Stain-resistant gene Di19 and application thereof in rape stain-resistant breeding

Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses a waterlogging-resistant gene Di19 and application thereof in waterlogging-resistant breeding of oilseed rape, the Di19 gene of Essel Russian mustard is transferred into oilseed rape for the first time by utilizing a transgenic technology, and the waterlogging resistance of the obtained transgenic oilseed rape is remarkably improved compared with that of wild type oilseed rape. Therefore, the invention provides a new gene resource for breeding the waterlogging-resistant rape.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a waterlogging-tolerant gene Di19 and an application thereof in waterlogging-tolerant breeding of rapeseed. Background Art

[0002] As the largest oil crop in my country, rapeseed oil accounts for more than 50% of the domestic oil crop production. However, global warming has led to an increase in extreme weather such as continuous rain and heavy rainfall, which often causes rapeseed to suffer from waterlogging. At the same time, the Yangtze River Basin and the Huanghuaihai Plain are the two main rapeseed production areas in my country. In the rice-oil and rice-rice-oil rotation modes promoted and implemented in the Yangtze River Basin, waterlogging-sensitive winter rapeseed varieties are mainly planted; therefore, the serious excess of moisture in rice fields and concentrated rainy weather in autumn and winter can easily cause rapeseed to suffer from waterlogging. Current studies have shown that waterlogging stress can cause rapeseed to suffer from severe hypoxia stress and ion toxicity, seriously affecting the development of rapeseed underground roots and the growth of aboveground plants. At the same time, it will also weaken rapeseed's disease resistance, make it easy to fall over, and reduce seed oil content, which seriously restricts the development of the rapeseed industry. Therefore, cultivating waterlogging-tolerant rapeseed varieties suitable for the situation of excess water in rapeseed planting areas will help promote the high-quality development of my country's rapeseed industry and have important strategic significance for ensuring the safety of the country's "oil bottle".

[0003] However, the genetic improvement of waterlogging-tolerant rapeseed varieties in my country is progressing slowly, there are few excellent waterlogging-tolerant germplasm resources available, and there is a lack of stable QTL intervals and available functional gene loci. Therefore, the discovery of excellent waterlogging-tolerant genes in rapeseed and the analysis of their regulatory mechanisms can provide excellent gene loci and germplasm resources for rapeseed waterlogging-tolerant breeding, and accelerate the promotion of more accurate and effective genetic improvement of waterlogging-tolerant rapeseed. Summary of the invention

[0004] On one hand, the present invention protects a gene Di19 for improving rapeseed's waterlogging tolerance. The coding sequence of the Di19 gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.2.

[0005] On the other hand, the present invention protects a method for improving the waterlogging tolerance of rapeseed by overexpressing the Di19 gene in rapeseed, comprising 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 transformation.

[0006] The present 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 by Agrobacterium transformation, and continuously screening to obtain homozygous Di19 gene-transformed rapeseed.

[0007] Compared with the prior art, the present invention has the following beneficial effects: the present invention utilizes transgenic technology to transfer the Di19 gene of Ethiopian mustard into rapeseed for the first time, and the obtained transgenic rapeseed has significantly improved waterlogging tolerance during the germination period compared with untransformed wild-type rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is the result of protein homology comparison of Di19.

[0009] Figure 2 Schematic diagram of the construction of recombinant plasmid pCAMBIA1305-Di19.

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

[0011] Figure 4 is the germination rate of rapeseed after waterlogging stress. WT represents wild-type rapeseed, and OE:Di19 represents Di19 transgenic rapeseed. DETAILED DESCRIPTION

[0012] In order to further illustrate the present invention, the waterlogging-tolerant gene Di19 provided by the present invention and its application in waterlogging-tolerant breeding of rapeseed are 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. Those skilled in the art 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 were purchased from Shanghai Weidi Biotechnology Co., Ltd.

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

[0014] In the early stage, gene loss analysis was conducted on rapeseed (B. napus) and Ethiopian mustard (B. carinata) at the whole genome level, and it was found that rapeseed lost the Bca4012B2G042770 gene during evolution; after blast analysis and comparison on the Arabidopsis genome, it was found that the sequence homology between the protein encoded by the Bca4012B2G042770 gene and the protein encoded by the Arabidopsis ATDIL9 gene was 74.87% (protein sequence comparison results are shown in Figure 2). Figure 1As shown), the present invention named the Bca4012B2G042770 gene as Di19 gene and used it in subsequent rapeseed genetic transformation experiments.

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

[0016] Using the CDS sequence of the Di19 gene as a reference sequence, the Di19 gene was artificially synthesized at Beijing Qingke Biotechnology Co., Ltd. (the sequence is shown in SEQ ID NO.1), and the pUC57-Di19 vector carrying the Di19 gene was obtained. Recombinant primers Di19-F (the sequence is shown in SEQ ID NO.3) and Di19-R (the sequence is shown in SEQ ID NO.4) were designed at both ends of the restriction site of the rapeseed transgenic backbone vector pCAMBIA1305, and the primer pair was used to perform a PCR experiment using the pUC57-Di19 plasmid as a template. The PCR reaction system is as follows: in 50 μL, preferably including: 2×Phanta Flash Master Mix (Dye Plus) 25 μL, Di19-F (concentration of 10 μM) 2 μL, Di19-R (concentration of 10 μM) 2 μL, pUC57-Di19 plasmid (10 ng) 1 μL and ddH 2 O20μL. The PCR amplification program was: 98℃ pre-denaturation for 30s; 94℃ denaturation for 10s, 65℃ annealing for 5s, 72℃ extension for 30s, 35 cycles; 72℃ extension for 1min, and finally stored at 4℃. 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.

