Tolerance gene Di19 and its application in breeding of rapeseed with tolerance to waterlogging

Abstract

The application belongs to the technical field of genetic engineering, and particularly discloses a salt-tolerant gene Di19 and application thereof in breeding of oilseed rape with salt tolerance, wherein the gene of Brassica carinata is first transferred into oilseed rape by using transgenic technology, and the transgenic oilseed rape obtained has significantly improved salt tolerance as compared with wild-type oilseed rape. Di19 Therefore, the application provides a new gene resource for breeding of oilseed rape with salt tolerance.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the waterlogging-resistant gene Di19 and its application in waterlogging-resistant breeding of rapeseed. Background Technology

[0002] Rapeseed is the most widely planted oilseed crop in my country, and rapeseed oil accounts for more than 50% of the country's domestic oilseed production. However, global warming has led to an increase in extreme weather events such as prolonged periods of rain and heavy rainfall, causing rapeseed to frequently suffer from waterlogging stress. Meanwhile, the Yangtze River Basin and the Huang-Huai-Hai Plain are my country's two major rapeseed producing areas. In the rice-oilseed and rice-rice-oilseed rotation models implemented in the Yangtze River Basin, waterlogging-sensitive winter rapeseed varieties are mainly planted. Therefore, excessive water in paddy fields and concentrated rainy weather in autumn and winter easily expose rapeseed to waterlogging stress. Current research shows that waterlogging stress causes severe hypoxia and ion toxicity in rapeseed, seriously affecting the development of underground roots and the growth of above-ground plants. It also weakens disease resistance, makes rapeseed more susceptible to lodging, and reduces seed oil content, severely restricting the development of the rapeseed industry. Therefore, cultivating waterlogging-tolerant rapeseed varieties suitable for the excessive water conditions in rapeseed planting areas will help promote the high-quality development of my country's rapeseed industry and is of significant strategic importance for ensuring the country's oil security.

[0003] However, the genetic improvement of waterlogged-tolerant rapeseed varieties in my country is currently progressing slowly, with limited available superior waterlogged-tolerant germplasm resources and a lack of stable QTL regions and usable functional gene loci. Therefore, the discovery of superior waterlogged-tolerant genes in rapeseed and the elucidation of their regulatory mechanisms can provide excellent gene loci and germplasm resources for waterlogged-tolerant rapeseed breeding, accelerating the advancement of more precise and effective genetic improvement of waterlogged-tolerant rapeseed. Summary of the Invention

[0004] This invention protects, in one aspect, a gene Di19 that improves the waterlogging resistance of rapeseed. The coding sequence of the Di19 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0005] Another aspect of this invention protects a method for improving the waterlogging resistance of rapeseed, which improves the waterlogging resistance of rapeseed by overexpressing the Di19 gene in rapeseed, including constructing a plant expression vector containing the Di19 gene (such as the plant expression vector pCAMBIA1305), and transferring the Di19 gene into rapeseed by Agrobacterium-mediated transformation.

[0006] This invention also protects a breeding method for waterlogging-tolerant rapeseed, comprising constructing a plant expression vector containing the Di19 gene, transferring the Di19 gene into rapeseed using Agrobacterium-mediated transformation, and continuously screening to obtain homozygous rapeseed transgenic with the Di19 gene.

[0007] Compared with the prior art, the present invention has the following advantages: The present invention utilizes transgenic technology to transfer the Di19 gene of Ethiopian mustard into rapeseed for the first time. The transgenic rapeseed obtained has significantly improved waterlogging tolerance during the germination period compared with the untransformed wild-type rapeseed. Attached Figure Description

[0008] Figure 1 The results show the protein homology comparison of Di19.

[0009] Figure 2 This is a schematic diagram of the construction of the recombinant plasmid pCAMBIA1305-Di19.

[0010] Figure 3 This shows the growth status of rapeseed seedlings after waterlogging stress. WT indicates wild-type rapeseed, and OE:Di19 indicates Di19 transgenic rapeseed.

