CN117535343A - Method for rapidly, efficiently and stably verifying functions of capsicum gene and application - Google Patents
Method for rapidly, efficiently and stably verifying functions of capsicum gene and application Download PDFInfo
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- 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
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8203—Virus mediated transformation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
Abstract
The invention provides a method for rapidly, efficiently and stably verifying the function of a capsicum gene and application thereof, and belongs to the technical field of gene function method research. The invention constructs a set of method for rapidly, efficiently and stably verifying the functions of the capsicum genes based on the VIGS technology developed by tobacco embrittlement virus (TRV), and obtains the important influence on the growth temperature, the agrobacterium infection concentration, the agrobacterium inoculation mode and the like of the capsicum after the infection in the capsicum type and the capsicum type through strict condition optimization, and finally discovers that the optimal conditions of a capsicum VIGS gene silencing system are as follows: the growth temperature after infection is 24 ℃, the OD600 value of the agrobacterium inoculation concentration is 0.01, and the optimal inoculation method is an injection method. The method of the invention has great significance for verifying the functions of various genes in the capsicum.
Description
Technical Field
The invention belongs to the technical field of gene function method research, and particularly relates to a method for rapidly, efficiently and stably verifying the function of a capsicum gene and application thereof.
Background
Gene silencing is commonly found in organisms, and is characterized in that the invasion of exogenous nucleic acids such as transposons, viruses and the like is resisted, the expression of exogenous genes is specifically recognized and inhibited, and the stability of the genome of the organisms is maintained. VIGS (virus-induced gene silencing) is a gene silencing technique induced by viral vectors, and has advantages of short cycle, low cost, high throughput, etc. compared with methods of transgene, gene knockout, antisense suppression, etc., and thus, VIGS is often used for studying functions of plant genes. VIGS is mainly characterized in that a plant is infected by using a virus vector to carry a target gene, double-stranded RNA (dsRNA) is formed in the replication and expression processes of the plant, the dsRNA is cut into small interfering RNA (siRNA) fragments of 21-24 nt by a specific endonuclease Dicer in cells, the siRNA is amplified by an RNA polymerase, and the siRNA is combined with some proteins in a single-stranded form to form RNA-induced complexes (RISC), and the complexes specifically interact with the target gene mRNA to cause degradation of the target gene mRNA, so that gene silencing at a post-transcriptional level occurs.
The genetic transformation of vegetable crops is generally difficult due to various reasons such as various varieties, research level and the like, particularly the genetic transformation of peppers has seriously affected the research of the gene functions of the peppers, and meanwhile, the efficiency and success rate of the detection of the actual application of VIGS technology of different host plants are greatly different, so that a method which is specially applicable to peppers and can rapidly, efficiently and stably verify the gene functions of the peppers is urgently needed at present.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for rapidly, efficiently and stably verifying the functions of a pepper gene and application thereof. The invention constructs a set of method for rapidly, efficiently and stably verifying the functions of the capsicum genes based on the VIGS technology developed by tobacco embrittlement virus (TRV), and obtains the important influence on the growth temperature, the agrobacterium infection concentration, the agrobacterium inoculation mode and the like of the capsicum after the infection in the capsicum type and the capsicum type through strict condition optimization, and finally discovers that the optimal conditions of a capsicum VIGS gene silencing system are as follows: the growth temperature after infection is 24 ℃, the OD600 value of the agrobacterium inoculation concentration is 0.01, and the optimal inoculation method is an injection method. The method of the invention has great significance for verifying the functions of various genes in the capsicum.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a method for rapidly, efficiently and stably verifying the functions of a capsicum gene, which comprises the following steps:
