CN114807222B - Application of cabbage type rape Bra040707 gene in drought stress - Google Patents
Application of cabbage type rape Bra040707 gene in drought stress Download PDFInfo
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Abstract
The invention relates to the technical field of plant genetic engineering, in particular to application of a cabbage type rape Bra040707 gene in improving drought stress resistance of cabbage type rape. RNAi vector of Bra040707 gene is constructed by utilizing 354bp fragment on second exon of Brassica napus Bra040707 gene and pHELLSGATE 12 vector, and OE vector is constructed by utilizing 2466bp of Brassica napus (cabbage 45) Bra040707 gene and pCAMBIAsuper 1300-EGFP. By constructing RNAi vector and OE vector containing the gene segment and transferring the gene segment into cabbage type rape by transgenic technology, drought stress tolerance of cabbage type rape can be effectively enhanced, and the cabbage type rape Bra040707 gene can be used as positive regulatory factor of drought resistance of cabbage type rape.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of a cabbage type rape Bra040707 gene in drought stress.
Background
The early-dry stress refers to that when the water absorbed by the plant is far lower than the water required for survival, the water in the plant is deficient, the water is excessively deficient due to the increase of the stress time, and the death of the plant is finally caused.
RNA interference is a gene silencing phenomenon induced by double-stranded RNA, and can play a role in transcriptional level and posttranscriptional level, small fragments homologous to target gene sequences interfere with RNA, efficiently and specifically degrade mRNA, thereby causing silencing of endogenous target genes and finally generating phenotype deletion of corresponding functions. Since the discovery of RNAi, RNAi technology has been widely used for genetic improvement of plants and crop breeding, and significant progress has been made.
Many DEAD-box helicases have been studied for many years, and found that many DEAD-box helicase genes not only enable plants to have tolerance to only single stress, but also participate in responses to various stresses, and that some genes promote the growth and development of plants through positive regulation to enhance the tolerance of the plants to stress, and some genes show the sensitivity of the plants to different stresses through negative regulation. At present, it has been demonstrated that DEAD-box helicase is stress resistant in a variety of plants, with extensive research on drought and salt stress. It is very ideal to use genetic engineering to improve stress resistance and productivity of plants, and the DEAD-box helicase Bra040707 gene can be used as a candidate gene for plants with different stress resistances.
The cabbage type rape is a domestic original vegetable, is various in variety and rich in nutrition, is a main leaf vegetable in daily life, and is also often deeply influenced by drought stress.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of a cabbage type rape Bra040707 gene in drought stress.
The invention adopts the technical scheme that:
the application of the cabbage type rape Bra040707 gene in drought stress comprises the following steps:
s1: selecting cabbage type rape, pHELLSGATE 12RNAi vector, pCAMBIAsuper1300-EGFP vector, escherichia coli DH5 alpha and Agrobacterium GV3101 as experimental objects;
s2: immediately placing fresh cabbage type rape leaves into liquid nitrogen, extracting total RNA of the cabbage type rape, and synthesizing a cDNA first strand;
s3: obtaining a reference sequence of Bra040707, designing a primer with 354bp of product on a second exon, adding a joint sequence of pHELLSGATE 12RNAi vector at the 5' end of the primer to form F1 and R1 primers, amplifying a target fragment by using the primers and performing agarose gel electrophoresis;
s4: the target fragment is subjected to gel recovery and connected with pHELLSGATE 12 carrier, 1 mu L of K protein is added after the target fragment is preserved for 2 hours at the ambient temperature of 25 ℃, and then the target fragment is reacted for 10 minutes at the ambient temperature of 37 ℃;