[0011] Figure 4 The germination rate of rapeseed after waterlogging stress is given. WT indicates wild-type rapeseed, and OE:Di19 indicates Di19 transgenic rapeseed. Detailed Implementation

[0012] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, illustrates the Di19 waterlogging tolerance gene provided by the present invention and its application in waterlogging tolerance breeding of rapeseed. However, this is only a part of the embodiments of the present invention, not all of them. Those skilled in the art can obtain other embodiments based on this embodiment without creative intent, and these embodiments all fall within the protection scope of the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the materials and reagents used can be obtained commercially. In the specific embodiments of the present invention, the rapeseed (Brassica napus L.) used is Brassica napus 862 (a spring rapeseed strain collected in the laboratory), and the Escherichia coli DH5α competent cells and Agrobacterium GV3101 competent cells used were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0013] Example 1: Screening of the Di19 gene and analysis of its protein sequence homology

[0014] Previous genome-wide gene loss analysis of rapeseed (B. napus) and Ethiopian mustard (B. carinata) revealed that rapeseed lost the Bca4012B2G042770 gene during evolution. BLAST analysis of the Arabidopsis genome showed that the protein encoded by the Bca4012B2G042770 gene shares 74.87% sequence homology with the protein encoded by the Arabidopsis ATDIL9 gene (protein sequence alignment results are shown in the figure). Figure 1As shown in the figure, the present invention names the Bca4012B2G042770 gene as the Di19 gene and uses it for subsequent rapeseed genetic transformation experiments.

[0015] Example 2: Cloning of the Di19 gene and construction of plant overexpression vector

[0016] Using the CDS sequence of the Di19 gene as a reference sequence, the Di19 gene (sequence shown in SEQ ID NO.1) was artificially synthesized at Beijing Qingke Biotechnology Co., Ltd., and the pUC57-Di19 vector carrying the Di19 gene was obtained. Recombinant primers Di19-F (sequence shown in SEQ ID NO.3) and Di19-R (sequence shown in SEQ ID NO.4) were designed at both ends of the restriction enzyme sites of the rapeseed transgenic backbone vector pCAMBIA1305, and PCR experiments were performed using this primer pair with the pUC57-Di19 plasmid as a template. The PCR reaction system was as follows: 50 μL, preferably including: 25 μL of 2×Phanta Flash Master Mix (Dye Plus), 2 μL of Di19-F (concentration 10 μM), 2 μL of Di19-R (concentration 10 μM), 1 μL of pUC57-Di19 plasmid (10 ng), and 20 μL of ddH2O. The PCR amplification program was as follows: 98℃ pre-denaturation for 30 s; 94℃ denaturation for 10 s, 65℃ annealing for 5 s, 72℃ extension for 30 s, repeated 35 times; 72℃ extension for 1 min; and finally, storage at 4℃. The amplified PCR products were purified using a PCR product purification kit and stored for later use. The plant binary expression vector pCAMBIA1305 was double-digested with SpeI and BamHI restriction endonucleases, and the residues were recovered and stored using a gel extraction kit after detection by 1% agarose gel electrophoresis.

[0017] The purified PCR product of the Di19 gene and the recovered restriction enzyme plasmid were recombined using the ClonExpress Recombinant Cloning Kit. The 20 μL reaction volume consisted of: 4 μL 5×CE II Buffer, 2 μL Exnase II, 50–200 ng linearized vector, 10–200 ng insert fragment, and finally ddH2O to 20 μL. The recombinant product was transformed into E. coli DH5α competent cells and plated on LB solid medium supplemented with 50 mg / L kanamycin (Kan). After incubation at 37°C for 12–16 h, the newly grown single clones were detected by PCR using the upstream primer of the 35S promoter of the pCAMBIA1305 vector (sequence shown in SEQ ID NO.5) and the downstream primer of the NOS terminator (sequence shown in SEQ ID NO.6). Single clones that were positive by PCR were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. Sequencing results showed that the full-length CDS of the Di19 gene was obtained in this invention, with its nucleotide sequence as SEQ ID NO.1 and its encoded amino acid sequence as shown in SEQ ID NO.2. Expression vector plasmids were extracted from correctly sequenced positive single colonies; this plasmid is the recombinant plasmid pCAMBIA1305-Di19 (vector construction as follows). Figure 2 (As shown).