step one: construction of viral vector TRV:
designing a primer according to NCBI published or cloned capsicum gene sequences, connecting a cloned fragment with a viral vector, converting the fragment into escherichia coli, and sending the fragment to be detected after shaking PCR detection; detecting DNA sequences, shaking bacteria, extracting plasmid, and double enzyme cutting to obtain target gene segment; simultaneously converting the empty vector grains of pTRV2 into escherichia coli, performing PCR detection, shaking bacteria, extracting plasmids, performing double enzyme digestion, precipitating chloroform and ethanol, then running gel, and recovering gel to obtain digested pTRV2 vector fragments; connecting a target gene fragment with a pTRV2 vector, then converting into escherichia coli, carrying out PCR detection, shaking after sequencing without errors, and extracting plasmids; finally, transferring the extracted constructed viral vector plasmid into agrobacterium;
step two: and (3) preparation of an infection Buffer:
firstly, adding 95-100 ml of sterilized double distilled water into 95-100 ml of sterilized distilled water, then adding 0.5-1.5 ml of 1MMgcl 2 0.5 to 1.5ml of 1M MES and 180 to 220ul of 200mM acetosyringone, and finally adjusting the PH value to 5 to 6;
step three: preparation of an infectious microbe liquid:
performing a capsicum VIGS experiment by using an agrobacterium-mediated method, firstly transferring two plasmid vectors of TRV1 and TRV2+ target gene fragments into agrobacterium competent GV3101 respectively, and culturing for 1-3 d at 25-30 ℃; then selecting positive agrobacterium monoclonals in a liquid LB culture medium containing 45-55 mg/mL kanamycin and 8-12 mg/mL rifampicin; then centrifugally collecting thalli at 2500-3500 rpm, discarding supernatant, suspending bacteria by using an infection Buffer, and adjusting the concentration to OD < 600 > = 0.005-0.015; finally uniformly mixing TRV1+TRV2 and TRV1+ (TRV2+ target gene fragment) in equal volume, and standing for 2-4 hours at room temperature or shaking and culturing for 2-4 by a shaking table at 80-120 rpm at 25-30 ℃;
step four: growing and preparing capsicum:
about 20d after sowing the peppers, and inoculating after two true leaves grow out; and watering is needed before inoculation, and water is needed to be controlled after inoculation;
step five: control of the infestation conditions:
including the choice of the mode of inoculation, the use of the inoculation site, and the control of environmental conditions after injection.
Preferably, the capsicum gene used in the first step is a capsicum PDS gene, and the sequence of the capsicum gene is shown as SEQ ID NO. 1; the sequence of the obtained target gene fragment is shown as SEQ ID NO. 2.
Preferably, the viral vector used in the first step is TRV, the cleavage sites are Xba I and Kpn I, the Escherichia coli strain is Trans-5a, and the Agrobacterium strain is GV3101.
Preferably, the specific process of the second step is as follows: 98ml of sterilized double distilled water was added to 98ml of sterilized distilled water, followed by 1ml of 1M Mgcl 2 1ml of 1M MES and 200ul of 200mM acetosyringone, and finally adjusting the pH to 5.6;
preferably, the specific process of the third step is as follows: firstly, respectively transferring two plasmid vectors of TRV1 and TRV2+ target gene fragments into agrobacterium competent GV3101, and culturing for 2d at 28 ℃; then, positive agrobacterium is selected and monoclonal in a liquid LB culture medium containing 50mg/mL kanamycin and 10mg/mL rifampicin; then, the thalli are collected by centrifugation at 3000rpm, the supernatant is discarded, bacteria are suspended by using an infection Buffer, and the concentration is adjusted to be OD600 = 0.01; finally uniformly mixing TRV1+TRV2, TRV1+ (TRV2+ target gene fragment) in equal volume, standing at room temperature for 3 hours or shaking and culturing at 100rpm by a shaking table at 28 ℃ for 3 hours;
preferably, in the fourth step, watering is performed 1d before inoculation, and water is controlled 3d after inoculation.
Preferably, the inoculation mode selected in the fifth step is injection inoculation, and the inoculation position used is the back of the true leaves.