s5: obtaining a reference sequence of Bra040707 as 2469bp, designing a primer with a product of 2466bp on the reference sequence, wherein the primer does not comprise a stop codon TAG, respectively adding an endonuclease PstI and a protecting base at the 5' end of the front primer and the rear primer, adding an endonuclease KpnI and a protecting base to the rear primer sequence to form F2 and R2 primers, amplifying a target fragment by using the primers, and performing agarose gel electrophoresis;
s6: carrying out double enzyme digestion of endonuclease PstI and endonuclease KpnI on an expression vector pCAMBIAsuper1300-EGFP, and connecting a target fragment recovered by glue with the expression vector pCAMBIAsuper1300-EGFP by using 2X Universal Ligation Mix;
s7: transforming RNAi vector connection products and OE vector connection products into escherichia coli DH5 alpha, performing inversion culture at 37 ℃ for 12 hours, and performing sequencing verification;
s8: respectively transforming agrobacterium GV3101 with two recombinant plasmids with correct sequence, picking up monoclonal and shaking in a test tube for 24 hours, and then turning into large shaking: placing RNAi recombinant plasmid bacterial liquid into 100mL of YEB culture medium containing SPE antibiotics, placing pCAMBIAsuper1300-Bra040707-EGFP recombinant plasmid bacterial liquid into 100mL YEB culture medium containing Kana and Rif antibiotics, and continuing shaking until OD600 = 0.8-1.0, wherein the concentration of Kana is 100 mu g/mL, and the concentration of Rif is 50 mu g/mL;
s9: subpackaging the cultured bacterial liquid into 50mL centrifuge tubes, centrifuging at 5000rpm for 10 minutes at room temperature, discarding supernatant, re-suspending the bacterial liquid precipitated in the 50mL centrifuge tubes by using the prepared re-suspension, and adjusting the bacterial liquid concentration to OD600 = 0.8-1.0 by using the re-suspension;
s10: the bacterial liquid obtained in the step S9 is dip-dyed on the cabbage type rape buds;
s11: screening and identifying RNAi-Bra040707 transgenic cabbage type rape and OE-Bra040707 transgenic cabbage type rape;
s12: and analyzing the phenotype and drought resistance of RNAi-Bra040707 transgenic cabbage type rape and OE-Bra040707 transgenic cabbage type rape under drought stress.
Preferably, in the step S1, the nucleotide sequence of the insert Bra040707 in the OE-Bra040707 transgenic brassica napus is: the nucleotide sequence shown in SEQ ID NO. 1 has a length of 2466bp, lacks a stop codon compared with the nucleotide sequence of the Bra040707 gene on the reference genome, and has only one A to G conversion at 1099bp, resulting in substitution of threonine for alanine in the protein sequence
I) the nucleotide sequence shown in SEQ ID NO. 1; or (b)
Ii) the nucleotide sequence shown in SEQ ID NO. 1 is substituted, deleted and/or added with one or more nucleotides and has the same function; or (b)
Iii) a nucleotide sequence which hybridizes under stringent conditions with the sequence shown in SEQ ID No. 1 and has the same function,
iv) a nucleotide sequence having a homology of 90% or more with the nucleotide sequence of i), ii) or iii) and having the same function.
Preferably, in the step S3, the nucleotide sequence of RNA interference in the RNAi-Bra040707 transgenic brassica napus is: SEQ ID NO. 2, 354bp in length, is completely identical to the reference sequence of the Bra040707 gene;
i) the nucleotide sequence shown in SEQ ID NO. 2; or (b)
Ii) the nucleotide sequence shown in SEQ ID NO. 2 is substituted, deleted and/or added with one or more nucleotides and has the same function; or (b)
Iii) a nucleotide sequence which hybridizes under stringent conditions with the sequence shown in SEQ ID No. 2 and has the same function,
iv) a nucleotide sequence having 90% or more homology with the nucleotide sequence of i), ii) or iii) and having the same function.
Preferably, in the step S3, the nucleotide sequences of the primers F1 and R1 are shown in SEQ ID NO. 3-4.
Preferably, in the step S5, the nucleotide sequences of the primers F2 and R2 are shown in SEQ ID NOS.5-6.
Preferably, in the step S7, the sequencing verification method is to select a monoclonal colony for shaking and extracting plasmids, design primers at two ends of the insertion position of the target fragment on the vector, and perform PCR to verify whether the target gene is successfully connected to the vector, and perform sequencing verification on the recombinant plasmids.
Preferably, in the step S9, the re-suspension is prepared by adding 50mg/L acetosyringone AS and 0.02% of surfactant Silwet L-77 in 1/2MS liquid medium containing 5% sucrose.
