Application of a drought-resistant gene in rapeseed to stabilize its yield

Abstract

The present invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of a rapeseed drought resistance gene in the stable yield of rapeseed. The rapeseed drought resistance gene is the BnaLEA41 gene, whose nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2; the encoded protein is localized in the semi-autonomous organelle mitochondria. By constructing an overexpression vector and introducing the BnaLEA41 gene into rapeseed and Arabidopsis, the drought resistance of the plant can be significantly improved; at the same time, the yield of the rapeseed overexpression strain under drought growth conditions is significantly higher than that of the control. The present invention provides important gene resources and germplasm resources for the genetic improvement of rapeseed's stress resistance and the breeding of new rapeseed varieties that are drought-resistant and high-yielding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of a rapeseed drought resistance gene in stabilizing the yield of rapeseed. Background Art

[0002] Drought is the most destructive abiotic stress factor. The loss of crop yield caused by drought stress alone each year is even greater than the impact of plant diseases on yield. Severe drought can lead to a significant reduction in crop yields or even a complete crop failure.

[0003] Rapeseed is an important oil crop and cash crop in my country, with an annual planting area of ​​more than 110 million mu and an annual oil production of 6 million tons. Rapeseed oil accounts for more than 50% of domestic vegetable oils and is recognized at home and abroad as the edible oil with the best fatty acid composition. As one of the largest winter crops and staple crops planted south of the Yangtze River Basin in my country, rapeseed is a core industry to maintain the security of the country's edible oil supply. Rapeseed requires a lot of water during the entire growth period (seedling stage, stalk stage, flowering stage, silique stage and maturity stage), but is susceptible to drought stress, which makes rapeseed seeds germinate poorly; the leaves of the aboveground part lose water and their fresh weight decreases, their size and thickness decrease, their photosynthetic capacity decreases, their stems shrink, and the plants shrink, resulting in delayed growth; even if the roots of the underground part increase in depth, it is difficult to meet the plant's demand for water, resulting in a decrease in root fresh weight. Therefore, drought can directly have a serious impact on the biomass, yield, oil content and quality of rapeseed.

[0004] When suffering from drought stress, genes related to plant drought resistance pathways will be induced to express, followed by a series of adaptive adjustments in physiological, biochemical and morphological structures to cope with adversity. So far, very few rapeseed drought resistance genes have been cloned, and there is a lack of research on the molecular and genetic mechanisms of rapeseed drought resistance, which seriously limits the genetic improvement of rapeseed drought resistance. Therefore, it is necessary to discover the key genes that control rapeseed drought stress, analyze its regulatory mechanism and genetic basis, and then use gene editing technology and genetic engineering to create new rapeseed drought-resistant germplasm and improve its stress resistance; and use modern molecular breeding technology to promote the process of rapeseed drought-resistant breeding, accelerate the selection of new rapeseed varieties that are drought-resistant, water-saving, and stable in yield and oil, and provide important scientific basis and application guidance for sustainable agricultural development. Summary of the invention

[0005] In order to solve the above problems, the present invention identified a rapeseed gene with unknown function and found that the gene was involved in regulating the drought resistance of the plant. The gene can be used to create new drought-resistant rapeseed germplasm, providing important genetic materials for the analysis of the molecular mechanism of rapeseed drought resistance and the improvement of drought resistance. At the same time, the gene positively regulates the growth potential and yield of the plant in a drought growth environment.

[0006] In a first aspect of the present invention, there is provided an application of a rapeseed drought resistance gene in stabilizing rapeseed yield. The rapeseed drought resistance gene is the BnaLEA41 gene, and its nucleotide sequence is shown in SEQ ID NO:1.

[0007] In a specific embodiment of the present invention, the CDS sequence of the BnaLEA41 / BnaA01T0192300ZS gene was obtained from the rapeseed database BnTIR (Brassica napus multi-omics information resource) and the corresponding primers were designed to clone the BnaLEA41 gene from the Brassica napus variety ZS11 (Zhongshuang 11). The nucleotide sequence of the gene coding region is shown in SEQ ID NO: 1, and the protein sequence encoded by it is shown in SEQ ID NO: 2. The protein encoded by the rapeseed BnaLEA41 gene is localized in semi-autonomous organelles: subcellular localization experiments show that the BnaLEA41 protein is localized in mitochondria.