[0018] Example 3: Agrobacterium transformation of the pCAMBIA1305-Di19 recombinant plasmid

[0019] 1. Agrobacterium-mediated transformation

[0020] (1) Place the Agrobacterium GV3101 competent cells stored at -80℃ into ice water mixture at room temperature and then insert them into ice.

[0021] (2) Use a pipette to aspirate about 100 ng of pCAMBIA1305-Di19 recombinant plasmid and add it to 100 μL of GV3101 competent cells. Place the cells on ice for 5 min, in liquid nitrogen for 5 min, at 37℃ for 5 min, and on ice for 5 min in sequence.

[0022] (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℃, and shake at 200 rpm for 2-3 h;

[0023] (4) Spread the bacterial culture onto LB solid medium containing 50 mg / L kanamycin (Kan), 50 mg / L gentamicin (Gent) and 50 mg / L rifampin (Rif), and incubate in an inverted incubator at 28°C for 36–48 h;

[0024] (5) The grown Agrobacterium single clones were detected by PCR using the upstream primer of the 35S promoter and the downstream primer of the NOS terminator in the plant overexpression vector pCAMBIA1305. Positive single clones were cultured to OD. 600 After reaching a concentration of 1.8 to 2.0, preserve the bacteria using 50% glycerol in an ultra-low temperature freezer at -80°C for later use.

[0025] 2. Preparation of Agrobacterium suspension carrying the Di19 target gene

[0026] (1) Inoculate 200 mL of LB liquid medium (containing 50 mg / L Kan, 50 mg / L Gent and 50 mg / L Rif) with the above Agrobacterium tumefaciens carrying the target gene at a volume ratio of 1:100, and incubate at 28 °C on a shaker with shaking at 200 rpm until OD. 600 The value is approximately 0.6;

[0027] (2) Place the bacterial culture in a high-speed centrifuge, centrifuge at 8000 rpm for 15 min, discard the supernatant and collect the bacterial cells;

[0028] (4) Resuspend Agrobacterium cells in a resuspension solution (5% sucrose and 0.02% surfactant Silwet L-77) until OD. 600 The concentration was approximately 1.0, and Agrobacterium suspension carrying the pCAMBIA1305-Di19 vector was prepared for subsequent rapeseed transformation.

[0029] Example 4: Transformation and Identification Screening of Rapeseed Overexpressing Di19

[0030] 1. The specific steps for converting rapeseed using the cotyledon petiole infection method are as follows:

[0031] (1) The seeds of rapeseed 862 (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.) were disinfected by soaking in 70% ethanol solution for 1 min, then disinfected by soaking in mercuric chloride (HgCl2) solution for 13-15 min, rinsed 5 times with sterile water, spread evenly on MS medium, and grown in an artificial climate culture chamber to obtain sterile rapeseed seedlings for use;

[0032] (2) Take the cotyledons of sterile seedlings that are 4 to 5 days old and soak them in the above-mentioned Agrobacterium suspension carrying the pCAMBIA1305-Di19 vector for 5 to 8 minutes, shaking them gently during the process. Then pour off the bacterial suspension and remove the residual bacterial suspension from the explants. Place the transformed cotyledons on a co-culture medium (MS solid medium containing 0.2 mg / L 6-benzyladenine (6-BA), 1 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and 200 μM acetylsylsyringone) and culture for 2 to 3 days.

[0033] (3) The co-cultured explants were transferred to differentiation medium (MS solid medium containing 400 mg / L penicillin (Car)) and cultured in a dark greenhouse for 5-7 days for sterilization and differentiation. Then they were transferred to selection medium supplemented with Kan (MS solid medium 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.