Preferably, the dip dyeing method in step S10 includes: each bud is opened by using tweezers, the inflorescences are immersed in the bacterial liquid for 20 seconds successively, the inflorescences in the bacterial liquid are rocked during the period, clear water is used for spraying the inflorescences and bagging is completed after the completion of the dip-dyeing, the dip-dyed plants are protected from light for 24 hours after the bagging is completed, normal culture conditions are restored after 24 hours, after five days, the newly-appearing buds to be opened are opened, the buds which are not suitable for dip-dyeing are removed, and then secondary infection is carried out according to the same method.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the drought-resistant Bra040707 gene in the cabbage type rape is provided, an RNAi vector containing the gene fragment and a full-length gene OE vector are constructed, and the gene fragment is transferred into the cabbage type rape by a transgenic technology, so that the obtained OE-Bra040707 transgenic cabbage type rape can effectively enhance the tolerance of plants under drought stress.
2. The invention shows that the cabbage type rape Bra040707 gene can be used as a cabbage type rape drought resistance factor, provides a reference for researching a plant drought resistance mechanism, and simultaneously the obtained drought resistance transgenic cabbage type rape is a good plant type variety which is resistant to abiotic stress and is suitable for environmental change.
Drawings
FIG. 1 is a graph showing the verification of the expression level of the Bra040707 gene in OE-Bra040707 transgenic brassica napus of the present invention;
FIG. 2 is a diagram showing the verification of the expression level of the Bra040707 gene in RNAi-Bra040707 transgenic cabbage type rape according to the present invention;
FIG. 3 is a plant root phenotype plot of OE-Bra040707 transgenic brassica napus and RNAi-Bra040707 transgenic brassica napus of the present invention at 8d under 1/2MS medium and 200mM Mannitol (Mannitol);
FIG. 4 is a major root length plot of plants of OE-Bra040707 transgenic brassica napus and RNAi-Bra040707 transgenic brassica napus of the present invention at 8d under 1/2MS medium and 200mM Mannitol (Mannitol);
FIG. 5 is a statistical plot of fresh weight of seedlings of OE-Bra040707 transgenic brassica napus and RNAi-Bra040707 transgenic brassica napus of the present invention at 8d under 1/2MS medium and 200mM Mannitol (Mannitol);
FIG. 6 is a leaf stomatal pattern of OE-Bra040707 transgenic brassica napus and RNAi-Bra040707 transgenic brassica napus of the present invention under normal conditions and 20% PEG-6000 treatment;
FIG. 7 is a statistical chart of leaf pore width to length ratio under normal conditions and 20% PEG-6000 treatment for OE-Bra040707 transgenic cabbage type rape and RNAi-Bra040707 transgenic cabbage type rape according to the present invention;
FIG. 8 is a phenotype diagram of OE-Bra040707 transgenic brassica napus and RNAi-Bra040707 transgenic brassica napus of the present invention at 10d drought;
FIG. 9 is a statistical plot of relative moisture content of OE-Bra040707 transgenic brassica napus and RNAi-Bra040707 transgenic brassica napus leaves of the present invention;
FIG. 10 is a graph showing statistics of water loss rates of OE-Bra040707 transgenic brassica napus and RNAi-Bra040707 transgenic brassica napus leaves of the present invention;
FIG. 11 is a graph showing the statistics of the proline content of the penetration regulators of OE-Bra040707 transgenic cabbage type rape and RNAi-Bra040707 transgenic cabbage type rape in the present invention when drought is 10 d;
FIG. 12 is a statistical plot of Malondialdehyde (MDA) content of OE-Bra040707 transgenic brassica napus and RNAi-Bra040707 transgenic brassica napus of the present invention at 10d drought;
FIG. 13 is a graph showing the analysis of the expression level of drought stress-related genes in E-Bra040707 transgenic cabbage type rape and RNAi-Bra040707 transgenic cabbage type rape of the present invention when drought is 10 d.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in fig. 1-13, the application of the brassica napus Bra040707 gene in drought stress comprises the following steps:
s1: the experimental object is selected from Brassica campestris, pHELLSGATE 12RNAi vector, pCAMBIAsuper1300-EGFP vector, escherichia coli DH5 alpha and Agrobacterium GV3101.