[0008] Furthermore, transgenic rapeseed was obtained by overexpressing the BnaLEA41 gene.

[0009] Furthermore, the transgenic rapeseed has higher drought resistance than the wild type.

[0010] Furthermore, under drought stress, the yield of the transgenic rapeseed is higher than that of the wild type.

[0011] The second aspect of the present invention provides a recombinant vector comprising the BnaLEA41 gene.

[0012] The third aspect of the present invention provides a recombinant bacterium comprising the recombinant vector.

[0013] The fourth aspect of the present invention provides the use of the recombinant vector or the recombinant bacteria in improving plant drought resistance, wherein the use is to transfer the recombinant vector or the recombinant bacteria into plants to obtain transgenic plants with improved drought resistance and stable yield under drought stress.

[0014] Furthermore, the plant is a dicotyledonous plant, a species of the family Cruciferae, including rapeseed and Arabidopsis thaliana.

[0015] The fifth aspect of the present invention provides a plant breeding method, which obtains transgenic plants with improved drought resistance and stable yield under drought stress by overexpressing the rapeseed drought resistance gene.

[0016] Furthermore, the plants include rapeseed and Arabidopsis thaliana.

[0017] In a specific embodiment of the present invention, an overexpression vector containing the rapeseed BnaLEA41 gene is constructed by isolating and cloning the target fragment, and the vector is introduced into Agrobacterium. Arabidopsis and rapeseed are transformed by Agrobacterium infection to obtain T0 generation materials containing the gene. The transformed plants are phenotypically identified, and the homozygous strains overexpressing the BnaLEA41 gene are identified by molecular biology techniques through three consecutive generations of breeding.

[0018] The rapeseed BnaLEA41 gene improves plant drought resistance: Arabidopsis and rapeseed lines overexpressing the BnaLEA41 gene grew better than the control under drought growth conditions.

[0019] Application of BnaLEA41 gene in stabilizing rapeseed yield: Rapeseed germplasm overexpressing BnaLEA41 gene had significantly higher yield than the control under drought growth conditions.

[0020] The present invention has the following beneficial effects:

[0021] The present invention identifies a new rapeseed drought resistance gene BnaLEA41, whose nucleotide sequence length is only 300bp, and the sequence of the encoded protein is only composed of 100 amino acids, with a molecular weight of about 10kDa, which is a small molecule protein, and is convenient for genetic transformation within species and across species. The function of the BnaLEA41 gene discovered in the present invention in improving plant resistance has not been reported, so studying the genetic basis of drought resistance regulation and the molecular mechanism of stable production of this gene is of great significance for analyzing the regulatory network of plant drought resistance. The cloning of this gene also provides important gene resources for the breeding of drought-resistant rapeseed and the genetic improvement of drought resistance of other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Electrophoresis of amplified rapeseed BnaLEA41 gene. Lanes 1-4 are PCR amplified products of BnaLEA41; Lane M is Trans5K Plus DNA Marker DNA (Beijing Quanshijin Biotechnology Co., Ltd.).

[0023] Figure 2 :Subcellular localization experiment of the protein encoded by the rapeseed BnaLEA41 gene. Among them, the GFP channel is the green fluorescence signal emitted by P3301-GFP-BnaLEA41; the mitochondrial probe is the red fluorescence signal emitted by the mitochondria; the chloroplast is the chloroplast autofluorescence; BF is bright field; Merged is the overlay of several channels.

[0024] Figure 3:Molecular identification of the T1 positive strains of the rapeseed BnaLEA41 gene transformed Arabidopsis in Example 3 of the present invention and the rapeseed in Example 4. Wherein, Figure A is the PCR detection result of the T1 generation of the transformed Arabidopsis, M is Trans2K PlusDNAMarker DNA (Beijing Quanshijin Biotechnology Co., Ltd.); 1-22 is the PCR product of the positive seedling; Yang is the P3301-GFP-BnaLEA41 plasmid as a positive control; WT is the wild-type control. Figure B is the PCR detection result of the T1 generation of the transformed rapeseed, M is Trans2KPlus DNA Marker DNA; 1-16 is the PCR product of the positive seedling; Yang is the P3301-GFP-BnaLEA41 plasmid as a positive control; Z6 is the wild-type control of double 6 in rapeseed.