Preferably, the control method of the environmental condition after injection in the fifth step is as follows: after injection, the cells were allowed to grow in the dark for 2 days at 18℃and then cultured normally at 24℃at 18℃under 16h day/8h light conditions.
Preferably, the method further comprises a step of detecting the effect of gene silencing after infection is complete.
The invention also provides application of the method in detecting the functions of other capsicum genes except the capsicum PDS gene.
Compared with the prior art, the invention has the following technical effects:
(1) In the invention, VIGS can be used to silence a target gene first, so that the expression of a plant under the condition that the gene is silent is observed, and researchers can infer the functions of the target gene in the aspects of plant growth, development, metabolism and the like by comparing the difference between the silent plant and the non-silent plant; at the same time VIGS can be used to screen a large number of genes rapidly to determine their role in a particular biological process, which is very useful for identifying key regulatory genes or new drug targets; secondly, VIGS can be used to study the resistance of plants to pathogens, and by silencing genes interacting with pathogens, the role of these genes in the immune response of plants can be understood, contributing to improving the disease resistance of plant varieties; finally, VIGS can also be used to verify the function of specific genes in genetic engineering, and the interaction between target genes and other genes can reveal gene regulation networks and signal transduction pathways, so that the complex gene regulation mechanism in organisms is understood more deeply. The VIGS can rapidly test the function of the target gene in the current generation of crops, and has remarkable advantages of high efficiency, stability and high flux in crop research.
(2) According to the invention, in the types of capsicum towards the sky and the thin skin peppers, the capsicum has obvious influence on the growth temperature, the agrobacterium infection concentration, the agrobacterium inoculation mode and the like after the infection of the agrobacterium through strict condition optimization, and finally, the optimal conditions of a capsicum VIGS gene silencing system are found as follows: the growth temperature after infection is 24 ℃, the OD600 value of the agrobacterium inoculation concentration is 0.01, and the optimal inoculation method is an injection method.
Drawings
FIG. 1 shows the growth of pepper plants before and after inoculation in example 1 of the present invention;
FIG. 2 shows the relative expression levels of PDS gene in the leaf of capsicum in example 1 of the present invention, which has undergone gene silencing to cause albino, including Wild Type (WT) and white-flowered pepper strain lines 1-4;
FIG. 3 is a graph showing the effect of different inoculation temperatures on the efficiency of silencing the PDS gene in example 2 of the present invention;
FIG. 4 is a graph showing the effect of different Agrobacterium infection concentrations on the efficiency of silencing of the PDS gene of capsicum according to example 3 of the present invention;
FIG. 5 shows the effect of different inoculation methods on the efficiency of silencing the PDS gene in example 4 of the invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The reagents, kits and instruments used in the following examples are commercially available, and the methods used in the examples are consistent with the methods conventionally used unless otherwise specified.
The pepper variety selected in this example was Zun a-1, the viral vector was TRV, the enzyme cleavage sites were Xba I and Kpn I, the E.coli (Escherichia coli) strain was Trans-5a, and the Agrobacterium strain was GV3101.
The technical scheme of the invention is further elaborated in the following in conjunction with examples.
Example 1 establishment of PDS Gene VIGS silencing System and silencing Effect verification
The TRV system mainly includes two vectors: TRV1 and TRV2.TRV1 is an helper vector, while TRV2 carries an interfering fragment of the gene of interest.
Phytoene Dehydrogenase (PDS) is a key enzyme in the carotenoid synthesis pathway. When the gene expression is silenced, the carotenoid synthesis pathway is blocked, and the plant loses the photoprotection of the carotenoid, so that the plant generates a photobleaching phenomenon, i.e., leaf whitening. Thus, the PDS gene is commonly used as a reporter gene in the VIGS silencing system. In order to better evaluate the silencing plant rate of the VIGS, the PDS gene in the capsicum plants is silenced by the VIGS, and the silencing plant rate of the VIGS is calculated by counting the number of plants with white flowers.
