgenic intermediate trials, random block experiments were performed experimentally. Each cell area is about 18m²The seeds are planted in 4 rows, the row spacing is 1.0m, the total number of the 15 plants in each row is 60, and the spacing between adjacent plants is 0.3 m. Each material was randomly planted in 3 plots. After the cotton bolls are mature and cracked, counting the number of single bolls (8 months end) of each plant by taking 2 lines in the middle of each cell, harvesting the cotton bolls, drying the cotton bolls to constant weight, weighing the total weight of seed cotton, delinting the cotton seeds by a delinting machine, weighing the total weight of fibers and the total weight of the cotton seeds, and calculating to obtain the single boll weight, the clothes fraction, the clothes finger, the number of single bolls, the seed finger, the seed cotton yield of the cell, the ginned cotton yield of the cell and the seed yield of the cell. The results are shown in table 4, the average number of single-boll seeds of the transgenic cotton is obviously increased compared with that of the wild type and 35S: GhCKX3bRNAi transgenic cotton, the single-boll weight of the transgenic cotton is obviously increased compared with that of the wild type, and the yield of cell cotton seeds, the yield of cell ginned cotton and the yield of cell seeds are all obviously increased and are simultaneously better than that of the 35S: GhCKX3bRNAi transgenic cotton to a certain extent.

TABLE 4 comparison of yield traits in transgenic Cotton and controls

3. Quality detection of transgenic cotton fibers

Fiber lengths were measured after picking 30 cotton seeds matured from AGIP, GhCKX3bRNAi transgenic and wild type cotton, and the fiber lengths were measured as shown in FIG. 10 (statistics of mature fiber lengths of AGIP, GhCKX3bRNAi transgenic cotton; wild type, Ji cotton No. 14 cotton from non-transgenic segregating line as control), which showed no significant difference between the fibers of the transgenic lines and the wild type.

Each cotton fiber sample is sent to the cotton quality supervision and detection test center (Anyang) of Ministry of agriculture, and the average length, the uniformity index, the breaking ratio strength, the elongation and the micronaire value 5 index of the upper half are tested by adopting HFT9000 under the environmental condition of 20 ℃ of temperature and 65% of relative humidity according to ASTM D5867-95 'HVI 900 large-capacity fiber tester test method'. The control material is Ji cotton No. 14 plant of non-transgenic segregation line. The results are shown in Table 5.

The 2017 detection result shows that the indexes of average length and uniformity of the upper half parts of the fibers of the ACR1 and ACR5 transgenic materials are not obviously different from those of wild type control. Compared with the wild type, the breaking ratio strength and the elongation of the ACR5 transgenic material are not obviously different, the ACR1 transgenic material is respectively reduced by 12.0 percent and 1.5 percent, and the micronaire value is increased by 3.7 percent compared with the wild type. The test result in 2019 shows that the average length, the uniformity index, the breaking ratio strength and the elongation of the upper half part of the ACR1 transgenic material are respectively reduced by 4.2%, 2.4%, 11.6 and 1.5%, and the micronaire value is not obviously changed. The micronaire and elongation of the ACR5 transgenic material decreased by 11.1% and 1.5%, respectively.

TABLE 5 comparison of fiber quality traits in transgenic Cotton and controls

The results show that the method for improving the yield of the cotton seeds and the cotton fibers can realize the down-regulation of the expression of the cotton cytokinin oxidase gene in the cotton flower primordium, increase the number of ovules and seeds and achieve the aim of increasing the yield of the seeds and the fibers. The test results prove that the cotton plants improved by the method for improving the cotton yield have normal growth and fiber development, the number of single-boll seeds, the weight of single bolls, the yield of cotton seeds in a cell, the yield of ginned cotton in the cell and the yield of seeds in the cell are all obviously increased, and the quality of the cotton fibers is still within an acceptable range although being influenced to a certain degree. The method is simple and easy to implement, has obvious effect and has good market prospect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of southwest

<120> fusion gene for improving cotton yield, plant expression vector, transformant and application

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aatattcttt tgtcctgttc acaaagattc caacaacaaa cttattttcc agcatttctt 480

tgggtaaaat taccataaac atcttactgc attttactca ttaatctatg tacgttagat 540

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cgtaaatttt ttaacaatat aattaaatga aatttttaat aaaaacaatc aatttacttt 720

aacttaaatt tataaaaatt ttaaataaaa aagataaaaa tactatttaa ctattgatat 780

aaatatttct atgtattttt tatccatttc ttagctggtg tctgatggtt catcatatgt 840

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gaacatcatc accatcatca tcatcattta tataatatta tatatatgtt ggttgcatca 1020