[0025] Figure 4 :Analysis of the relative expression of the BnaLEA41 gene in the homozygous T3 transgenic lines of Arabidopsis and rapeseed. Figure A shows the relative expression of the gene in 19 independent Arabidopsis overexpression lines of the T3 transgenic BnaLEA41 gene. The expression value of the wild-type Arabidopsis (WT) was set to 1, and the data are the averages of three technical replicates plus the standard error; Figure B shows the relative expression of the gene in 15 independent rapeseed overexpression lines of the T3 transgenic BnaLEA41 gene. The expression of the gene in the control (Z6) was set to 1, and the data are the averages of three technical replicates plus the standard error.

[0026] Figure 5 :The drought resistance of Arabidopsis plants overexpressing BnaLEA41 gene is better than that of wild type control WT. The phenotypes of Arabidopsis under normal growth conditions, drought conditions and after rehydration are shown respectively.

[0027] Figure 6 :The drought resistance of rapeseed plants overexpressing BnaLEA41 gene is better than that of wild-type control Z6. The phenotypes of rapeseed under normal growth conditions, drought conditions and after rewatering are shown respectively.

[0028] Figure 7 :The yield of rapeseed plants overexpressing BnaLEA41 gene is significantly higher than that of wild-type control Z6. Figure A is a phenotype of rapeseed at maturity under normal growth conditions and drought conditions. Figure B is a statistical chart of the yield of rapeseed per plant, and the data are the average of 5 individual plants plus standard error. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0030] The primer sequence information involved in the following examples is shown in Table 1:

[0031] Table 1 Primer sequence information

[0032]

[0033]

[0034] Note: The horizontal line marks the vector linker.

[0035] Example 1: Cloning of rapeseed drought resistance gene BnaLEA41 and construction of overexpression vector

[0036] The total RNA was extracted from the Brassica napus variety "Zhongshuang 11" (ZS11) using the plant RNA extraction kit TaKaRaMiniBEST Plant RNA Extraction Kit (9769Takara), and the quantitative PCR-specific reverse transcription kit PrimeScript TM cDNA was synthesized using RT reagent Kit with gDNA Eraser (RR047A TAKARA). The cDNA was used as a template to amplify the BnaLEA41 gene of Brassica napus using primers P3301-BnaLEA41-F and P3301-BnaLEA41-R and high-fidelity DNA polymerase. The amplification system included 2×Phanta Flash Master Mix (Dye Plus) 25 μL, Forward Primer (10 μM) 2 μL, Reverse Primer (10 μM) 2 μL, cDNA template 1 μL, and ddH 2 O to 50 μL.

[0037] The amplification program was: 98°C, 30 sec; 98°C, 5 sec, 58°C, 10 sec, 72°C, 5 sec, 35 cycles; 72°C, 10 min. The PCR amplification product was detected by agarose gel electrophoresis, and the band size was about 300 bp ( Figure 1 ), the sequence is shown in SEQ ID NO.1, the amino acid sequence of the encoded protein is shown in SEQ ID NO.2, and the PCR product is recovered using a gel recovery kit.

[0038] SEQ ID NO.1:

[0039] ATGGCAGCTGCTCGTTCACTCTCCGGCCGTTAAATCTCTTTGCTCCGCCGCATCCCACAACATTTCTGGTTCCATTGTCTTGAGGAGGAGTTACGTTGCGACAGTACCAGGATTTGGTAAGGGAGGTTCCACAAGAGTTACGGTGGGGAAGATGGAACAAAGAGCAAATCAAGAGGCAGAGTCTGCGTGGGCCCCGGATCCAGTTACGGGATACTACAGGCCATCTAATCGTGCGGATGAGATTG ATCCAGCGGAGCTGCGTGAGATGCTTTTGAAAAACAAAGCCAAGCCTTTCTGA.

[0040] SEQ ID NO.2:

[0041] MAAARSLSGAVKSLCSAASHNISGSIVLRRSYVATVPGFGKGGSTRVTV GKMEQRANQEAESAWAPDPVTGYYRPSNRADEIDPAELREMLLKNKAKPF.

[0042] The plant binary expression vector Pcambia3301-GFP was selected, which was transformed from the commercial vector Pcambia3301 (Solarbio: VT000215) and the transient expression vector PAN580 (BT Lab: VT4118). The specific implementation plan was as follows: the vector Pcambia3301 was double-digested with Sac I and Pml I, and the linearized vector was recovered by agarose gel electrophoresis and gel cutting; the PAN580 plasmid was used as a template, and the primers Sac IF and Pml IR were used to amplify the 35S-GFP fragment, and the fragment of about 1500 bp was recovered by agarose gel electrophoresis and gel cutting. The amplification procedure was the same as above.

