CN116286876B - Application and method of BnaWRKY25.C04 gene of brassica napus - Google Patents
Application and method of BnaWRKY25.C04 gene of brassica napus Download PDFInfo
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- CN116286876B CN116286876B CN202310443802.9A CN202310443802A CN116286876B CN 116286876 B CN116286876 B CN 116286876B CN 202310443802 A CN202310443802 A CN 202310443802A CN 116286876 B CN116286876 B CN 116286876B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8267—Seed dormancy, germination or sprouting
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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Abstract
The invention belongs to the field of plant genetic engineering and biotechnology, and particularly relates to application and a method of a BnaWRKY25.C04 gene of brassica napus. The BnaWRKY25.C04 gene is cloned, an over-expression vector pK7FWG2.0-BnaWRKY25.C04 of the gene is constructed, the cabbage type rape is transformed, an over-expressed stable transformant line is obtained in the rape, and a transgenic plant with high temperature resistance and bacterial sclerotinia resistance is obtained.
Description
Technical Field
The invention belongs to the field of plant genetic engineering and biotechnology, and particularly relates to application and a method of a BnaWRKY25.C04 gene of brassica napus.
Background
Cabbage type rape (Brassica napus L.) is a cold-resistant crop, which is cool and is prohibited from being heated and is greatly limited by temperature. There are also a number of diseases and pests that are encountered during the growth and development process, of which sclerotinia is the most common, most harmful, most studied, and most studied for the longest period of time. However, the rape sowing area in China is in a decreasing trend in the last decade, and the rape yield is seriously supplied under. At present, 14 hundred million people face the serious situation that the population is highly dependent on imported oil, and the maintenance of rape industry development has important economic significance and important strategic significance in guaranteeing national edible oil safety.
First, temperature is one of important abiotic stresses affecting the growth and development of rape, and when the environmental temperature exceeds the adaptation range of plants, temperature stress is formed, which is an abiotic stress with a great influence on the growth and development of rape. The high temperature stress is used as one of main abiotic stress, and can directly influence the yield and quality of plants by destroying the permeability of biological membranes, influencing enzyme activity, changing photosynthetic rate and the like.
Secondly, rape is also subject to many biotic stresses throughout its short and lengthy lifetime, with sclerotinia sclerotiorum being the most common in the rape growing process and one of the biotic stresses that are the most detrimental to rape growth. The incidence rate of the plant in the common year is 10% -30%, and the incidence rate of the plant in the high-incidence year is up to more than 80%. After the disease, the yield is generally reduced by 10% -70%, the quality of rapeseeds is further influenced, the quality of the rapeseeds is further influenced, and the yield of the rapeseeds and the income of farmers are seriously threatened.
In recent years, the growth conditions of rape have changed due to the influence of varied climates and high occurrence of various diseases. Wherein the temperature stress in abiotic stress has a remarkable effect on rape yield, and the temperature stress in abiotic stress not only affects the germination of rape seeds, but also directly affects the later growth of rape and the formation of yield. Sclerotinia in biotic stress can seriously affect the yield of rape, resulting in a significant decrease in the oil content and quality of rape seed. Therefore, research on high temperature stress and antibacterial nuclear disease stress is important for germination of rape seeds and growth and development of rape.
Under high temperature stress, the germination rate, the growth of the sprouts and the young roots of the rice are improved after the seeds are soaked by ABA; the exogenous brassinolide seed soaking can obviously promote the growth of rice radicle under high temperature stress, increase the fresh root weight, fresh bud weight, bud dry weight and the like; the combined solution of different medicaments (sodium selenate, sodium bisulphite, salicylic acid, potassium dihydrogen phosphite, brassinolide and water) can improve the activity of enzymes related to seed germination after seed soaking of rape seeds, promote the growth of radicle and improve the capability of seedlings to absorb and transport water. However, these methods only promote the high temperature resistance of plants by means of exogenous application, and high temperature resistant rape germplasm resources cannot be obtained.
For sclerotinia, chemical control is the most common method for controlling sclerotinia caused by sclerotinia, and is mostly used for the ascomycete soil emergence period or the initial disease onset period of sclerotinia; however, chemical control is too costly and not particularly effective, and is also harmful to the environment. Since sclerotinia sclerotiorum often survives in soil for a long period of time in the form of sclerotium, the control effect of various agricultural techniques employed, such as rotation, cultivation soil cleaning, etc., is not particularly remarkable. Therefore, the chemical prevention and biological prevention of sclerotinia are not particularly ideal, and the breeding of new varieties of rape resistant to sclerotinia is still a fundamental way to overcome sclerotinia.
At present, the excavation of important functional genes related to the biological stress resistance and the abiotic stress of rape, so as to perform germplasm innovation and culture of new varieties of rape resistant to biological stress and abiotic stress, is one of important measures for improving the mechanized problem of rape.
Disclosure of Invention
Aiming at some defects existing in the prior art, the invention provides application and a method of a brassica napus BnaWRKY25.C04 gene in resisting biotic stress and/or abiotic stress, in particular application and a method of the BnaWRKY25.C04 gene in resisting high temperature and/or antibacterial nucleopathy. In the invention, bnaWRKY25.C04 gene is cloned and overexpressed in rape to obtain a cabbage type rape transgenic strain for promoting high temperature resistant germination and/or bacterial nuclear disease resistance of seeds.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention firstly provides a BnaWRKY25.C04 gene for promoting high-temperature germination and/or bacterial nuclear disease resistance of rape seeds, and the nucleotide sequence of the BnaWRKY25.C04 gene is shown as SEQ.ID.NO. 1.