[0017] The PCR purified product of Di19 gene and the recovered restriction plasmid were recombined using ClonExpress recombination cloning kit. The 20μL reaction system was: 4μL 5×CE II Buffer, 2μL Exnase II, 50-200ng linearized vector, 10-200ng inserted fragment, and finally supplemented with ddH 2O to 20μL. The recombinant product was transformed into Escherichia coli DH5α competent cells, spread on LB solid culture medium supplemented with 50mg / L kanamycin (Kan), and inverted in a 37°C incubator for 12 to 16 hours. The newly grown single clones were PCR tested with the upstream primers of the 35S promoter of the pCAMBIA1305 vector (the sequence is shown in SEQ ID NO.5) and the downstream primers of the NOS terminator (the sequence is shown in SEQ ID NO.6). The single clones that were verified as positive by PCR were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results showed that the present invention obtained the full-length CDS of the Di19 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-Di19 (the vector construction is shown in Figure 2 shown).

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

[0019] 1. Agrobacterium Transformation

[0020] (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;

[0021] (2) Use a pipette to draw about 100 ng of pCAMBIA1305-Di19 recombinant plasmid and add it to 100 μL of GV3101 competent cells, and 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.

[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 °C and shake at 200 rpm for 2 to 3 h;

[0023] (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;

[0024] (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.

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

[0026] (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 Rif) 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;

[0027] (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;

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

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

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

[0031] (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 disinfected by soaking in 70% ethanol solution for 1 min, and then mercuric chloride (HgCl 2 ) solution for 13-15 minutes, washed with sterile water for 5 times, spread on MS culture medium, and placed in an artificial climate culture room for growth to obtain rapeseed sterile seedlings for use;

[0032] (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-Di19 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 (MS solid 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;

[0033] (3) The explants after co-culture were transferred to differentiation medium (MS solid medium containing 400 mg / L penicillin (Car)) and sterilized and differentiated in a dark greenhouse for 5 to 7 days; then they were 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 (AgNO 3 ), 400 mg / L Car and 15 mg / L Kan on MS solid medium) for screening;

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

[0035] (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 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. Use hygromycin to screen homozygous positive transgenic rapeseed plants. The specific steps are as follows:

[0038] (1) Referring to the experimental operation steps of the above-mentioned rapeseed sterile seedlings, the T1 generation transgenic rapeseed seeds were disinfected;

[0039] (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;

[0040] (3) When two true leaves grow, transplant the positive seedlings into nutrient pots in an artificial climate greenhouse;

[0041] (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;

[0042] (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;

[0043] (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.

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

[0045] Dry and plump T3 homozygous transgenic rapeseed seeds and untransformed wild-type rapeseed seeds (862 rapeseed) were selected for germination on moist filter paper; after the radicle grew about 1-2 mm, 100 seeds with uniform germination were selected and placed in a flooded anaerobic environment for 12 hours, and the control group was not subjected to flooded anaerobic treatment; after treatment, the seeds were placed on moist filter paper again for growth, and the rooting of the rapeseed in the experimental group and the control group was observed after 3 days. The results showed that after flooding treatment, the wild-type rapeseed did not take root and sprout, while the transgenic rapeseed took root and sprouted normally (the growth of the seedlings was shown in Figure 2). Figure 3 At the same time, after statistically analyzing the germination rates of the seedlings in the experimental group and the control group, it was found that compared with the wild-type rapeseed, the germination rates of the seedlings of the two different transgenic rapeseed lines after flooding treatment increased by 12.6 times and 27 times, respectively (the germination rate statistical results are shown in Figure 4 Therefore, it was confirmed that overexpression of Di19 gene could improve the waterlogging tolerance of rapeseed.

Claims

1. Genes regulating rapeseed waterlogging tolerance Di19 , characterized in that, The coding sequence of the gene is shown in SEQ ID NO.

1.

2. The gene according to claim 1 Di19 The encoded protein is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Containing the gene according to claim 1 Di19 The biomaterial is characterized in that Including expression vectors and recombinant bacteria.

4. The gene according to claim 1 Di19 Or the use of the protein according to claim 2 or the biomaterial according to claim 3 in improving the waterlogging tolerance of rapeseed.

5. A method for improving the waterlogging tolerance of rapeseed, characterized in that: Expressing the method of claim 1 in rapeseed Di19 Gene.

6. A method for breeding waterlogging-tolerant rapeseed, characterized in that: Include build containing Di19 The plant expression vector of the gene is transformed into Di19 The gene was transferred into rapeseed and the homozygous transgenic plants were obtained through continuous screening. Di19 Genetic rapeseed.

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