[0043] The linearized vector and PCR fragment were recombined using the ClonExpress MμLtiS One Step Cloning Kit from Norvegic. The specific recombination reaction was as follows: the reaction system was prepared on ice, with 1 μL of the linearized vector (100 ng / μL), 1 μL of the 35S-GFP PCR fragment (40 ng / μL), 2 μL of 5×CE MμLtiS Buffer, 1 μL of Exnase MμLtiS, and ddH 20 to 10μL; use a pipette to gently mix (do not oscillate), briefly centrifuge to collect the reaction solution to the bottom of the tube; react at 37℃ for 30min, then cool on ice. Transform the recombinant product into E. coli DH5α, pick several single clones on the transformation plate, and use primers 35S and GFP-R for colony PCR identification. The identification system: 2×Es Taq MasterMix (Dye) 10μL, Forward Primer (10μM) 0.8μL, Reverse Primer (10μM) 0.8μL, DNA template 1μL, supplemented with ddH 2 O to 20μL; amplification program: 95℃, 4min; 95℃, 30sec, 58℃, 30sec, 72℃, 1min, 35 cycles; 72℃, 5min. The remaining bacterial liquid of the colony identified as positive by colony PCR can be inoculated into liquid LB medium containing appropriate antibiotics and cultured overnight for first-generation sequencing. After sequencing confirmation, the plasmid with a completely consistent sequence alignment was named Pcambia3301-GFP.

[0044] The prokaryotic resistance of the vector Pcambia3301-GFP is kanamycin resistance, and the screening marker is phosphinothricin (Bar). The vector was linearized by single digestion with the restriction endonuclease BamH I in the vector. The linearized vector and PCR fragment were recombined using the ClonExpress MμLtiS OneStep Cloning Kit from Norvegic. The specific recombination reaction was as follows: the reaction system was prepared on ice, 1μL of the linearized vector (100ng / μL), 1μL of the BnaLEA41 gene PCR fragment (30ng / μL), 2μL of 5×CE MμLtiS Buffer, 1μL of Exnase MμLtiS, and ddH 2 0 to 10μL; use a pipette to gently mix (do not oscillate), briefly centrifuge to collect the reaction solution to the bottom of the tube; react at 37℃ for 30min, then cool on ice. Transform the recombinant product into E. coli DH5α, pick several single clones on the transformation plate, and use primers P3301-BnaLEA41-F and GFP-R for colony PCR identification. The identification system is: 2×Es Taq MasterMix (Dye) 10μL, Forward Primer (10μM) 0.8μL, Reverse Primer (10μM) 0.8μL, DNA template 1μL, supplemented with ddH 2O to 20μL; amplification program: 95℃, 4min; 95℃, 30sec, 58℃, 30sec, 72℃, 1min, 35 cycles; 72℃, 5min. For colonies identified as positive by colony PCR, the remaining bacterial liquid can be inoculated into liquid LB medium containing appropriate antibiotics and cultured overnight for first-generation sequencing. After sequencing confirmation, the plasmid with a completely consistent sequence alignment was named P3301-GFP-BnaLEA41.

[0045] Example 2: Agrobacterium transformation and subcellular localization

[0046] The constructed plasmid P3301-GFP-BnaLEA41 was transferred into Agrobacterium competent GV3101 (Weidi), cultured at 28℃ for 2 days, single clones were selected for colony PCR identification, positive colonies were selected and inoculated into 10ml LB liquid medium, cultured at 200rpm overnight (16-18h). Centrifuged at 5000rpm for 5min, removed the supernatant, and collected the bacteria. Use AS suspension (containing 10mMMgCl 2 , 10 mM MES, 150 μM AS) to resuspend the cells and adjust the OD 600 To about 0.6.

