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

Abstract

The application belongs to the technical field of genetic engineering, and particularly discloses AtDi19 Application of the gene in drought resistance and high-oil rape breeding AtDi19 The gene is first transferred into the rape by using the transgenic technology, and the transgenic rape obtained has significantly improved drought resistance compared with the wild type rape; and the oil content of the rape seed is significantly increased under the drought stress. Therefore, the application provides a new gene resource for drought-resistant and stable high-oil rape breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to application of AtDi19 gene in drought resistance and high-oil rape breeding. BACKGROUND

[0002] Drought stress seriously affects the growth and development of plants, and is one of the most serious survival challenges currently faced by plants. In production, drought leads to a large reduction in crop yield, causing huge economic losses to China. How to realize stable yield and supply of crops under limited water resources is a huge challenge currently faced by the agricultural field of China. Improving the response capacity of plants to drought stress and cultivating crop varieties with strong drought resistance are important ways to solve this problem.

[0003] Improving the oil yield of oil crops is a strategic requirement for ensuring the safety of national oil supply. As the largest oil crop in China, rapeseed accounts for more than 50% of the oil yield of domestic oil crops. In recent years, China has been promoting the expansion of rapeseed area, but with global warming, the rapeseed planting area (especially in the northern region) often suffers from severe drought. Therefore, cultivating new drought-resistant rapeseed varieties is of great significance for promoting the development of the rapeseed industry.

[0004] Rapeseed oil content is a quantitative trait controlled by multiple genes and is greatly affected by external environmental factors; current research shows that drought stress seriously affects the yield, oil content, and oil quality of rapeseed, causing huge economic losses to farmers. Therefore, excavating genes and sites 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 the rapeseed planting area, improving the oil yield of rapeseed, and maintaining the safety of national oil supply. SUMMARY

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

[0006] In one aspect, the present application protects the application of AtDi19 gene or the protein encoded thereby in improving the drought resistance of rapeseed and the oil content of rapeseed, the CDS sequence of the AtDi19 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the AtDi19 gene-encoding protein is shown as SEQ ID NO. 2. Preferably, the primer sequence for amplifying the AtDi19 gene is shown as SEQ ID NO. 3 and 4.

[0007] Another aspect of the present application protects a method for improving drought resistance of Brassica napus and oil content of seeds of Brassica napus under drought stress, by overexpressing AtDi19 gene in Brassica napus to improve drought resistance of Brassica napus and oil content of seeds of Brassica napus under drought stress, comprising constructing a plant expression vector containing AtDi19 gene (such as plant expression vector pCAMBIA1305), and transforming the AtDi19 gene into Brassica napus by Agrobacterium transformation method.

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

[0009] Compared with the prior art, the present application has the beneficial effects that: the present application uses transgenic technology to first transfer drought-resistant gene AtDi19 of an exogenous species into Brassica napus, and the obtained transgenic Brassica napus has significantly improved drought resistance compared with wild-type Brassica napus that has not been transformed; and under drought stress conditions, the oil content of the transgenic Brassica napus is significantly higher than that of wild-type Brassica napus that has not been transformed. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 Protein homology alignment results of AtDi19 in Brassica napus.

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

[0012] Figure 3 Transgenic Brassica napus drought resistance identification results.

[0013] Figure 4 Survival rate of transgenic Brassica napus after drought treatment.

[0014] Figure 5 Oil content of seeds of transgenic Brassica napus after drought treatment. DETAILED DESCRIPTION

[0015] In order to further illustrate the present application, the application of AtDi19 gene in drought resistance and high oil breeding of Brassica napus provided by the present application is described in detail below in combination with the drawings and examples, but it is only a part of the embodiments of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, which all belong to the protection scope of the present application. If no special description, the technical means used in the following examples are all conventional means known by those skilled in the art, and the materials and reagents used can be obtained from commercial channels. In the specific embodiments of the present application, the Brassica napus L. used is Brassica napus L. 862 (a spring rape strain collected in the laboratory), and the E. coli DH5a 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 Brassica napus

[0017] Previous studies in Arabidopsis thaliana showed that AtDi19 gene is an important transcription factor in the process of Arabidopsis thaliana responding to drought stress, and overexpression of AtDi19 gene in Arabidopsis thaliana can significantly improve the drought resistance of Arabidopsis thaliana (Liu, W. X., et al., Arabidopsis Di19 Functions as a Transcription Factor and Modulates PR1, PR2, and PR5 Expression in Response to Drought Stress. Molecular Plant, 2013.). In the present application, the amino acid sequence of Arabidopsis thaliana AtDi19 gene is used as a template to perform blast comparison and sequence homology analysis on the Brassica napus genome, and it is found that the similarity of Arabidopsis thaliana AtDi19 protein sequence and Brassica napus protein sequence is less than 40% ( Figure 1 ). Therefore, based on the great difference of AtDi19 protein sequence between Arabidopsis thaliana and Brassica napus, it is temporarily impossible to infer the function of AtDi19 gene in Brassica napus.

