Application of AtDi19 gene in breeding of drought-resistant and high-oil rape

Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses application of an AtDi19 gene in breeding of drought-resistant and high-oil oilseed rape, the AtDi19 gene of an exogenous species is transferred into oilseed rape for the first time by utilizing a transgenic technology, and the drought resistance of the obtained transgenic oilseed rape is remarkably improved compared with that of wild oilseed rape; under the drought stress condition, the oil content of the rape seeds is obviously increased. Therefore, the invention provides a new gene resource for breeding drought-tolerant and stable high-oil rape.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to the application of AtDi19 gene in drought-resistant and high-oil rapeseed breeding. Background Art

[0002] Drought stress seriously affects the growth and development of plants and is one of the most significant challenges to their survival. In production, drought has led to a large reduction in crop yields, causing huge economic losses to my country. How to achieve stable production and supply of crops under limited water resources is a huge challenge currently facing my country's agricultural sector. Improving the ability of plants to respond to drought stress and cultivating crop varieties with strong drought resistance are important ways to solve this problem.

[0003] Increasing the oil yield of oil crops is a strategic need to ensure the security of the country's oil supply. Rapeseed is the largest oil crop in my country, and rapeseed oil accounts for more than 50% of the domestic oil crop production. In recent years, my country has been promoting the expansion of rapeseed areas, but with global warming, rapeseed planting areas (especially in the north) often suffer from severe droughts. Therefore, cultivating new drought-resistant rapeseed varieties is of great significance to promoting the development of the rapeseed industry.

[0004] The oil content of rapeseed is a quantitative trait controlled by multiple genes and greatly affected by external environmental factors. Current studies have shown that drought stress seriously affects agronomic traits such as rapeseed yield, oil content, and oil quality, causing huge economic losses to farmers. Therefore, exploring the genes and loci that regulate rapeseed oil content under drought stress conditions and cultivating stable high-oil rapeseed varieties that are not affected by drought stress are of great significance for expanding rapeseed planting areas, increasing rapeseed oil production, and maintaining national oil supply security. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the AtDi19 gene in drought-resistant and high-oil rapeseed breeding, and to improve the drought resistance of rapeseed and its oil content under drought conditions by overexpressing the AtDi19 gene in rapeseed.

[0006] On the one hand, the present invention protects the use of the AtDi19 gene or the protein encoded by it in improving the drought resistance and oil content of rapeseed, the CDS sequence of the AtDi19 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the AtDi19 gene is shown in SEQ ID NO.2. Preferably, the primer sequences used to amplify the AtDi19 gene are shown in SEQ ID NOs.3 and 4.

[0007] On the other hand, the present invention protects a method for improving the drought resistance of rapeseed and the oil content of rapeseed seeds under drought stress, by overexpressing the AtDi19 gene in rapeseed to improve the drought resistance of rapeseed and the oil content of rapeseed seeds under drought stress, comprising constructing a plant expression vector containing the AtDi19 gene (such as the plant expression vector pCAMBIA1305), and transferring the AtDi19 gene into rapeseed by Agrobacterium transformation.

[0008] The present invention also protects a breeding method for drought-resistant and high-oil rapeseed, comprising constructing a plant expression vector containing the AtDi19 gene, transferring the AtDi19 gene into rapeseed by Agrobacterium transformation, and continuously screening to obtain homozygous AtDi19 gene-transformed rapeseed.

[0009] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses transgenic technology to transfer the drought-resistant gene AtDi19 of an exogenous species into rapeseed for the first time, and the drought resistance of the obtained transgenic rapeseed is significantly improved compared with that of the untransformed wild-type rapeseed; and under drought stress conditions, the oil content of the transgenic rapeseed is significantly increased compared with that of the untransformed wild-type rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the result of protein homology comparison of AtDi19 in rapeseed.

[0011] Figure 2 Schematic diagram of the construction of recombinant plasmid pCAMBIA1305-AtDi19.

[0012] Figure 3 This is the result of drought resistance identification of transgenic rapeseed. 862 represents wild-type rapeseed, OE:AtDi19 represents AtDi19 transgenic rapeseed;

[0013] Figure 4 is the survival rate of transgenic rapeseed after drought treatment. 862 represents wild-type rapeseed, and OE:AtDi19 represents AtDi19 transgenic rapeseed.