The invention also provides a protein coded by BnaWRKY25.C04 gene for promoting high temperature germination and/or bacterial nuclear disease resistance of rape, and the amino acid sequence of the protein is shown as SEQ.ID.NO. 2.
The invention also provides an application of the BnaWRKY25.C04 gene for promoting high-temperature germination and/or anti-bacterial sclerotinia of rape seeds in the breeding of new varieties of rape for promoting high-temperature germination and/or anti-bacterial sclerotinia of rape seeds, resisting biological or abiotic stress, breeding of high-temperature resistant seed resources or breeding of anti-bacterial sclerotinia rape.
Further, the application is realized by overexpression of the BnaWRKY25.C04 gene.
The invention also provides a recombinant expression vector which comprises the nucleotide sequence of the BnaWRKY25.C04 gene, a 35S promoter and eGFP enhanced green fluorescent protein.
Further, the recombinant expression vector comprises vector pk7fwg2.0.
The invention also provides a recombinant engineering bacterium, which comprises the BnaWRKY25.C04 gene or the recombinant expression vector.
Further, the host bacterium of the engineering bacterium is agrobacterium GV3101.
The invention also provides application of the recombinant expression vector or the recombinant engineering bacteria in promoting rape seed high temperature germination and/or bacterial nuclear disease resistance, biological or abiotic stress resistance rape new variety breeding, high temperature seed resource breeding or bacterial nuclear disease resistance rape breeding.
The invention also provides a method for promoting high-temperature-resistant germination and/or antibacterial nuclear disease of rape seeds, which comprises the following specific steps:
Constructing a recombinant expression vector containing BnaWRKY25.C04 genes, and transforming the recombinant expression vector into a receptor bacterium to obtain a recombinant engineering bacterium; performing amplification culture on the obtained recombinant engineering bacteria, and infecting rape hypocotyl with the obtained bacterial liquid; the rape transformant which promotes the high temperature resistant germination and/or the antibacterial nuclear disease of rape seeds is obtained through the steps of inducing the callus of the hypocotyl of rape, re-differentiating, rooting culture and the like.
The invention has the beneficial effects that:
(1) The agrobacterium-mediated hypocotyl transformation method is used for transforming the brassica napus, and has the advantages of high reliability, high speed and high efficiency.
(2) The BnaWRKY25.C04 gene is excessively expressed in rape, so that germination of seeds in a high-temperature environment can be promoted, and under the condition that the seeds are treated for 6 hours at 45 ℃, the germination rate of the seeds can still reach 94%; the invention can also promote the rape to resist the infection of sclerotinia by overexpressing BnaWRKY25.C04 gene in rape, and the result shows that the growth area of sclerotinia lesions in BnaWRKY25.C04 overexpressing transgenic lines is much smaller than that of non-transgenic lines by taking plant true leaves and inoculating sclerotinia in vitro; meanwhile, the BnaWRKY25.C04 gene is excessively expressed in the rape, so that the elongation of plant roots can be promoted, and the yield of the rape and the quality of the rapeseed oil are improved.
(3) The application of the gene BnaWRKY25.C04 in improving the quality of rape has important guiding and reference significance for breeding, growing and developing oil crops such as rape and the like. The recombinant expression vector pK7FWG2.0-Bna WRKY25.C04 constructed by the invention provides a raw material for rape production, provides a new gene source for improving the resistance of rape to biotic and abiotic stress, and is beneficial to cultivating new varieties with better quality.
Drawings
FIG. 1 is a graph of predicted amino acid conserved domain encoded BnaWRKY.
FIG. 2 is a phylogenetic analysis of BnaWRKY genes.
FIG. 3 is a diagram of an alignment of the corresponding coding sequences of LOC106407015 and BnaWRKY25.C04 in canola, where LOC106407015 is the BnaWRKY gene searched at NCBI; bnaWRKY25.C04 is the target gene BnaC g41050D amplified by the invention.
FIG. 4 is a schematic diagram of pK7FWG2.0-BnaWRKY25.C04 vector.
FIG. 5 is a PCR characterization of BnaWRKY-OE plants obtained by hypocotyl tissue culture.
FIG. 6 is an expression level measurement of BnaWRKY-OE strain, where WT is wild-type, #1, #2, #4, #6 are independent over-expressed transgenic lines.
FIG. 7 is a statistical plot of germination rates for wild type versus BnaWRKY-OE at high Wen Mengfa over different time periods, where WT is wild-type and BnaWRKY-OE is transgenic.
FIG. 8 is a statistical plot of root length and root length of seeds of wild type and BnaWRKY-OE after germination at high temperature, where WT is wild-type and BnaWRKY-OE is transgenic.
FIG. 9 is a plot of plaque area and plaque area statistics of leaves of wild type and BnaWRKY-OE 24 hours after inoculation with sclerotinia, where WT is wild-type strain, #1, #4, #6 are independent transgenic strains.
FIG. 10 is a plot of plaque area and plaque area statistics of leaves of wild type and BnaWRKY-OE 48 hours after inoculation with sclerotinia sclerotiorum, where WT is wild-type strain, #1, #4, #6 is an independent transgenic strain.
Detailed Description
In order to enable those skilled in the art to better understand the technical scheme of the present invention, the following detailed description of the preferred embodiments of the present invention is provided, but the following embodiments do not limit the protection scope of the present invention.
In the examples of the present invention, which are not described in detail, conventional experimental methods are adopted, and the procedures involved in the examples are understood and easily implemented by those skilled in the art based on the product specification or the basic knowledge in the art.
In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the components of the reagents are shown when the reagents are first shown, and the reagents are the same when the reagents are first shown; the reagents, materials, etc. referred to herein are commercially available as specified.