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

[0019] According to the backbone vector pCAMBIA1305, recombination primers AtDi19-F (SEQ ID NO. 3: 5'-ggacagcccagatcaactagtatggacgctgattccaagag-3') and AtDi19-R (SEQ ID NO. 4: 5'-gcccttgctcaccatggatccgacttcatcgaaaatggatg-3') are designed at the recombination site at both ends of the enzyme cutting site for PCR. The PCR reaction system is as follows: 50 μL, preferably comprising: 2x Phanta Flash Master Mix (Dye Plus) 25 μL, AtDi19-F 2 μL, AtDi19-R 2 μL, Arabidopsis thaliana cDNA 1 μL and ddH2O 20 μL. The PCR amplification program is: 98 ℃ pre-denaturation for 30 s; 94 ℃ denaturation for 10 s, 65 ℃ annealing for 5 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 1 min, and finally 4 ℃ preservation. The amplified PCR product is purified using a PCR product purification kit and stored for standby. The plant binary expression vector pCAMBIA1305 is double enzyme cut using SpeI and BamH I restriction endonucleases, detected by 1% agarose gel electrophoresis, and recovered using a gel recovery kit and stored.

[0020] The PCR purified product of the AtDi19 gene and the recovered enzyme cut plasmid are used for plasmid recombination using a ClonExpress recombination cloning kit, and the 20 μL reaction system is: 5x CE II Buffer 4 μL, Exnase II 2 μL, linearized vector 50-200 ng, insert 10-200 ng, and finally supplemented with ddH2O to 20 μL. The recombination product is transformed into E. coli DH5α competent cells, spread on LB solid medium added with 50 mg / L kanamycin (Kan), and incubated in a 37 ℃ incubator for 12-16 h. The newly grown single colonies are detected by PCR using the upstream primer (SEQ ID NO. 5: 5'-gacgcacaatcccactatcc-3') of the 35S promoter of the pCAMBIA1305 vector and the downstream primer (SEQ ID NO. 6: 5'-gataatcatcgcaagaccgg-3') of the NOS terminator. The single colonies that are positive in PCR verification are sent to Shanghai Sangon Biological Technology Co., Ltd. for Sanger sequencing. The sequencing results show that the full-length CDS of the AtDi19 gene is cloned, the nucleotide sequence is SEQ ID NO. 1, and the encoded amino acid sequence is shown as SEQ ID NO. 2. The expression vector plasmid of the positive single colony with correct sequencing is extracted, and the plasmid is the recombinant plasmid pCAMBIA1305-AtDi19 (vector construction as shown in FIG. 1).Figure 2

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

[0022] 1. Agrobacterium transformation

[0023] (1) Agrobacterium GV3101 competent cells stored at -80°C were thawed to an ice water mixture state at room temperature and then inserted into ice;

[0024] (2) About 100 ng of pCAMBIA1305-AtDi19 recombinant plasmid was taken up with a pipette and added to 100 μL of GV3101 competent cells, which were sequentially placed 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) About 700 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) was then added, and the mixture was cultured at 28°C on a shaker at 200 rpm for 2-3 h;

[0026] (4) The bacterial solution was spread on LB solid medium added with 50 mg / L kanamycin (Kan), 50 mg / L gentamycin (Gent), and 50 mg / L rifampicin (Rif), and cultured in an inverted culture box at 28°C for 36-48 h;

[0027] (5) The grown Agrobacterium monoclonal was subjected to PCR detection with primers upstream of the 35S promoter and downstream of the NOS terminator in the plant overexpression vector pCAMBIA1305, and the positive monoclonal was shaken to an OD 600 value of 1.8-2.0, and preserved in a -80°C ultra-low temperature refrigerator using 50% glycerol.

[0028] 2. Preparation of Agrobacterium suspension with AtDi19 target gene

[0029] (1) 200 mL of LB liquid medium (containing 50 mg / L Kan, 50 mg / L Gent, and 50 mg / L Rif) was inoculated with the above Agrobacterium solution carrying the target gene at a volume ratio of 1:100, and cultured at 28°C on a shaker at 200 rpm until the OD 600 value was about 0.6;

[0030] (2) The bacterial solution was centrifuged in a high-speed centrifuge at 8000 rpm for 15 min, and the supernatant was discarded, and the bacterial body was collected;

[0031] (3) The Agrobacterium bacterial body was resuspended with a resuspension solution (5% sucrose and 0.02% surfactant Silwet L-77) to an OD 600 ​about 1.0, to prepare Agrobacterium suspension carrying pCAMBIA1305-AtDi19 vector for subsequent rape transformation.