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cttaatctac gcttaaatct gcattttgat aagtaataaa aaataaaatt gatagggtca 60

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cttttgagtc agaagaaatg caacttaatg tgacacatga tgtgaagaaa aaacaaaagt 180

aatataagaa aagggaacaa ttaaatagtt aataaaatat ttccttaaag ttgtaacaaa 240

taaagaatca ttttatgaaa caatatgaac cctaaataaa ttaaaattcc tctgaaacct 300

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tagaaggcag accaaactct taaactattg gatccgtact aaaaatctca ctttcctctc 780

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aagcaattcc aatgctatac ttctatgcca aatctattta ttcagtgatc attaatcttt 960

ttacttccaa gaaatatgaa caatttagta tccttataat ttttgtctct atatatgtaa 1020

tatgaacatt gggtattgac caaatgagaa atctaatatt aaatggtcaa aagtagtaat 1080

atgatgacat ttttgaattt ataaataggt tacaaattaa ttcattatga cataaaacct 1140

tcttgtcaga agtcaagaac tgaaactaac aaaactttat aataaattag taaaaataca 1200

agtgaaaaat aaaaagaaat aatatctgag tgatgacgtg atcaaagatt ctttaacaaa 1260

gacaacaaat cttacagacc caaaacctaa tcttgcgctc aattccaacc tctgaaaaaa 1320

cctcaaaaat cttataaaag aaaataaata aagaaacgaa actctgattt cgtagagtac 1380

ccatcggata tataaaaaga aattagtagg taaatgaaga ctaattttga ttgactgatt 1440

taatttgaag tcgttgttag cttttcttgt tttggacatg agaattatat atttcaggac 1500

atgagagttg acaactgtaa acgattaaga aaattgatct tttaattttc aaacaccatt 1560

taatcttgac atgttttatg ttttggtgga gaagaaagta atcacgtggg actctctact 1620

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gtcgacgcta ctccagttca gttcct 26

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gtcgacgtcg actagcttca gcgtgtcctc tccaaatgaa atgaacttcc ttatatagag 60

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gaagcctcat cgataccgtc 20

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ctaccactac catcatggc 19

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ggatgatagg atgtcagctg ta 22

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ctctggcctg gtgacaacaa ca 22

Claims

1. A fusion gene for improving cotton yield, wherein the fusion gene comprises a cotton cytokinin oxidase gene containing RNA interferenceGhCKX3bRNAiAnd a floral primordium-specific promoterAGIP(ii) a The above-mentionedGhCKX3bRNAiThe nucleotide sequence of (A) is shown as SEQ ID NO. 1; the floral primordium specific promoterAGIPThe nucleotide sequence of (A) is shown in SEQ ID NO. 2.

2. A plant expression vector for increasing the yield of cotton, which contains the cytokinin oxidase gene containing RNA interferenceGhCKX3bRNAiAnd a floral primordium-specific promoterAGIP(ii) a The above-mentionedGhCKX3bRNAiThe nucleotide sequence of (A) is shown as SEQ ID NO. 1; the floral primordium specific promoterAGIPThe nucleotide sequence of (A) is shown in SEQ ID NO. 2.

3. The plant expression vector of claim 2, wherein the backbone vector used to construct the plant expression vector is a p5 vector.

4. A method for preparing transgenic cotton based on the fusion gene of claim 1, comprising the following steps:

1) construction of a Cotton cytokinin oxidase Gene containing the RNA interference-containing Gene of claim 1GhCKX3bRNAiAnd a floral primordium-specific promoterAGIPThe plant expression vector of (1);

5. Use of the fusion gene of claim 1 or the plant expression vector of claim 2 or 3 to increase the cytokinin content in cotton flower buds.

6. Use of the fusion gene of claim 1 or the plant expression vector of claim 2 or 3 for increasing the number of cotton single boll seeds.

7. Use of the fusion gene of claim 1 or the plant expression vector of claim 2 or 3 for increasing cotton yield.

8. Use of the fusion gene of claim 1 or the plant expression vector of claim 2 or 3 for improving cotton seed and fiber yield.

9. Use of the fusion gene of claim 1 or the plant expression vector of claim 2 or 3 for breeding new varieties of plants.