S2: immediately placing fresh cabbage type rape leaves into liquid nitrogen, extracting total RNA of the cabbage type rape, and synthesizing a cDNA first strand;
s3: obtaining a reference sequence of Bra040707, designing a primer with 354bp of product on a second exon, adding a joint sequence of pHELLSGATE 12RNAi vector at the 5' end of the primer to form primers F1 and R1 (SEQ ID NO: 3-4), amplifying a target fragment by using the primer and performing agarose gel electrophoresis;
wherein, the F1 and R1 primer sequences are as follows:
s4: the target fragment is subjected to gel recovery and connected with pHELLSGATE 12 carrier, 1 mu L of K protein is added after the target fragment is preserved for 2 hours at the ambient temperature of 25 ℃, and then the target fragment is reacted for 10 minutes at the ambient temperature of 37 ℃;
wherein the connection system is as follows:
s5: obtaining a reference sequence of Bra040707 as 2469bp, designing a primer (excluding a stop codon TAG) with a product of 2466bp on the reference sequence, adding an endonuclease PstI and a protecting base at the 5' end of the front and rear primers respectively, adding an endonuclease KpnI and a protecting base to the rear primer sequence to form F2 and R2 primers (SEQ ID NO: 5-6), amplifying a target fragment by using the primer, and performing agarose gel electrophoresis;
s6: carrying out double enzyme digestion of endonuclease PstI and endonuclease KpnI on an expression vector pCAMBIAsuper1300-EGFP, and connecting a target fragment recovered by glue with the expression vector pCAMBIAsuper1300-EGFP by using 2X Universal Ligation Mix;
wherein the connection system is as follows:
s7: transforming E.coli DH5 alpha with the OE vector connection product and the RNAi vector connection product, culturing for 12 hours at 37 ℃ in an inversion way, and performing sequencing verification to obtain the OE-Bra040707 vector with the insertion sequence of SEQ ID NO. 1 and the RNAi-Bra040707 vector with the insertion sequence of SEQ ID NO. 2;
s8: transforming recombinant plasmids with correct sequence respectivelyAgrobacterium GV3101, single clone was picked up and shaken in test tubes for 24h, followed by shaking up: placing RNAi recombinant plasmid bacterial liquid into 100mL of YEB culture medium containing SPE antibiotics, placing pCAMBIAsuper1300-Bra040707-EGFP recombinant plasmid bacterial liquid into 100mL of YEB culture medium containing Kana (100 mug/mL) and Rif (50 mug/mL) antibiotics, and continuing shaking until OD 600 ＝0.8-1.0；
S9: the cultured bacterial liquid is packed in 50mL centrifuge tubes, centrifuged at 5000rpm for 10 minutes at room temperature, the supernatant is removed, the bacterial cells precipitated in the 50mL centrifuge tubes are resuspended in the prepared resuspension liquid, and the bacterial liquid concentration is adjusted to OD by the resuspension liquid 600 ＝0.8-1.0；
S10: the bacterial liquid obtained in the step S9 is dip-dyed on the cabbage type rape buds;
s11: screening and identifying OE-Bra040707 transgenic cabbage type rape and RNAi-Bra040707 transgenic cabbage type rape;
transgenic seedlings initially screened by the resistant culture medium are transplanted into conventional soil culture, and the culture medium at the root of the seedlings is cleaned during seedling transplanting. DNA of positive soil culture seedlings is extracted, and PCR detection is carried out by using EGFP sequence of pCAMBIAsuper1300-EGFP and pHELLSGATE 12RNAi vector skeleton sequence design primer. And extracting RNA from the 3 RNAi-Bra040707 transgenic cabbage type rapes (cabbage heart 45) and the 4 OE-Bra040707 transgenic cabbage type rapes (cabbage heart 45), and performing fluorescent quantitative PCR to detect the expression quantity of the Bra040707 gene, wherein the result is that the expression quantity of OE-Bra040707 is increased, the expression quantity of RNAi-Bra040707 is reduced, and the construction of the transgenic Bra040707 transgenic cabbage type rapes is successful as shown in the figure 1-2.