[0032] Example 4 Transformation and identification screening of overexpression AtDi19 rape

[0033] 1. The rape transformation was carried out by using cotyledon petiole infection method, and the specific operation steps were as follows:

[0034] (1) The 862 rape seeds (which have been disclosed in the article Fan, S. H., 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.) were soaked with 70% ethanol solution for 1 min, then soaked with mercury chloride (HgCl2) solution for 13-15 min, washed with sterile water for 5 times, then plated on MS medium and placed in an artificial climate incubator for growth to obtain rape sterile seedlings for standby;

[0035] (2) The 4-5 day old sterile seedling cotyledon petiole was taken and soaked in the above-mentioned Agrobacterium suspension carrying pCAMBIA1305-AtDi19 vector for 5-8 min, with light shaking in between, then the bacterial solution was poured out and the residual bacterial solution on the explant was absorbed; the transformed cotyledon petiole was placed on 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 acetyl-syringone) for 2-3 days;

[0036] (3) The co-cultured explants were transferred to differentiation medium containing penicillin (400 mg / L Car) for decontamination and differentiation culture in dark conditions for 5-7 days; then transferred to selection medium containing Kan (containing 3 mg / L 6-BA, 0.1 mg / L α-naphthaleneacetic 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 1 cm long 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 are rooted and grow leaves, DNA of the transformed seedlings is extracted, and PCR identification is performed with the upstream primer of the plant expression vector 35S promoter and the downstream primer of the AtDi 19 gene;

[0039] (6) The positive seedlings identified by PCR are transplanted into pots in the artificial climate greenhouse, and after the seeds mature, Tl generation transgenic rape seeds are obtained by seed collection of single plants.

[0040] 2. Homozygous positive transgenic rape plants are screened by using hygromycin, and the specific operation steps are as follows:

[0041] (1) The Tl generation transgenic rape seeds are sterilized according to the experimental operation steps of the above sterile rape seedlings;

[0042] (2) The sterilized seeds are spread on MS solid medium containing 25 mg / L hygromycin, and are cultured in the dark for about 3 days in the incubator, and then are cultured under normal light;

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

[0044] (4) After the positive transgenic rape seeds mature, T2 generation transgenic rape seeds are obtained by seed collection of single plants and are sequentially numbered and stored;

[0045] (5) The T2 generation transgenic rape seeds are screened for resistance to hygromycin according to the above experimental steps, and the strain is separated at a ratio of 3:1, is transplanted into the artificial climate greenhouse, is harvested by single plant, and is sequentially numbered and stored to obtain T3 generation transgenic rape seeds;

[0046] (6) The T3 generation transgenic rape seeds are screened for resistance to hygromycin according to the above experimental steps, and all strains that grow normally are screened to obtain homozygous T3 generation transgenic rape.

[0047] Example 5 Drought resistance identification of AtDi 19 transgenic rape

[0048] The screened T3 generation homozygous transgenic rape and untransformed wild type rape (862 rape) are sown in pots in the artificial climate greenhouse, and after the seeds germinate for two weeks, drought treatment is started. The specific implementation scheme is as follows: one day before the drought treatment, the two-week-old rape is watered to make the soil completely absorb water; then the excess water in the pot is poured out, and the drought treatment is started; after 3 weeks of drought treatment, the wild type rape appears obvious drought withering phenotype, and then is rewatered; after 5 days of rewatering, the growth state and survival rate of the experimental group and the control group are observed and recorded. The results show that after drought treatment, the wild type rape is basically withered and dead, and the AtDi 19 transgenic rape grows well Figure 3According to statistics, compared with wild-type rapeseed, the survival rate of AtDi19 transgenic rapeseed significantly increased after drought treatment. Figure 4 ).

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

[0050] The selected T3 generation homozygous transgenic rapeseed and untransformed wild-type rapeseed (862 rapeseed) were sown in nutrient pots in an artificial climate greenhouse. Two weeks after seed germination, drought treatment was initiated. The specific implementation plan was as follows: one day before the drought treatment, the two-week-old rapeseed was fully watered until the soil was completely saturated; then, excess water was drained from the pots, and the drought treatment began; two weeks after the drought treatment, the soil was re-watered for a rehydration experiment. After the seeds were fully mature, they were threshed, and the oil content of the rapeseed seeds was determined using nuclear magnetic resonance spectroscopy. The results showed that compared with untransformed wild-type rapeseed, the oil content of AtDi19 transgenic rapeseed seeds was significantly increased after drought treatment. Figure 5 ).

Claims

1. The application of the AtDi19 gene or its encoded protein in improving the drought resistance and oil content of rapeseed seeds under drought stress, characterized in that, 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.

2. A method for improving the drought resistance and oil content of rapeseed seeds under drought stress, characterized in that, Express the AtDi19 gene of claim 1 in rapeseed.

3. The method according to claim 2, characterized in that, This includes constructing a plant expression vector containing the AtDi19 gene and transferring the AtDi19 gene into rapeseed using Agrobacterium-mediated transformation.

4. The method according to claim 3, characterized in that, The plant expression vector is pCAMBIA1305.

5. A breeding method for drought-resistant rapeseed and for increasing the oil content of rapeseed seeds under drought stress, characterized in that, The method includes constructing a plant expression vector containing the AtDi19 gene, transforming the AtDi19 gene into rapeseed using Agrobacterium-mediated transformation, and continuously screening to obtain homozygous rapeseed transgenic with the AtDi19 gene. The CDS sequence of the AtDi19 gene is shown in SEQ ID NO.1.

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