[0014] Figure 5 is the oil content of transgenic rapeseed seeds after drought treatment. 862 represents wild-type rapeseed, and OE:AtDi19 represents AtDi19 transgenic rapeseed. DETAILED DESCRIPTION

[0015] In order to further illustrate the present invention, the application of the AtDi19 gene provided by the present invention in the breeding of drought-resistant and high-oil rapeseed is described in detail below in conjunction with the accompanying drawings and examples, but it is only a part of the embodiments of the present invention, not all of the embodiments, and people can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials and reagents used can be obtained from commercial channels. In a specific embodiment of the present invention, the rapeseed (Brassica napus L.) used is Brassica napus spring rapeseed 862 (a spring rapeseed line collected in the laboratory), and the Escherichia coli DH5α competent cells and Agrobacterium GV3101 competent cells used are purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0016] Example 1 Homology analysis of AtDi19 gene in rapeseed

[0017] Previous studies in Arabidopsis have shown that the AtDi19 gene is an important transcription factor in Arabidopsis in response to drought stress. Overexpression of the AtDi19 gene in Arabidopsis can significantly improve the drought resistance of Arabidopsis (Liu, WX, et al., Arabidopsis Di19 Functions as a Transcription Factor and Modulates PR1, PR2, and PR5 Expression in Response to Drought Stress. Molecular Plant, 2013.). The present invention uses the amino acid sequence of the Arabidopsis AtDi19 gene as a template to perform blast alignment and sequence homology analysis on the rapeseed genome, and finds that the similarity between the Arabidopsis AtDi19 protein sequence and the rapeseed protein sequence is less than 40% ( Figure 1 ). Therefore, based on the huge difference in AtDi19 protein sequence between Arabidopsis and rapeseed, it is currently impossible to infer the function of AtDi19 gene in rapeseed.

[0018] Example 2 Cloning of AtDi19 gene and construction of plant overexpression vector

[0019] According to the backbone vector pCAMBIA1305, the recombination primers AtDi19-F (SEQ ID NO.3: 5'-ggacagcccagatcaactagtatggacgctgattccaagag-3') and AtDi19-R (SEQID NO.4: 5'-gcccttgctcaccatggatccgacttcatcgaaaatggatg-3') were designed at the recombination sites at both ends of the restriction site for PCR. The PCR reaction system is as follows: 50 μL, preferably including: 2×Phanta Flash Master Mix (Dye Plus) 25 μL, AtDi19-F 2 μL, AtDi19-R 2 μL, Arabidopsis cDNA 1 μL and ddH2O 20 μL. The PCR amplification procedure was as follows: 98°C pre-denaturation for 30s; 94°C denaturation for 10s, 65°C annealing for 5s, 72°C extension for 30s, 35 cycles; 72°C extension for 1min, and finally storage at 4°C. The amplified PCR product was purified using a PCR product purification kit and stored for later use. The plant binary expression vector pCAMBIA1305 was double-digested with SpeI and BamH I restriction endonucleases, and after detection by 1% agarose gel electrophoresis, it was recovered and stored using a gel recovery kit.

[0020] The PCR purified product of the AtDi19 gene and the recovered restriction plasmid were recombined using the ClonExpress recombination cloning kit. The 20 μL reaction system was: 4 μL 5×CE II Buffer, 2 μL Exnase II, 50-200 ng of linearized vector, 10-200 ng of inserted fragment, and finally ddH2O was added to 20 μL. The recombinant product was transformed into Escherichia coli DH5α competent cells, spread on LB solid culture medium supplemented with 50 mg / L kanamycin (Kan), and inverted in a 37°C incubator for 12 to 16 hours. The newly grown single clones were PCR detected using the upstream primer of the 35S promoter of the pCAMBIA1305 vector (SEQ ID NO.5: 5'-gacgcacaatcccactatcc-3') and the downstream primer of the NOS terminator (SEQ ID NO.6: 5'-gataatcatcgcaagaccgg-3'). The single clones that were positive after PCR verification were sent to Shanghai Shenggong Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results showed that the present invention cloned the full-length CDS of the AtDi19 gene, and its nucleotide sequence was SEQ ID NO.1, and the amino acid sequence encoded by it was shown in SEQ ID NO.2. The expression vector plasmid of the positive single colony with correct sequencing was extracted, and the plasmid was the recombinant plasmid pCAMBIA1305-AtDi19 (the vector construction is shown in Figure 2 shown).