[0047] Select tobacco plants with good growth conditions, use a 1ml syringe without a gun tip to inject from the lower epidermis of tobacco leaves, and mark them; culture the injected tobacco plants under weak light for 2 days. Before microscopic observation, inject the mitochondrial marker MARKER into the lower epidermis of tobacco leaves injected with labeled Agrobacterium. After the leaves are protected from light for 15 minutes, cut the leaves at the injection site and make slides, observe under a laser confocal microscope, and take pictures (see Figure 2 ).from Figure 2 It can be seen that the green fluorescence emitted by the P3301-GFP-BnaLEA41 vector and the red fluorescence emitted by the mitochondrial probe can be completely superimposed, thereby confirming that rapeseed BnaLEA41 is localized in the mitochondria.

[0048] Example 3: Screening and identification of Arabidopsis overexpression strains

[0049] The colony containing the recombinant plasmid P3301-GFP-BnaLEA41 was inoculated into 100 ml LB liquid medium and cultured overnight at 200 rpm (16-18 h). Centrifuge at 5000 rpm for 15 min, remove the supernatant, and collect the bacteria. Prepare Agrobacterium infection solution (containing 5% sucrose, 0.01% SILWETL-77), and use the flower dipping method to infect the Arabidopsis inflorescence. Then, cover the infected plants with plastic film and culture them in the dark for 24 hours, then move them to the greenhouse for conventional culture. After the Arabidopsis matures, the seeds are harvested. After the harvested Arabidopsis seeds are dried, they are sown and planted in the culture matrix (vermiculite: nutrient soil mixed in a volume ratio of 1:1). After the seedlings grow up, spray the herbicide Bar (Liberty, Invitrogen), cut the leaves of the plants to extract DNA, and use primers 35S and P3301-BnaLEA41-R for PCR detection and verification (see Figure 3 A).

[0050] PCR identification: Take about 100 mg of leaves, add 600 μL extraction buffer (0.2M Tris-Cl, 0.25M NaCl, 25mM EDTA, 0.5% SDS, pH 7.5), grind quickly at room temperature; vortex mix in a 1.5mL Ependorff tube for 5-10 seconds; centrifuge at 12000rpm at room temperature for 25 minutes. Take the supernatant, add an equal volume of isopropanol, and precipitate overnight at -20℃. Centrifuge at 12000rpm at room temperature for 15 minutes. Add 200 μL of 70% ethanol solution by volume to wash the DNA precipitate. Centrifuge at 12000rpm at room temperature for 15 minutes. Remove the ethanol. Invert on a paper towel and wait for the ethanol to evaporate; add 100 μL of sterile water to dissolve the crude DNA precipitate. Determine its concentration with a spectrophotometer or estimate its concentration by electrophoresis, and perform PCR using total DNA as a template.

[0051] Amplification system: 2×Es Taq MasterMix (Dye) 10μL, forward primer (10μM) 0.8μL, reverse primer (10μM) 0.8μL, DNA template 1μL, supplemented with ddH 2 O to 20μL; amplification program: 95℃, 4min; 95℃, 30sec, 58℃, 30sec, 72℃, 30sec, 35 cycles; 72℃, 5min.

[0052] The transgenic families identified as positive by PCR were harvested and continued to be planted, and herbicides were continuously used for screening until the T3 generation of homozygous Arabidopsis thaliana families with BnaLEA41 gene were obtained. The homozygous Arabidopsis thaliana families with BnaLEA41 gene were named BnaLEA41-OE-At. RNA was extracted from leaves of the homozygous BnaLEA41-OE-At families at the age of 3 weeks, and the cDNA obtained by reverse transcription (the method is the same as that in Example 1) was subjected to real-time fluorescence quantitative RT-PCR (see Figure 4 A). The expression level of BnaLEA41 in homozygous families was detected by RT-PCR, and three families with higher expression levels, BnaLEA41-OE-At-4, BnaLEA41-OE-At-9, and BnaLEA41-OE-At-12, were selected for subsequent drought experiments.

[0053] Real-time fluorescence quantitative RT-PCR for gene expression analysis: qPCR amplification was performed using 10-fold diluted cDNA as a template and gene-specific primers BnaLEA41-qp-F and BnaLEA41-qp-R. The Arabidopsis Actin2 / 8 gene was used as an internal reference, and the internal reference primers were Actin2 / 8-qp-F and Actin2 / 8-qp-R. The reaction system was: cDNA 2μL, 2×SYBR Green Master Mix 10μL, primers 0.8μL each, ddH 2 O 6.4μL. The program was: 95℃ pre-denaturation for 30sec, 95℃ denaturation for 5sec, 60℃ annealing for 30sec, 72℃ extension for 30sec, for a total of 40 cycles. Wild-type Arabidopsis thaliana (WT) was used as the control.