S12: and analyzing the phenotype and drought resistance of RNAi-Bra040707 transgenic cabbage type rape and OE-Bra040707 transgenic cabbage type rape under drought stress.
Example 1 drought resistance test of cabbage type rape transformed with Bra040707 Gene under simulated drought conditions
Under simulated drought stress, three strains of wild type cabbage 45, OE-Bra040707 transgenic cabbage type rape (cabbage 45) and RNAi-Bra040707 transgenic cabbage type rape (cabbage 45) are simultaneously planted in a 1/2MS culture medium, the root growth condition of plants and the pore morphology of each plant leaf are observed under normal conditions and simulated drought stress, as shown in figures 3-7, the root length and the pore morphology of each strain are observed, and the root length, the fresh weight of seedlings and the pore width-length ratio of each strain are counted. The results show that the OE-Bra040707 strain is more environmentally compatible and moisture absorptive compared to the wild-type strain, while the RNAi-Bra040707 strain is more sensitive to the external environment and less moisture absorptive.
Example 2 drought resistance detection of Bra040707 Gene transferred cabbage type rape under true drought conditions
Three lines of wild cabbage 45 and OE-Bra040707 transgenic cabbage type rape (cabbage 45) and RNAi-Bra040707 transgenic cabbage type rape (cabbage 45) are simultaneously planted and cultivated under the same conditions. When the plant grows to 4 weeks, drought treatment is started for 10 days, and the phenotype, the relative water content of the leaves and the water loss rate of the leaves before and after drought are counted, as shown in figures 8-10; the OE-Bra040707 strain has good drought resistance, and the RNAi-Bra040707 strain is sensitive to drought compared with a wild plant. Further, quantitative analysis of partial drought-resistant physiological indexes (proline content, malondialdehyde content), the expression quantity of partial drought-resistant related genes and the like is carried out, and the quantitative analysis is shown in figures 11-13. The results indicate that the OE-Bra040707 transgenic plants exhibit greater drought stress tolerance than wild type plants, while the RNAi-Bra040707 transgenic plants have less drought tolerance. This indicates that the Bra040707 gene positively regulates drought resistance in plants, and has a positive effect on drought stress response.
In step S1, the cabbage type rape selection line is cabbage 45. The vector selected pHELLSGATE 12RNAi and pCAMBIAsuper1300-EGFP vectors, and the strain selected E.coli DH5 alpha and Agrobacterium GV3101.
In the step S7, the sequencing verification method is to select a monoclonal colony for shaking bacteria and extracting plasmids, design primers at two ends of the insertion position of a target fragment on the vector, verify whether the target gene is successfully connected into the vector by PCR, and sequence and verify the recombinant plasmids, wherein the result is shown as a sequence 1 and a sequence 2.
In step S9, the resuspension is prepared by adding 50mg/L acetosyringone AS into 1/2MS liquid medium containing 5% sucrose, and adding 0.02% surfactant Silwet L-77.
In the step S10, the dip dyeing mode is that each bud is opened by using tweezers, inflorescences are immersed into bacterial liquid for 20 seconds, inflorescences in the bacterial liquid are rocked during the process, and after the dip dyeing is finished, inflorescences are sprayed by clear water, and bagging is finished.
After the bagging is completed, the impregnated plant is protected from light for 24 hours, normal culture conditions are restored after 24 hours, after five days, small openings are opened for newly appeared buds to be opened, and buds which are not suitable for the impregnation are removed.
After removing the buds unsuitable for the dip-dyeing, a second infestation is carried out in the same way.
The preferred embodiments of the present invention have been described in detail, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention, and the various changes are included in the scope of the present invention.