The primer sequences used in the examples were all synthesized by the company Shanghai, inc. of Biotechnology.
The culture medium and the formula thereof are as follows:
LB liquid medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride are weighed and dissolved in 800mL of ddH 2 O, the volume is fixed to 1L, then the mixture is packaged into 10 conical flasks and sealed by sealing films, the mixture is sterilized at the high temperature of 121 ℃ for 15min, and the mixture is cooled and then stored at the temperature of 4 ℃.
LB solid medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar powder are weighed and dissolved in 800mL of ddH 2 O, then the volume is fixed to 1L, and then the mixture is packaged into 10 conical flasks and sealed by sealing films, sterilized at the high temperature and high pressure of 121 ℃ for 15min, cooled and then stored at the temperature of 4 ℃. When in use, the mixture is put into a microwave oven to be heated until the mixture is melted, the antibiotics are added when the liquid is cooled to about 50 ℃, and the mixture is immediately poured into a sterile plate after shaking, wherein each plate is about 10mL.
M0 Medium (1L): MS powder 4.4g, sucrose 30g,800mL ddH 2 O are fully dissolved, the pH value is regulated to 5.84-5.88, the volume is fixed to 1L, finally coagulant agent Agar 10g is added, and the mixture is packaged into a sterile plate after sterilization and placed in a refrigerator at 4 ℃ for standby.
DM medium (1L): MS powder 4.4g, sucrose 30g,800mL ddH 2 O are fully dissolved, the pH value is regulated to 5.84-5.88, the volume is fixed to 1L, sterilization is carried out, AS is added after the culture medium is cooled, 1mL AS (mother liquor 100 mu mol/mL) is added into 1L, and the mixture is put into a refrigerator at 4 ℃ for standby.
M1 Medium (1L): MS powder 4.4g, sucrose 30g, mannitol 18g,2, 4-D1mg,KT 0.3mg,800mL ddH 2 O are fully dissolved, the pH value is regulated to 5.84-5.88, the volume is fixed to 1L, finally coagulant agent Agar 10g is added, AS is added after the culture medium is quickly cooled after sterilization, 1mL AS (mother liquor 100 mu mol/mL) is added into 1L, and then the mixture is packaged into a sterile plate and placed in a refrigerator at 4 ℃ for standby.
M2 medium (1L): MS powder 4.4g, sucrose 30g, mannitol 18g,2, 4-D1mg,KT 0.3mg,800mL ddH 2 O are fully dissolved, the pH value is regulated to 5.84-5.88, the volume is fixed to 1L, coagulant agent Agar 10g is added, and after sterilization and other culture mediums are rapidly cooled, the culture mediums are added: temeitin 300mg, STS 150. Mu. Mol, kanamycin 25mg, and then split charged into sterile dishes and placed in a refrigerator at 4℃for use.
M3 Medium (1L): MS powder 4.4g, glucose 10g, xylose 0.25g,MES 0.6g,800mL ddH 2 O is fully dissolved, the pH value is regulated to 5.84-5.88, the volume is fixed to 1L, coagulant agent Agar 10g is added, and after sterilization and other culture mediums are rapidly cooled, the culture mediums are added: ZT 2mg, IAA 0.1mg, timentin 300mg, agNO 3. Mu. Mol, kanamycin 25mg, and then split into sterile plates and placed in a refrigerator at 4℃for use.
M4 medium (1L): MS powder 4.4g, sucrose 10g,800mL ddH 2 O are fully dissolved, the pH value is adjusted to 5.84-5.88, the volume is fixed to 1L, coagulant agent Agar 10g, and after sterilization and other culture mediums are rapidly cooled, added: 300mg of timentin is packaged and put in a refrigerator at 4 ℃ for standby.
STS: [ Ag (SO 3) 2] 3-is prepared in the prior art, and precipitates after too long; mother liquor: sodium thiosulfate, 0.1M (2.48 g in 100mL ddH 2O);AgNO3, 0.1M (1.7 g in 100mL ddH 2O)VNa2SO3:VAgNO3 =4:1, agNO 3 in sodium thiosulfate).
2,4-D (1 mg/mL): 1mL of 10mg/mL of the mother solution was taken in a 12mL sterile tube, 9mL of sterilized double distilled water was added, and the mixture was mixed well and stored at-20 ℃.
KT (1 mg/mL): 0.02g KT is firstly dissolved in 1M HCL, water is added to fix the volume to 20mL, and the mixture is stored at the temperature of minus 20 ℃;
AS (100 umol/mL): 0.392g AS is firstly dissolved in a small amount of methanol, then dimethyl sulfoxide is added, the volume is fixed to 20m, and the mixture is filtered and split charging is carried out, and is stored at the temperature of minus 20 ℃.
ZT (2 mg/mL): 0.02g ZT powder is weighed and dissolved in a small amount of 75% alcohol, water is added to a volume of 10mL, suction filtration and split charging are carried out, and the mixture is stored at the temperature of minus 20 ℃.
IAA (1 mg/mL) 0.1gIAA was dissolved in a small amount of 1mol/L NaOH, and ddH 2 O was added to a volume of 100mL, and the mixture was filtered and packaged with suction and stored at-20deg.C.
TMT (300 mg/mL) 3.2g of timentin powder was dissolved in 10.66mL of sterilized ddH 2 O, mixed by shaking, filtered and sub-packaged, and stored at-20deg.C.
HYG (50 mg/mL): 1g of hygromycin B is dissolved in 20mL of sterilized ddH 2 O, mixed evenly by shaking, filtered and split-packed and stored at-20 ℃.
Kan (100 mg/mL): 10g of kana powder is dissolved in 100mL of sterilized ddH 2 O, mixed evenly by shaking, filtered and split-packed and stored at-20 ℃.