The following method was used to establish a VIGS silencing system based on PDS gene:
step one: construction of viral vector TRV:
firstly, designing a primer according to a chilli PDS gene with a sequence shown as SEQ ID NO.1, connecting a cloned fragment with a TRV virus vector, converting the fragment into escherichia coli Trans-5a, and sending and measuring after shaking PCR detection; detecting DNA sequences, performing bacterium shaking, extracting plasmids, selecting enzyme cutting sites Xba I and Kpn I, and performing double enzyme cutting to obtain target gene fragments; simultaneously converting the empty vector grains of pTRV2 into escherichia coli, performing PCR detection, shaking bacteria, extracting plasmids, performing double enzyme digestion, precipitating chloroform and ethanol, then running gel, and recovering gel to obtain digested pTRV2 vector fragments; connecting a target gene fragment with a pTRV2 vector, transferring into escherichia coli competence, performing PCR detection, shaking after sequencing, and extracting plasmids; finally, transferring the extracted constructed viral vector plasmid into agrobacterium GV3101 (Kan); the sequence of the obtained target gene fragment is shown as SEQ ID NO. 2.
Step two: and (3) preparation of an infection Buffer:
98ml of sterilized double distilled water was added to 98ml of sterilized distilled water, followed by 1ml of 1M Mgcl 2 1ml of 1M MES and 200ul of 200mM acetosyringone, and finally adjusting the pH to 5.6;
step three: preparation of an infectious microbe liquid:
firstly, respectively transferring two plasmid vectors of TRV1 and TRV2+ target gene fragments into agrobacterium competent GV3101, and culturing for 2d at 28 ℃; then, positive agrobacterium is selected and monoclonal in a liquid LB culture medium containing 50mg/mL kanamycin and 10mg/mL rifampicin; then, the thalli are collected by centrifugation at 3000rpm, the supernatant is discarded, bacteria are suspended by using an infection Buffer, and the concentration is adjusted to be OD600 = 0.01; finally uniformly mixing TRV1+TRV2, TRV1+ (TRV2+ target gene fragment) in equal volume, standing at room temperature for 3 hours or shaking and culturing at 100rpm by a shaking table at 28 ℃ for 3 hours;
step four: growing and preparing capsicum:
about 20d after sowing the capsicum, inoculating after two true leaves grow out, and in order to enable bacterial liquid infection to be more efficiently completed, water control treatment is needed to be carried out on capsicum seedlings, namely 1d watering is carried out before inoculation, and 3d water control treatment after inoculation is more beneficial to the infection of agrobacterium;
step five: control of the infestation conditions:
about 20d of pepper seedlings grow out, the back of the true leaves of the peppers are injected by a disposable injector, so that the whole leaves are filled with agrobacterium solution, but in order to improve the infection effect, 1d of watering is performed before inoculation infection, 3d of water control is performed after inoculation, the infection of the agrobacterium is facilitated, the pepper seedlings grow for 2 days in dark conditions at 18 ℃ after injection, and then the normal culture is most favorable for the efficient and stable function of the VIGS under the conditions of 24 ℃/18 ℃ and 16h day/8h right.