SEQUENCE LISTING
<110> university of Zhengzhou
INSTITUTE OF HORTICULTURE, HENAN ACADEMY OF AGRICULTURAL SCIENCES
Application of <120> cabbage type rape Bra040707 gene in drought stress
<130> 2022
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 2466
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atgacatcct tgctcctccc aaatcccgac tcgattacca ccacacccct cgtttccaac 60
ctacgctcct tccggaggtt cttctctctc agacgctcct ccttgccccg aaactcatca 120
tcatcatctc ttccactagt cgctctttgc tctctctccg caactgccgc aaaacctact 180
acagcgacca ggtggagaga gaagcatgag ttagctgaaa gcgattcaat ctccatcctc 240
aacgagagga ttcggcgcga cctcggaaag agagagactg ctaggccggc catggactct 300
aaggaggccg aaaagtacat tcagatggtg aaggaacaac aagagagggg tcttcagaag 360
ctcaaaggag ttaggccagg ctcagacggt ggtttcagct acaaggttga cccttacact 420
ctcctttccg gtgattatgt ggtgcacaag aaggttggca ttggccgttt tgtcggaatt 480
aagttggatg tccccaagga ttcttctgag cccctcgaat atgtcttcat tgagtatgct 540
gatggcatgg ccaagcttcc cctcaagcag gcctcccgtc tcctctaccg atacaacctt 600
cctaatgagt ccaaacggcc tcggacttta agtcggctga gcgacactag tgtttgggag 660
agaagaaaga ccaaaggaaa agtcgctatt cagaaaatgg ttgttgactt gatggagctc 720
tatcttcata ggcttagaca gaagagattt ccttatccca aaaaccccgt catggctgat 780
ttcgctgctc aatttcctta taacgctaca cccgaccaga agcaggcttt ccttgatgtt 840
gacaaggatt tgactgagag ggaaacgcct atggaccgat tgatatgtgg agacgttgga 900
tttggtaaaa ctgaggttgc tctacgtgcc atcttttgtg tggtctccgc tggcaaacaa 960
gctatggttt tggcacccac tattgttttg gccaagcaac attacgatgt catctctcag 1020
cggttttcct tgtatcccca aatcaaagtc ggtcttttaa gtcggtttca gaccaaagca 1080
gagaaggagg cgtatttgga aatgataaaa cacggtcatc tcaatatcat tgtaggtact 1140
cactcccttc tcggaagccg tgttgtgtac agcaatctag gccttcttgt cgtcgatgag 1200
gaacagagat ttggggtcaa gcagaaagaa aagattgcat ctttcaaaac gtcagtggat 1260
gtgcttaccc tctccgcaac acctatacca aggacgttat acttagcttt gactggattc 1320
cgggatgcca gtttaatctc cacacctcca ccggagagga ttccaataaa aacccatctt 1380
tcatcgttcc gtaaggaaaa ggttatcaaa gcaataaaaa atgaactgaa tcgtggtggc 1440
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gaggaaacca tggagagatt tgcgcaagga aagatcaaaa tcctcatatg cactaatatt 1620
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catgcctacc tgttttatcc tgataaatca ctgctctctg atcaagcact ggaaaggctt 1800
agtgctcttg aagagtgtcg cgagcttgga caaggtttcc aacttgcgga gaaagacatg 1860
ggtataagag gttttgggac aatttttggt gaacagcaga caggagatgt tggaaatgtc 1920
ggcatcgatc tcttctttga aatgcttttt gagagtctat ccaaggtgga ggaactccgt 1980
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tccgagtatg taaattacct ggaaaatccg atggagatca tcaatgaagc tgaaaaagca 2100
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gatcttggag taaacagaat ttatgcatca gggaagatgg ttgtgatgaa aacaaatatg 2280
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catggccaag cttcccctca agcaggcctc ccgtctcctc taccgataca accttcctaa 180
tgagtccaaa cggcctcgga ctttaagtcg gctgagcgac actagtgttt gggagagaag 240
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Claims (4)
1. The application of the cabbage type rape Bra040707 gene in improving the drought stress resistance of the cabbage type rape is characterized in that: the method comprises the following steps:
s1: selecting cabbage type rape, pCAMBIAsuper1300-EGFP vector, escherichia coli DH5 alpha and agrobacterium GV3101 as experimental objects;
s2: immediately placing fresh cabbage type rape leaves into liquid nitrogen, extracting total RNA of the cabbage type rape, and synthesizing a cDNA first strand;
s3: obtaining a nucleotide sequence of a Bra040707 gene, designing a primer, adding an endonuclease PstI and a protective base at the 5 'end of an upstream primer, adding an endonuclease KpnI and a protective base at the 5' end of a downstream primer to form F2 and R2 primers, amplifying a target fragment by using the primers, performing agarose gel electrophoresis, and recovering the target fragment;
s4: carrying out double enzyme digestion of endonuclease PstI and endonuclease KpnI on an expression vector pCAMBIAsuper1300-EGFP, and connecting the target fragment recovered by glue with the expression vector pCAMBIAsuper1300-EGFP by using 2X Universal Ligation Mix to obtain an expression vector over-expressing cabbage type rape Bra040707 gene;