[0054] Example 4: Screening and identification of rapeseed overexpression strains

[0055] The seeds of Zhongshuang 6 (Z6) were successively soaked in 70% ethanol solution for 1 min and mercuric chloride (HgCl 2 ) Soak for 13-15 min in ddH 2 After washing 5 times with O, the cells were spread in MS medium (pH 5.8) with an agar concentration of 0.8% to obtain a seeding medium. The seeding medium was cultured in a dark environment at 24° C. for 5 to 6 days.

[0056] Agrobacterium tumefaciens GV3101 containing the recombinant plasmid P3301-GFP-BnaLEA41 was inoculated on the solid culture medium LB. Two days later, a single colony was picked and cultured in 50 mL of LB liquid culture medium (containing 25 mg / L rifampicin, 50 mg / L gentamicin and 50 mg / L kanamycin).

[0057] Use sterile forceps and a razor blade to cut the hypocotyls of the seedlings 6 days after sowing, each with a length of 0.8 to 1.0 cm. 600 = about 0.8) and then resuspended once with an equal volume of DM (MS + 3% sucrose + 100 μM acetosyringone) as the bacterial liquid, centrifuged again, and then resuspended with an equal volume of DM to obtain a suspension containing P3301-GFP-BnaLEA41.

[0058] The explants were placed in a suspension containing P3301-GFP-BnaLEA41 for 30 minutes. The explants were gently shaken, and then the bacterial solution was removed. The residual bacterial solution on the explants was removed with sterile filter paper. The explants were placed in a co-culture medium (MS + 0.2 mg / L 6-benzyladenine (6-BA) + 1 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 200 μm acetosyringone (AS), pH 5.8) and cultured for 2 to 3 days.

[0059] The co-cultured explants were transferred to a differentiation medium containing only penicillin (Car) but no Kan, and sterilized and differentiated in a dark greenhouse for 5-7 days. The explants were then subcultured in a selection medium (MS + 3mg / L 6-BA + 0.1mg / L α-naphthylacetic acid (NAA) + 5mg / L silver nitrate + 400mg / L Car + 15mg / L Bar; pH 5.8) supplemented with Bar screening pressure, and screened to differentiate regenerated green shoots.

[0060] When the regenerated buds grow to 1 cm, cut the small buds and transfer them to the rooting medium (MS+0.2mg / L NAA+10mg / L Bar+400mg / L Car; pH value is 5.8), and screen them with solid MS medium with Car and Bar. When the green seedlings produce a relatively developed root system (including taproot and lateral roots) in the rooting medium, they can be transferred to the soil for growth. Before being transferred to the soil, they need to go through the bud hardening stage, that is, open the lid of the triangular bottle and grow for a few days. After being transferred to the soil, they also need to be covered with a bag and grown for two or three days before they can adapt.

[0061] Analysis of relative expression of BnaLEA41 gene in transformed plants. The transformed plants were screened by applying herbicide Bar (Liberty, Invitrogen) to their leaves, and DNA was extracted for PCR identification ( Figure 3B). Finally, it was confirmed that the gene had been introduced into the positive plants. The herbicide was used for continuous screening until the T3 generation BnaLEA41 gene-transformed rapeseed homozygous family was obtained. The rapeseed BnaLEA41-transformed rapeseed homozygous family was named BnaLEA41-OE-Bn. RNA was extracted from leaves of the 4-week-old BnaLEA41-OE-Bn homozygous family, and the cDNA obtained by reverse transcription was used for RT-PCR. The gene-specific primers were BnaLEA41-qp-F and BnaLEA41-qp-R, the internal reference was the BnaTMA7 gene, and the internal reference primers were BnaTMA7-qp-F and BnaTMA7-qp-R. The two families with the highest expression levels, BnaLEA41-OE-Bn-3 and BnaLEA41-OE-Bn-7 (see Figure 4 B), and then conduct subsequent drought experiments. Real-time fluorescence quantitative RT-PCR was used to analyze gene expression as above, and the steps were the same as those described for identifying Arabidopsis overexpression lines.