[0034] (4) After the explants differentiate into 1cm long regenerated green shoots, cut off the shoots and transfer them to rooting medium (MS solid medium containing 0.2mg / L NAA, 10mg / L Kan and 400mg / L Car) for screening;

[0035] (5) After the transformed seedlings have taken root and grown leaves, the DNA of the transformed seedlings was extracted and PCR identification was performed using the upstream primer of the 35S promoter of the plant expression vector and the downstream primer of the Di19 gene.

[0036] (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.

[0037] 2. The specific steps for screening homozygous positive transgenic rapeseed plants using hygromycin are as follows:

[0038] (1) Following the experimental procedure for sterile rapeseed seedlings described above, T1 generation transgenic rapeseed seeds were sterilized.

[0039] (2) Spread the sterilized seeds evenly on MS solid medium containing 25 mg / L hygromycin, and culture them in the dark for about 3 days in an incubator, then switch to culture under normal light.

[0040] (3) When two true leaves have grown, transplant the positive seedlings into the nutrient pots of the artificial climate greenhouse.

[0041] (4) After the positive transgenic rapeseed seeds mature, each plant is harvested and numbered sequentially to obtain T2 generation transgenic rapeseed seeds.

[0042] (5) The T2 generation transgenic rapeseed seeds were screened for resistance to hygromycin according to the above experimental steps. The lines with a separation ratio of 3:1 were screened and transplanted into an artificial climate greenhouse. Individual plants were harvested and numbered sequentially to obtain T3 generation transgenic rapeseed seeds.

[0043] (6) The T3 generation transgenic rapeseed seeds were screened for hygromycin resistance according to the above experimental steps. All normally growing lines were screened out to obtain homozygous T3 generation transgenic rapeseed.

[0044] Example 5: Identification of waterlogging tolerance during the germination period of Di19 transgenic rapeseed seeds

[0045] Dry, plump T3 generation homozygous transgenic rapeseed seeds and untransformed wild-type rapeseed seeds (862 rapeseed) were selected and germinated on moist filter paper. After the radicles grew to approximately 1-2 mm, 100 uniformly germinated seeds were placed in a water-submerged anaerobic environment for 12 hours. The control group did not receive this treatment. After treatment, the seeds were placed back on moist filter paper for further growth. Rooting was observed in both groups after 3 days. The results showed that the wild-type rapeseed did not root or emerge after water submersion, while the transgenic rapeseed rooted and emerged normally (seedling growth was as shown in the image). Figure 3 (As shown); Meanwhile, after statistically analyzing the germination rates of seedlings in the experimental and control groups, it was found that compared to wild-type rapeseed, the germination rates of the two different transgenic rapeseed lines increased by 12.6 times and 27 times, respectively, after waterlogging treatment (germination rate statistics are shown in the figure). Figure 4 As shown in the figure, this confirms that overexpression of the Di19 gene can improve the waterlogging resistance of rapeseed.

Claims

1. Gene Di19 Use in increasing tolerance to waterlogging in Brassica napus, characterized in that, The coding sequence of the gene is shown in SEQ ID NO.

1.

2. gene Di19 application of the encoded protein in improving the tolerance of oilseed rape to waterlogging, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Contains genes Di19 The application of expression vectors and recombinant bacteria in improving the waterlogging resistance of rapeseed is characterized by, The coding sequence of the gene is shown in SEQ ID NO.

1.

4. A method for improving the waterlogging resistance of rapeseed, characterized in that, Expression in rapeseed Di19 The gene, the coding sequence of which is shown in SEQ ID NO.

1.

5. A breeding method for waterlogging-tolerant rapeseed, characterized in that, Including the construction of containing Di19 The plant expression vector of the gene was transformed using Agrobacterium-mediated transformation. Di19 Genes were transferred into rapeseed, and homozygous transgenic individuals were obtained through continuous screening. Di19 The rapeseed with the gene, the described Di19 The coding sequence of the gene is shown in SEQ ID NO.1.

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