[0021] Example 3 Agrobacterium transformation of pCAMBIA1305-AtDi19 recombinant plasmid

[0022] 1. Agrobacterium Transformation

[0023] (1) Thaw the competent cells of Agrobacterium tumefaciens GV3101 stored at -80°C to an ice-water mixture at room temperature and place them on ice;

[0024] (2) Use a pipette to draw about 100 ng of pCAMBIA1305-AtDi19 recombinant plasmid and add it to 100 μL of GV3101 competent cells. Place it on ice for 5 min, in liquid nitrogen for 5 min, at 37°C for 5 min, and on ice for 5 min.

[0025] (3) Then add about 700 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L), place in a shaker at 28 °C and shake at 200 rpm for 2 to 3 h;

[0026] (4) Spread the bacterial solution on LB solid medium supplemented with 50 mg / L kanamycin (Kan), 50 mg / L gentamicin (Gent), and 50 mg / L rifampicin (Rif), and culture in an inverted incubator at 28°C for 36 to 48 h;

[0027] (5) The grown Agrobacterium single clones were tested by PCR using the 35S promoter upstream primer and NOS terminator downstream primer in the plant overexpression vector pCAMBIA1305. The positive single clones were shaken to OD 600 =1.8-2.0, and then preserve the bacteria in 50% glycerol and store in -80℃ ultra-low temperature refrigerator for later use.

[0028] 2. Preparation of Agrobacterium Suspension with AtDi19 Target Gene

[0029] (1) Inoculate the Agrobacterium carrying the target gene in 200 mL of LB liquid medium (containing 50 mg / L Kan, 50 mg / L Gent, and 50 mg / L R Fif) at a volume ratio of 1:100, and place in a 28°C shaker at 200 rpm until OD 600 The value is about 0.6;

[0030] (2) Place the bacterial solution in a high-speed centrifuge and centrifuge at 8000 rpm for 15 min, discard the supernatant, and collect the bacterial cells;

[0031] (3) Resuspend the Agrobacterium cells in a resuspension solution (5% sucrose and 0.02% surfactant Silwet L-77) to an OD of 600The concentration was about 1.0, and an Agrobacterium suspension carrying the pCAMBIA1305-AtDi19 vector was prepared for subsequent rapeseed transformation.

[0032] Example 4 Transformation and Identification Screening of Rapeseed Overexpressing AtDi19

[0033] 1. Transformation of rapeseed using the cotyledon petiole infection method. The specific steps are as follows:

[0034] (1) 862 rapeseed (disclosed in the article, Fan, SH, et al., CRISPR / Cas9-targeted mutagenesis of the BnaA03.BP gene confers semi-dwarf and compact architecture to rapeseed (Brassica napus L.). Plant Biotechnology Journal, 2021.) seeds were soaked and disinfected in 70% ethanol solution for 1 min, then soaked and disinfected in mercuric chloride (HgCl2) solution for 13-15 min, washed with sterile water 5 times, and then spread on MS culture medium and placed in an artificial climate culture room for growth to obtain rapeseed sterile seedlings for use;

[0035] (2) Take the cotyledon petioles of sterile seedlings of 4 to 5 days old, place them in the above-mentioned Agrobacterium suspension carrying the pCAMBIA1305-AtDi19 vector and soak them for 5 to 8 minutes, shake them gently, then pour out the bacterial solution and absorb the residual bacterial solution on the explants; place the transformed cotyledon petioles on a co-culture medium (containing 0.2 mg / L 6-benzyladenine (6-BA), 1 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and 200 μM acetosyringone) and culture them for 2 to 3 days;

[0036] (3) The co-cultured explants were transferred to a differentiation medium containing penicillin (400 mg / L Car) and sterilized and differentiated in a dark greenhouse for 5 to 7 days; then transferred to a selection medium supplemented with Kan (containing 3 mg / L 6-BA, 0.1 mg / L α-naphthylacetic acid (NAA), 5 mg / L silver nitrate (AgNO3), 400 mg / L Car and 15 mg / L Kan) for screening;

[0037] (4) After the explants differentiated into 1 cm long regenerated green shoots, the small shoots were cut and transferred to rooting medium (containing 0.2 mg / L NAA, 10 mg / L Kan and 400 mg / L Car) for screening;

[0038] (5) After the transformed seedlings have taken root and grown leaves, the DNA of the transformed seedlings is extracted and PCR identification is performed using the upstream primer of the 35S promoter of the plant expression vector and the downstream primer of the AtDi19 gene;

[0039] (6) The positive seedlings identified by PCR were transplanted into nutrient pots in an artificial climate greenhouse for growth. After the seeds matured, individual plants were harvested to obtain T1 generation transgenic rapeseed seeds.