Example 1: identification and acquisition of BnaWRKY25.C04 Gene
The gene was designated LOC106407015 based on brassica napus BnaWRKY retrieved in NCBI (https:// www.ncbi.nlm.nih.gov/nucleic /). According to the amino acid sequence of BnaWRKY genes, NCBI website (https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) is used for carrying out the conserved domain analysis, as shown in figure 1, the gene contains two WRKY conserved domains, and transcription factors of the WRKY family can widely regulate the host's defense against various plant pathogens, so that the preliminary judgment BnaWRKY has a certain relationship with plant stress resistance.
6 Members of the blast BnaWRKY gene from the brassica napus reference genome website (https:// www.genoscope.cns.fr/brassicanapus /), the 6 member genes are analyzed by a bioinformatics method to form a evolutionary tree, the analysis result is shown in figure 2, the gene with the highest similarity with the BnaWRKY gene retrieved from NCBI is determined to be the target gene-BnaC g41050D of the embodiment, and the target gene is named BnaWRKY25.C04.
The method comprises the following specific steps:
(1) Culturing plant materials: cabbage type rape plants (which are given away from the Hong Dengfeng topic group of the agricultural university in China and are known as public materials) are used as experimental materials, and the growth conditions are as follows: the temperature is 24+/-2 ℃; humidity is 40-6%; the light period is 16 hours of illumination and 8 hours of darkness every day; the illumination intensity was 3000Lux.
(2) Extraction of total RNA of rape and cDNA synthesis: extracting rape total RN A by adopting a Trizol reagent rapid extraction method: taking 100mg of leaf tissue, rapidly and fully grinding by liquid nitrogen until the leaf tissue is in a powder state, adding 1mL of precooled Trizol, fully mixing, standing at room temperature for 5min, adding 200 mu L of chloroform, shaking vigorously, standing at room temperature for 2min, and centrifuging at 12,000r/min for 15min; adding equal volume of isopropanol into 400 mu L of supernatant, shaking gently, standing at room temperature for 10min after reversing and mixing uniformly, and centrifuging at 4 ℃ for 10min at 12,000 r/min; adding 75% ethanol into the supernatant, washing thoroughly, and centrifuging at 4deg.C for 5min at 12,000 r/min; removing supernatant, drying the precipitate, adding DEPC water for dissolving, and preserving at-70deg.C for use. cDNA synthesis is carried out by taking total RNA as template and adopting reverse transcription kitQ RT Super Mix for Q-PCR, purchased from Nanjinopran Biotechnology Co., ltd.) was reverse transcribed to synthesize the first strand. The reaction system is as follows: 4X gDNA wiperMix. Mu.L; an RNA template; RNase-free was completed to 16. Mu.L at 42℃for 2min. Then, 16. Mu.L of the first-step reaction solution; 5X HiScript IIIqRT Mix 4. Mu.L, 15min at 50℃and 2min at 85 ℃.
(3) Cloning of BnaWRKY25.C04 Gene cDNA sequence: primers were designed based on the BnaWRKY25.C04 gene sequence, and were synthesized by the company limited by the biological engineering (Shanghai) Co., ltd.) and were:
BnaWRKY25-F(SEQ.ID.NO.3):5’-ATGTCTTCGACATCTTTCACC-3’
BnaWRKY25-R(SEQ.ID.NO.4):5’-CCGCGCCACGTCGCTCA-3’
The full-length coding sequence of BnaWRKY25.C04 gene was amplified using cDNA of Brassica napus as a template and using high-fidelity enzyme 2X Phanta Max Master Mix (available from Nanjinouzan Biotechnology Co., ltd., cat. No. P525-01/02/03), and the PCR reaction system was as shown in Table 1.
TABLE 1 high fidelity enzyme PCR amplification reaction system
| PCR reaction system | Volume of |
| 2×Phanta Max Master Mix | 25μL |
| Upstream primer (10. Mu.M) | 2μL |
| Downstream primer (10. Mu.M) | 2μL |
| Template cDNA (50-400 ng) | 1μL |
| dd H2O | To 50μL |
Reaction conditions: 94℃for 5min, then 35cycles (95℃30s,56℃15s,72℃15 s), 72℃for 10min. Then, PCR amplification product was recovered from the agarose gel with reference to FastPure Gel DNA Extraction MINI KIT DNA gel recovery kit (available from Nanjinouzan Biotechnology Co., ltd.), and then the gel recovery product, i.e., amplified target gene BnaWRKY25.C04, was cloned into pMD19-T vector (available from Takara doctor Material technology (Beijing Co., ltd.), in the following connection system: 4.6. Mu.L of the fragment of interest, 0.4. Mu.L of pMD19-T vector, 5. Mu.L of Solution I were ligated overnight at 16℃and transformed into E.coli DH5a competent cells (available from Nanjinouzan Biotechnology Co., ltd.). It is sent to the engineering and bioengineering (Shanghai) Inc. for sequencing, and the sequencing results are completely correct. Amplifying and culturing the bacterial liquid with correct sequence, preserving the bacterial liquid by a glycerol preservation method and storing the bacterial liquid in an ultralow temperature refrigerator at the temperature of minus 70 ℃. The full-length sequence of the target gene BnaWRKY25.C04 is shown as SEQ ID NO.1, and the amino acid sequence of the gene obtained through CDS translation is shown as SEQ ID NO. 2.
As shown in FIG. 3, the accession number of the gene of BnaWRKY searched on NCBI was LOC106407015, and the similarity was found to be 94.49% by amino acid sequence alignment with the obtained BnaWRKY25.C04.