[0062] Example 5: Analysis of drought resistance of Arabidopsis thaliana and rapeseed BnaLEA41 gene overexpression strains

[0063] The wild-type Arabidopsis WT and three homozygous transgenic Arabidopsis lines BnaLEA41-OE-At-4, BnaLEA41-OE-At-9 and BnaLEA41-OE-At-12 that had grown normally in soil for 3 weeks were subjected to drought treatment (i.e., watering was stopped). After 7 days, the rosette leaves of the WT line were severely dried up, while the rosette leaves of the BnaLEA41-OE-At-4, BnaLEA41-OE-At-9 and BnaLEA41-OE-At-12 lines were only slightly wilted. After 2 days of rehydration, the WT phenotype was still wilted and could not grow normally, while the transgenic lines BnaLEA41-OE-At-4, BnaLEA41-OE-At-9 and BnaLEA41-OE-At-12 could resume normal growth (see Figure 5 The drought resistance experiment showed that the drought resistance of the Arabidopsis thaliana overexpression strain transferred with the BnaLEA41 gene of the present invention was better than that of the control, proving that the rapeseed BnaLEA41 gene can improve the drought resistance of Arabidopsis thaliana plants.

[0064] Z6 and two homozygous transgenic rapeseed lines BnaLEA41-OE-Bn-3 and BnaLEA41-OE-Bn-7 that had grown normally in soil for 4 weeks were subjected to drought treatment (i.e., watering was stopped). After 14 days, the basal leaves of Z6 were severely dried up and the new leaves were completely wilted, while the basal leaves of BnaLEA41-OE-Bn-3 and BnaLEA41-OE-Bn-7 were partially dried up, and the new leaves were still able to grow. After 3 days of rehydration, Z6 still showed a dry state and could not grow normally, while the transgenic lines BnaLEA41-OE-Bn-3 and BnaLEA41-OE-Bn-7 could resume normal growth (see Figure 6 The rapeseed drought resistance experiment showed that the rapeseed overexpression strains transferred with the BnaLEA41 gene of the present invention had better drought resistance than the control, proving that the rapeseed BnaLEA41 gene can improve the drought resistance of rapeseed plants.

[0065] Example 6: Yield statistics of rapeseed BnaLEA41 gene overexpression strains under drought growth conditions

[0066] Z6 and two homozygous transgenic rapeseed lines, BnaLEA41-OE-Bn-3 and BnaLEA41-OE-Bn-7, were germinated on filter paper and transplanted to a greenhouse drought pond 5 days later. They were grown normally until the middle and late stages of flowering and then subjected to drought treatment (i.e., watering was stopped) until seeds were mature and harvested ( Figure 7 A), the yield of a single plant was counted, at least 5 plants were counted for each family, and the final data was the average value of the data of the 5 seedlings plus the standard error. The results are as follows Figure 7 As shown in B, the single-plant yield of transgenic rapeseed lines BnaLEA41-OE-Bn-3 and BnaLEA41-OE-Bn-7 under drought treatment conditions was significantly higher than that of Z6, indicating that the BnaLEA41 gene can stabilize rapeseed under drought conditions.

[0067] The research and development process related to the present invention has been supported by the National Key R&D Program "Molecular Regulatory Network for the Formation of High-Yield, High-Quality, High-Resistant and Mechanically Harvestable Traits of Rapeseed" (Project No. 2023YFF1000700).

[0068] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0069] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of a rapeseed drought resistance gene in stabilizing rapeseed yield, characterized in that: The rapeseed drought resistance gene is BnaLEA41 The gene has a nucleotide sequence as shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Through overexpression BnaLEA41 Gene, obtain transgenic rapeseed.

3. The use according to claim 2, characterized in that: The drought resistance of the transgenic rapeseed is higher than that of the wild type.

4. The use according to claim 2, characterized in that: Under drought stress, the yield of the transgenic rapeseed is higher than that of the wild type.

5. Use of a recombinant vector or recombinant bacteria comprising the rape drought resistance gene of claim 1 in improving plant drought resistance, characterized in that: The recombinant vector or recombinant bacteria is transferred into plants to obtain transgenic plants with improved drought resistance and stable yield under drought stress. The plants are rapeseed or Arabidopsis.

6. A plant breeding method, characterized in that: By overexpressing the rapeseed drought resistance gene described in claim 1, a transgenic plant with improved drought resistance and stable yield under drought stress is obtained, wherein the plant is rapeseed or Arabidopsis.

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