[0040] 2. Use hygromycin to screen homozygous positive transgenic rapeseed plants. The specific steps are as follows:

[0041] (1) Referring to the experimental operation steps of the above-mentioned rapeseed sterile seedlings, the T1 generation transgenic rapeseed seeds were disinfected;

[0042] (2) Spread the sterilized seeds on MS solid medium containing 25 mg / L hygromycin, culture them in the dark in an incubator for about 3 days, and then switch to culture under normal light;

[0043] (3) When two true leaves grow, transplant the positive seedlings into nutrient pots in an artificial climate greenhouse;

[0044] (4) After the positive transgenic rapeseed seeds mature, the seeds are harvested individually and numbered and stored in sequence to obtain T2 generation transgenic rapeseed seeds;

[0045] (5) The T2 transgenic rapeseed seeds were screened for hygromycin resistance according to the above experimental steps, and strains with a separation ratio of 3:1 were screened and transplanted into an artificial climate greenhouse. Individual plants were harvested and numbered and stored in sequence to obtain T3 transgenic rapeseed seeds;

[0046] (6) The T3 transgenic rapeseed seeds were screened for hygromycin resistance according to the above experimental steps, and all the strains with normal growth were screened out to obtain homozygous T3 transgenic rapeseed.

[0047] Example 5 Identification of drought resistance of AtDi19 transgenic rapeseed

[0048] The screened T3 generation homozygous transgenic rapeseed and untransformed wild-type rapeseed (862 rapeseed) were sown in nutrient pots in an artificial climate greenhouse, and drought treatment was started two weeks after the seeds germinated. The specific implementation plan is as follows: one day before the drought treatment, the two-week-old rapeseed was watered to allow the soil to completely absorb water; then, the excess water in the pot was poured out and the drought treatment was started; after the drought treatment for about 3 weeks, the wild-type rapeseed showed obvious drought wilting phenotypes and then rewatered; after 5 days of rewatering, the growth status and survival rate of the experimental group and the control group were observed and recorded. The results showed that after the drought treatment, the wild-type rapeseed basically wilted and died, while the AtDi19 transgenic rapeseed was in good growth condition ( Figure 3); According to statistics, compared with wild-type rapeseed, the survival rate of AtDi19 transgenic rapeseed increased significantly after drought treatment ( Figure 4 ).

[0049] Example 6 Determination of oil content of AtDi19 transgenic rapeseed under drought stress

[0050] The screened T3 generation homozygous transgenic rapeseed and untransformed wild-type rapeseed (862 rapeseed) were sown in nutrient pots in an artificial climate greenhouse, and drought treatment was started two weeks after the seeds germinated. The specific implementation plan is as follows: the two-week-old rapeseed was watered one day before the drought treatment so that the soil was completely filled with water; then the excess water in the pot was poured out and the drought treatment was started; after two weeks of drought treatment, the seeds were watered again for a rehydration experiment, and the seeds were threshed after they were fully mature, and the oil content of the rapeseed seeds was determined using a nuclear magnetic resonance instrument. The results showed that compared with the untransformed wild-type rapeseed, the oil content of AtDi19 transgenic rapeseed seeds after drought treatment was significantly increased ( Figure 5 ).

Claims

1. AtDi19 The use of a gene or a protein encoded by the gene in improving the drought resistance and oil content of rapeseed is characterized in that: Said AtDi19 The CDS sequence of the gene is shown in SEQ ID NO.

1. AtDi19 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

2. The method according to claim 1 AtDi19 A primer pair for amplifying a gene, characterized in that: The amplification primer pair comprises AtDi19 -F and AtDi19 -R, AtDi19 -F nucleotide sequence is shown in SEQ ID NO.3, AtDi19 The nucleotide sequence of -R is shown in SEQ ID NO.

4.

3. A method for improving the drought resistance of rapeseed and the oil content of rapeseed seeds under drought stress conditions, characterized in that: Expressing the method of claim 1 in rapeseed AtDi19 Gene.

4. The method according to claim 3, characterized in that: Include build containing AtDi19 The plant expression vector of the gene is transformed into AtDi19 The gene was transferred into rapeseed.

5. The method according to claim 4, characterized in that The plant expression vector is pCAMBIA1305.

6. A method for breeding drought-resistant and high-oil rapeseed, characterized in that: Include build containing AtDi19 The plant expression vector of the gene is transformed into AtDi19 The gene was transferred into rapeseed and homozygous strains were obtained through continuous screening. AtDi19 Genetic rapeseed.

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