SEQ ID NO.1：
ATGTCTTCGACATCTTTCACCGACCTCCTTGCTTCCTCCGGCGTTGACCCCTACGAGCAAGACGAAGACTTTCTTGGTGGGTTTTTCCCGGAGACAACTGGGTCGGGTTTACCCAAGTTCAAGACGGCTCAACCGTCGCCTCTTCCGATCTCGCAATCTTCTCGCAGCTTCGCCTTCTCCGAGTTGCTTGACTCTCCTCTTCTCCTCAGCTCCTCACATAGTTTGATATCTCCAACGACAGGAGCGTTTCCATTTCAAGGCTTCAACGGATCAGATTTTCCCTGGCAGTTACCATCGCAAACGCAAACGCAAACGCAAACGCCAAACGCTGCTTCCGCTTTGCAAGAAGAGACGTATGGTGTTCAAGATCTCCAGAAGAAGCAGGAGGATCCGGTTCCTCGTGAGTTTGCGGATCGCCAGGTTAAGGTACCATCGTACATGGTGAGTAGGAACTCTAACGACGGTTACGGTTGGAGAAAATACGGTCAGAAACAAGTGAAGAAGAGCGAGAACCCTAGGAGTTACTTCAAGTGCACGTATCCCAACTGTGTTTCCAAGAAGATTGTTGAGACTACTTCTGATGGACAGATCACTGAGATCATCTATAAAGGTGGTCATAACCATCCTAAGCCTGAGTTCACCAAGAGACCATCATCATCGTCGGCTAATGCCAGAAGAATGCTTAATCCTTCTTCTGTTGTTAGTGAACAATCAGAGAGTTCATCGATCTCGTTTGATTATGGAGAGGTAGATGAAGAGAAGGAACAGCCTGAGATTAAGAGACTGAAAAGAGAAGGTGGAGATGAAGGGATGTCTGTAGAAGTAAGCAGAGGAGTGAAGGAACCAAGAGTTGTTGTTCAGACAATAAGTGAGATTGATGTTCTTATAGATGGCTTTAGATGGAGGAAGTATGGTCAAAAAGTTGTCAAGGGGAATACCAACCCAAGGAGCTATTACAAGTGCACATACCAAGGCTGTGGAGTGAGGAAGCAAGTGGAAAGATCAGCAGAAGACGAGAGAGCGGTTCTCACTACCTATGAAGGAAGGCACAATCATGATGTCCCAACCGCGCCACGTCGCTCATGA
SEQ ID NO.2：
MSSTSFTDLLASSGVDPYEQDEDFLGGFFPETTGSGLPKFKTAQPSPLPISQSSRSFAFSELLDSPLLLSSSHSLISPTTGAFPFQGFNGSDFPWQLPSQTQTQTQTPNAASALQEETYGVQDLQKKQEDPVPREFADRQVKVPSYMVSRNSNDGYGWRKYGQKQVKKSENPRSYFKCTYPNCVSKKIVETTSDGQITEIIYKGGHNHPKPEFTKRPSSSSANARRMLNPSSVVSEQSESSSISFDYGEVDEEKEQPEIKRLKREGGDEGMSVEVSRGVKEPRVVVQTISEIDVLIDGFRWRKYGQKVVKGNTNPRSYYKCTYQGCGVRKQVERSAEDERAVLTTYEGRHNHDVPTAPRRS
Example 2: construction of BnaWRKY25.C04 Gene overexpression vector
This example uses the Gateway method, adding a CACC sequence to the 5' end of the upstream primer, and removing the terminator when amplifying the BnaWRKY25.C04 gene sequence for the expression of green fluorescent protein. The primer sequences were designed as follows:
2.0-BnaWRKY25.C04-F(SEQ.ID.NO.5)：5’-CACCATGTCTTCGACATCTTTCACC-3’
2.0-BnaWRKY25.C04-R(SEQ.ID.NO.6)：5’-CCAACCGCGCCACGTCGCTCA-3’
First, the positive bacterial liquid obtained in example 1 was taken out from an ultralow temperature refrigerator at-70℃and activated to extract plasmids. The BnaWRKY25.C04 gene sequence was amplified using Hi-Fi enzyme 2X Phanta Max Master Mix (available from Nanjinouzan Biotechnology Co., ltd.) using the extracted plasmid as a template, and the fragment obtained by this amplification was four more bases than CACC at the front of the ATG initiation codon and lacked TAA termination codon at the sequence end, unlike example 1. After amplification was completed, 1% agarose gel was prepared, agarose gel electrophoresis was performed at 120V,400mA, then gel recovery was performed according to the procedure of example 1, and then an entry clone BP reaction was performed.
The total system of the entry clone BP reaction is 3 mu L, and the specific components are as follows:
Entry vector pENTR-D-ToPo 0.5.5. Mu.L, salt Solution 0.5. Mu.L, molar ratio of PCR recovery product to entry vector pENT R-D-ToPo was 2:1, ddH 2 O was added to 3. Mu.L. After 2h reaction at 22 ℃, DH 5. Alpha. Competent cells were transformed, several monoclonals were picked up into 1.5mL EP tubes, 400. Mu.L of Kan-resistant liquid LB was added, and after 4h shaking culture, rTaq enzyme, universal primers were used:
M13-F(SEQ.ID.NO.7)：5’-GTTGTAAAACGACGGCCAG-3’
M13-R(SEQ.ID.NO.8)：5’-CAGGAAACAGCTATGAC-3’
performing bacterial liquid PCR identification, carrying out agarose gel electrophoresis on a target band of about 1100bp, performing imaging, performing amplification culture on bacterial liquid with correct band size, performing bacteria preservation by a glycerol preservation method, extracting plasmids by using the residual bacterial liquid, and performing LR recombination reaction on the plasmids and a final vector pK7FWG2.0.