s5: transforming the expression vector into escherichia coli DH5 alpha, performing inversion culture at 37 ℃ for 12h, and performing sequencing verification;
s6: the expression vector with correct sequencing is transformed into agrobacterium GV3101, and monoclonal shaking 24h in a test tube is selected, and then shaking is performed: inoculating the bacterial liquid into 100mL YEB culture medium containing Kana and Rif antibiotics, and shaking until OD 600 =0.8-1.0, the Kana concentration is 100 μg/mL, the Rif concentration is 50 μg/mL;
s7: sub-packaging the cultured bacterial liquid into a 50mL centrifuge tube, centrifuging at 5000rpm for 10 min at room temperature, discarding supernatant, re-suspending the bacterial liquid precipitated in the 50mL centrifuge tube with the prepared heavy suspension, and adjusting bacterial liquid concentration to OD with the heavy suspension 600 =0.8-1.0; the preparation method of the heavy suspension comprises the steps of adding 50mg/L acetosyringone AS into a 1/2MS liquid culture medium containing 5% of sucrose, and adding 0.02% of a surfactant Silwet L-77;
s8: the bacterial liquid obtained in the step S7 is subjected to dip dyeing on cabbage type rape buds;
s9: screening and identifying the cabbage type rape of the over-expression cabbage type rape Bra040707 gene;
s10: analyzing the phenotype and drought resistance of the transgenic cabbage type rape under drought stress,
the nucleotide sequence of the Bra040707 gene is shown as SEQ ID NO. 1.
2. The use of the brassica napus Bra040707 gene according to claim 1 for improving drought stress tolerance of brassica napus, wherein the brassica napus Bra040707 gene is characterized in that: in the step S3, the nucleotide sequences of the primers F2 and R2 are shown in SEQ ID NO. 5-6.
3. The use of the brassica napus Bra040707 gene according to claim 1 for improving drought stress tolerance of brassica napus, wherein the brassica napus Bra040707 gene is characterized in that: in the step S5, the sequencing verification method comprises the steps of selecting monoclonal colony shaking bacteria and plasmid extracting, designing primers at two ends of the insertion position of a target fragment on the vector, verifying whether the target gene is successfully connected into the vector by PCR, and sequencing and verifying the recombinant plasmid.
4. The use of the brassica napus Bra040707 gene according to claim 1 for improving drought stress tolerance of brassica napus, wherein the brassica napus Bra040707 gene is characterized in that: the dip dyeing mode in the step S8 comprises the following steps: each bud is opened by using tweezers, the inflorescences are immersed in the bacterial liquid for 20 seconds successively, the inflorescences in the bacterial liquid are rocked during the period, clear water is used for spraying the inflorescences and bagging is completed after the completion of the dip-dyeing, the dip-dyed plants are protected from light for 24 hours after the bagging is completed, normal culture conditions are restored after 24 hours, after five days, the newly-appearing buds to be opened are opened, the buds which are not suitable for dip-dyeing are removed, and then the secondary dip-dyeing is carried out according to the same dip-dyeing mode.
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| CN101638658A (en) * | 2008-07-29 | 2010-02-03 | 四川贝安迪生物基因工程有限公司 | Gene and polypeptide for improving heat resistance of plants and microorganisms and application thereof |
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| CN104745609A (en) * | 2015-03-20 | 2015-07-01 | 河南大学 | Method for high-flux rapidly cloning of rape draught-resistant gene |
| CN110804614A (en) * | 2019-10-08 | 2020-02-18 | 湖南农业大学 | Cabbage type rape drought-resistant gene BnatZF1A, primer, expression vector and application thereof, and method for improving drought resistance |
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