The LR recombination reaction system is 10 mu L in total, and the specific components are as follows:
150ng of plasmid extracted from the residual bacterial liquid, and final vector pK7FWG2.0 150ng,LR Clonase TM II Enzy me 2 mu L, and TE buffer were added to 10 mu L. After 1h of reaction at 25 ℃,1 mul of protein kinase K solution is added, the reaction is carried out for 10min at 37 ℃, the competent cells of escherichia coli DH5 alpha are transformed again, 35S-F (SEQ. ID. No. 9)/2.0-BnaWRK Y25.C04-R (SEQ. ID. No. 6) is used for carrying out bacterial liquid PCR identification, bacterial liquid with correct strip size and bright and no impurity band is selected and sent to a biological engineering (Shanghai) stock company for sequencing, the bacterial liquid with correct sequencing is amplified for culture, bacteria are preserved and plasmids are extracted, and the vector pK7FWG2.0-BnaWRKY25.C04 which overexpresses the BnaWRKY25.C04 gene is successfully constructed, and the schematic diagram of the vector is shown in figure 4. The recombinant vector pK7FWG2.0-BnaWRKY25.C04 is transformed into Agrobacterium GV3101 competent cells (purchased from Shanghai Weidi biotechnology Co., ltd.) and the transformed positive clone is amplified and cultured, and then the bacterial solution is stored in a ultralow temperature refrigerator at-70 ℃ for standby by using a glycerol preservation method.
Example 3: transformation of Brassica napus (Brassica napus) with pK7FWG2.0-BnaWRKY25.C04 recombinant vector
(1) Seed disinfection and sowing
Selecting full-grain brassica napus (presented by Hong Dengfeng subject group of agricultural university in China, known public materials), placing the brassica napus in a12 mL sterile tube, sterilizing the brassica napus in an ultra clean bench with 75% alcohol for 1min, then cleaning seeds with sterile ddH 2 O for 2-3 times, then sterilizing the brassica napus with 15% Bleach solution (prepared as 8.115mL sterile water+1.875 mL sodium hypochlorite+10 mu L triton) for 7min, then cleaning the seeds with sterile ddH 2 O until no obvious foam exists in the water, finally uniformly sowing the sterilized seeds on an M0 culture medium, and culturing the seeds in an incubator for 7 days under the following growth conditions: the temperature is 24+/-2 ℃; humidity is 40-60%; dark for 24h.
(2) Preparation of bacterial liquid
On day 7 after sowing, the GV 3101 agrobacterium containing the pk7fwg2.0-bnwrky25.c04 recombinant vector obtained in example 2 was subjected to expansion culture: mu.L of the stock solution was added with 5mL of resistant LB (50 mg/L Kan+50mg/L Gen+50mg/L Rif) and cultured at 28℃for about 14-16 hours in a shaker at 220 rpm.
(3) Infection and co-cultivation
And (3) measuring the OD value of the bacteria in the resistant LB medium in the step (2) by using a spectrophotometer, wherein the bacterial liquid is better when the OD value is about 0.4, and the bacteria are generally shaken for 14-16 hours. Sucking 2mL of the cultured bacterial liquid into a 2mL sterile centrifuge tube, centrifuging at 5000rpm for 10min, and discarding the supernatant; then 2mL of DM (AS+) solution was added to suspend, centrifuged at 5000rpm for 10min, and the supernatant was discarded; then, 2mL of DM (AS+) was added to the suspension, and the suspension was kept in a refrigerator at 4℃for further use.
Taking out seedlings which are dark-cultured for 7 days, cutting hypocotyls by using forceps and scissors which are sterilized at high temperature, placing the seedlings in a disposable plate filled with 18mL DM (AS+) and cutting the seedlings downwards in an inclined way AS fast AS possible during cutting, and ensuring that the cut is flat and the length is 0.8-1 cm. After cutting, adding a prepared bacterial liquid, dip-dyeing for 13min, and shaking for 1 time at intervals for 3-4 times; the DM (AS+) and bacterial liquid are sucked by a gun head, the explant is paved on filter paper sterilized in advance, and the bacterial liquid with excessive surface is sucked. The explants are evenly placed on an M1 culture medium by forceps, and when the explants are placed, two ends of the explants are fully contacted with the culture medium, and the explants are subjected to dark culture at 24 ℃ for 36-48 hours.
(4) Selective culture and callus induction
Transferring the explant in the M1 after co-culture to an M2 culture medium for selecting and inducing callus, and culturing the explant in the M2 culture medium under inverted light normally, wherein the culturing condition is that the explant is cultured alternately in a mode of 16h in daytime and 8h in evening at 24 ℃ and the callus is induced for 2-3 weeks.
(5) Redifferentiation
Selected explants are transferred to differentiation medium M3, and subcultured every 2-3 weeks, during which dead explants are eliminated, preferably explants with enlarged ends and green spots until green buds appear.
(6) Rooting culture
Cutting a part of wounds on the base of the bud seedlings by using a high-temperature sterilized scalpel, and transferring the bud seedlings into an M4 culture medium for rooting culture. After rooting, the seedlings can be placed in a culture room for hardening, after the seedling state is stable, the seedlings are taken out from a culture medium, the root systems of the plants are not damaged as much as possible in the seedling taking process, then the root systems are soaked with 1% carbendazim for one time, finally the seedlings are moved into soil for culture, and the transgenic rape waiting for identification can be obtained by using a preservative film to preserve moisture for about 1 week during the culture.
Example 4: PCR identification of BnaWRKY25.C04 gene transformed plant and expression quantity detection of positive plant
After the transformed rape plant in example 3 grows stably, extracting DN A in transgenic rape leaves by adopting a CTAB method, and specifically comprises the following steps: a small amount of leaves was placed in a 1.5mL centrifuge tube, ground with liquid nitrogen to a dry powder, 600. Mu.L CTAB was added, and the sample was then placed in a 65℃water bath for 60min.
After waiting for the incubation to complete, 600. Mu.L of chloroform/isoamyl alcohol (24:1 by volume) solution was added to the tube, vigorously shaken, and the protein was removed thoroughly, and then placed in a centrifuge for centrifugation at 12000rpm for 10min. After centrifugation, the centrifuge tube was gently removed, at which time the solution was divided into three layers, the aqueous phase, the leaf debris impurity layer, and the organic phase, 400 μl of supernatant was aspirated, transferred to a new centrifuge tube, and then an equal volume of isopropanol was added to the supernatant, gently inverted and mixed, and then the sample was placed in a-20deg.C refrigerator for precipitation for at least 10min. The tube was placed in a centrifuge and centrifuged at 12000rpm for 10min at room temperature. After centrifugation, the supernatant was discarded, and 700. Mu.L of pre-chilled 70% ethanol was added to wash, flick the pellet, gently invert the wash, and spin at 12000 rpm. After centrifugation, the supernatant was discarded, the ethanol solution was aspirated with a pipettor, and then the pellet was air-dried in an ultra clean bench to remove the volatile organic solution. Adding 50-100 mu L of ddH 2 O into a centrifuge tube to dissolve the precipitate, and placing the precipitate into a water bath kettle at 37 ℃ to incubate for 30min to obtain a genome sample. And 1 mu L of genome sample is taken for measuring the concentration, and after the genome sample is detected to be qualified, the genome sample is put into a refrigerator at the temperature of minus 20 ℃ for standby.
Taking the genome sample obtained in the above steps as a template, taking the pK7FWG2.0-BnaWRKY25.C04 plasmid as a positive control, taking DNA of a receptor material which is not subjected to genetic transformation as a negative control, taking ddH 2 O as a blank control, carrying out PCR identification according to the 35S promoter and eGFP on the pK7FWG2.0 vector as identification primers, wherein the annealing temperature is 57 ℃, and the primer sequences are as follows:
35S-F(SEQ.ID.NO.9):5’-CTTCGCAAGACCCTTCCTC-3’
eGFP-R(SEQ.ID.NO.10):5’-TCCACACAACATACGAGCCG-3’
After the PCR is completed, the amplified products are electrophoresed in 1% agarose gel, imaged by using an ultraviolet gel imager, and the result is recorded. And selecting plants with clear and bright bands and correct sizes to detect the expression quantity of the BnaWRKY25.C04 genes. FIG. 5 shows a PCR identification gel diagram of plant leaf genome obtained by transformation and an expression level analysis of positive plants, and it can be seen from the diagram that 4 PCR positive lines are obtained after PCR identification, wherein the diagram shows that the stripe brightness is higher and the stripe size accords with expectations. The expression level of BnaWRKY25.C04 genes in #1, #2, #4, #6 transgenic plants and wild type plants is detected by adopting qRT-PCR technology, and the method specifically comprises the following steps: fresh leaves of each plant were collected and immediately frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator at-70 ℃ for later use. RNA and synthetic cDNA of transgenic and wild type canola leaf tissue were obtained as in example 1. The primer sequences are as follows:
BnaWRKY25.C04-QF(SEQ.ID.NO.11)：5'-CCCCTACGAGCAAGACGAAG-3'
BnaWRKY25.C04-QR (SEQ. ID. NO. 12): 5'-GAGATCGGAAGAGGCGACG-3', and plotting the obtained gene expression amount data, and plotting the data result by GRAPH PAD PRISM software.
The expression level of WRKY25.C04 in BnaWRKY25.C04 transgenic plants is shown in FIG. 6, and the expression level of WRKY25.C04 in wild type cabbage type rape leaves is used as a control, so that the expression level of BnaWRKY25.C04 genes in transgenic plants is remarkably increased. These results show that the BnaWRKY25.C04 gene is successfully over-expressed in the transgenic plant, and the results provide a material basis for the subsequent application of BnaWRKY25.C04.
Example 5: positive transformant line T1 generation seed high temperature resistant germination of BnaWRKY25.C04 gene
And reserving seeds of the cultivated positive plants, and harvesting T1 generation seeds. The heat resistance identification method of the cabbage type rape comprises the following specific steps: selecting transgenic early-maturing spring rape seeds and wild type seeds which are harvested in the same period, have robust and full seeds and have the same size, cleaning the seeds with sterile water, placing the seeds in a culture dish paved with two layers of filter paper, soaking the filter paper in distilled water, performing normal-temperature treatment, placing the filter paper in a 24 ℃ incubator for germination, performing high-temperature treatment, placing the filter paper in a 45 ℃ illumination incubator for 6 hours, and then placing the filter paper in the 24 ℃ incubator for germination; treating 16 seeds at each temperature, repeating three times; judging that the radicle length is larger than the seed diameter, and calculating the number of germinated seeds once every 24 hours; the germination percentage is calculated by dividing the total number of germinated seeds at that time point by the total number of seeds.
And carrying out germination experiments on the transgenic type and the wild type of the over-expressed BnaWRKY25.C04 according to the cabbage type rape heat resistance identification method. As shown in FIG. 7, the germination rate of the over-expressed BnaWRKY25.C04 is consistent with that of the wild type at 24 ℃, but the germination rate of the wild type is only about 70% under the treatment condition of 45 ℃ for 6 hours, and the germination rate of the transgene type of the over-expressed BnaWRKY25.C04 can reach about 94% and is obviously higher than that of the wild type. Therefore, the BnaWRKY25.C04 gene overexpression can improve the germination rate and the survival rate of seeds in the high-temperature stress of the cabbage type rape, and the germination of the seeds in the rape crops at high temperature can be changed by regulating and controlling the BnaWRKY25.C04, so that the mechanization degree is improved.
Example 6: measurement of root length of seed after high temperature germination of untransformed plants and positive transformant T1 generation seed overexpressing BnaWRKY25.C04 Gene
Transplanting the germinated seeds of example 5 subjected to high temperature treatment at 45 ℃ for 6 hours into soil, and controlling the temperature to be 24+/-2 ℃; humidity is 40-60%; the light period is 16 hours of illumination and 8 hours of darkness every day; growing in an environment with the illumination intensity of 2400Lux for 10 days, and observing the growth condition. As shown in FIG. 8, the wild type and the overground parts of the overrepresented BnaWRKY25.C04 of the seeds subjected to high-temperature germination have no obvious difference, but the root length of the overrepresented BnaWRKY25.C04 is obviously higher than that of the wild type, and the strong root system is beneficial to better absorbing nutrition in the later period of the plant. Therefore, the over-expression of the BnaWRKY25.C04 gene can improve the elongation of main roots in the high-temperature stress of the brassica napus, and the growth and development conditions of the brassica napus crops at high temperature can be hopefully changed by regulating and controlling the BnaWRKY25.C04.
Example 7: evaluation of BnaWRKY25.C04 over-expressed transgenic rape sclerotinia resistance
Taking true leaves of the four-leaf one-heart stage BnaWRKY25.C04 positive plant which is detected as positive by PCR in the example 4 and has the increased expression level for in vitro inoculation; cutting the leaves, placing the leaves in a box which is made to be moisturized, placing the leaves of each transgenic plant in a box side by side with a non-transgenic contrast, paving a layer of wet gauze under the leaves, sealing the box, making the box to be moisturized, and culturing in the dark for 24 hours, so that the vitality of each leaf tends to be consistent, and the error is reduced; after the dark culture is completed, taking prepared 3mm mycelium blocks (Jiangsu university rape test field), inoculating the mycelium blocks with the mycelium face downwards to a position slightly deviated from a main vein in the middle of the leaf, and inoculating one mycelium block on each leaf; moisturizing the box, and then culturing in dark at 22 ℃; plaque growth was recorded at 24h intervals, starting 24h after inoculation. The results of the in vitro leaf inoculation show that the over-expressed transgenic plants significantly enhance sclerotinia resistance. As shown in fig. 9 and 10, the growth area of the sclerotinia sclerotiorum plaques in the bnwrky 25.c04 over-expressed transgenic strain was found to be much smaller than that in the non-transgenic strain 24 hours and 48 hours after inoculation, then the Image J software was used for data statistics of the plaque areas, prism 9 software analysis was used to test whether the data results were significantly different, and the t-text test results showed that the plaque area of BnaWRKY over-expressed transgenic strain was significantly smaller than that of the non-transgenic strain. P <0.0001, P <0.001, P <0.01, P <0.05, more representing a greater significant difference).
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (4)
- The BnaWRKY25.C04 gene has a nucleotide sequence shown as SEQ.ID.NO.1, and can be used for promoting high-temperature germination of rape seeds and/or bacterial nuclear disease resistance, breeding of high-temperature resistant seed resources or breeding of bacterial nuclear disease resistance rape.
- 2. The use according to claim 1, wherein the amino acid sequence of the protein encoded by the bnwrky 25.c04 gene is shown in seq id No. 2.
- 3. The use according to claim 1 or 2, characterized in that the use is achieved by overexpression of the bnwrky 25.c04 gene.
- 4. A method for promoting high temperature germination and/or bacterial nuclear disease resistance of rape seeds is characterized in that a recombinant expression vector containing the BnaWRKY25.C04 gene in claim 1 is constructed, and the recombinant expression vector is transformed into recipient bacteria to obtain recombinant engineering bacteria; amplifying and culturing the obtained recombinant engineering bacteria, and transforming the rape hypocotyl by the obtained bacterial liquid; culturing the callus of the hypocotyl of the rape, inducing and re-differentiating to obtain rape transformant for promoting the high temperature germination of rape seed and/or resisting bacterial sclerotinia.
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| CN103911384A (en) * | 2014-01-21 | 2014-07-09 | 江苏大学 | Gene for controlling Sclerotinia sclerotiorum (Lib.) de Bary of Brassica napus L. and use thereof |
| CN105063085A (en) * | 2015-07-31 | 2015-11-18 | 江苏大学 | Cabbage type rape gene BnMPK3 and application thereof in resisting sclerotinia rot of colza |
| CN111500593A (en) * | 2020-04-13 | 2020-08-07 | 山西大学 | Cabbage type rape gene BnTN L R1 and sclerotinia resistant application thereof |
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| CN103911384A (en) * | 2014-01-21 | 2014-07-09 | 江苏大学 | Gene for controlling Sclerotinia sclerotiorum (Lib.) de Bary of Brassica napus L. and use thereof |
| CN105063085A (en) * | 2015-07-31 | 2015-11-18 | 江苏大学 | Cabbage type rape gene BnMPK3 and application thereof in resisting sclerotinia rot of colza |
| CN111500593A (en) * | 2020-04-13 | 2020-08-07 | 山西大学 | Cabbage type rape gene BnTN L R1 and sclerotinia resistant application thereof |
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