The PDS gene sequence (SEQ ID No. 1) of capsicum:
ATGCCCCAAATTGGACTTGTTTCTGCTGTCAACTTGAGAGTCCAAGGTAATTCAGCTTATCTTTGGAGCTCGAGGTCTTCTTTGGGAACTGATAGTCAAGATGGTTGCTCGCAAAGGAATTCGTTATGTTTTGGTGGTAGTGACTCAATGAGTCATAGGTTAAAGATTCGTAATCCCCATTCCATAACGAGAAGATTGGCTAAGGATTTCCGGCCTTTAAAGGTTGTTTGCATTGATTATCCAAGGCCAGAGCTAGACAATACAGTTAACTATTTGGAGGCTGCATTCTTATCATCATCATTCCGATCTTCTCCGCGCCCAACCAAACCACTGGAGATTGTTATTGCTGGTGCAGGTTTGGGTGGTTTGTCTACAGCAAAATATTTGGCAGATGCTGGTCACAAACCAATACTGCTGGAGGCAAGGGATGTTCTAGGTGGAAAGGTAGCTGCATGGAAAGATGATGATGGAGATTGGTATGAGACTGGTTTGCACATATTCTTTGGGGCTTACCCAAATATGCAGAACCTATTTGGAGAATTAGGGATAAATGATCGATTGCAATGGAAGGAACATTCGATGATATTTGCAATGCCAAACAAGCCAGGAGAATTCAGCCGCTTTGATTTCCCCGAAGCTTTACCTGCTCCTTTAAATGGAATTTTGGCAATCCTAAAGAACAATGAAATGCTTACATGGCCAGAAAAAGTCAAATTTGCAATTGGACTCTTGCCAGCAATGCTTGGTGGGCAATCTTATGTTGAAGCTCAAGACGGGATAAGTGTTAAGGACTGGATGAGAAAACAAGGTGTGCCGGATAGGGTGACGGATGAGGTGTTCATCGCCATGTCAAAGGCACTTAACTTCATAAATCCTGATGAGCTTTCGATGCAGTGCATCTTGATCGCGTTGAACAGATTTCTTCAGGAGAAACATGGTTCAAAAATGGCCTTTTTAGATGGTAATCCTCCTGAGAGACTTTGCATGCCGATTGTTGAACATATCGAGTCAAAAGGTGGACAAGTCAGACTGAACTCACGAATAAAAAAGATTGAGCTGAATGAGGATGGAAGTGTCAAGTGTTTTATACTGAACGATGGTAGTACAATTGAGGGAGATGCTTTTGTGTTTGCGACTCCAGTGGATATTTTCAAGCTTCTTTTGCCTGAAGACTGGAAAGAGATTCCATATTTCCAAAAGTTGGAGAAGTTAGTCGGAGTACCTGTGATAAATGTCCATATATGGTTTGACAGAAAACTGAAGAACACATCTGATAATTTGCTCTTCAGCAGAAGCCCACTGCTCAGTGTGTATGCTGACATGTCCGTCACATGTAAGGAATATTACGACCCCAACAAGTCCATGTTGGAATTGGTCTTTGCGCCTGCAGAAGAGTGGGTATCTCGCAGTGACTCTGAAATTATTGATGCTACAATGAAGGAACTAGCAAAGCTATTTCCTGATGAAATTTCGGCGGATCAGAGCAAAGCAAAAATATTGAAGTATCATGTTGTCAAAACTCCAAGGTCTGTATATAAAACTGTGCCAGGTTGTGAACCCTGTCGGCCCTTGCAAAGATCCCCTGTAGAGGGGTTTTATTTAGCTGGTGACTACACGAAACAGAAATACTTGGCTTCAATGGAAGGTGCTGTCTTATCAGGAAAGCTTTGTGCACAAGCTATTGTACAGTTAGCAGTCTCCGTTTCTCATGAATCTGGTGTGAGGAGAGCGGTTGTCAAG
the sequence of the target gene fragment (SEQ ID NO. 2):
AGGTCTTCTTTGGGAACTGATAGTCAAGATGGTTGCTCGCAAAGGAATTCGTTATGTTTTGGTGGTAGTGACTCAATGAGTCATAGGTTAAAGATTCGTAATCCCCATTCCATAACGAGAAGATTGGCTAAGGATTTCCGGCCTTTAAAGGTTGTTTGCATTGATTATCCAAGGCCAGAGCTAGACAATACAGTTAACTATTTGGAGGCTGCATTCTTATCATCATCATTCCGATCTTCTCCGCGCCCAACCAAACCACTGGAGATTGTTATTGCTGGTGCAGGTTTGGGTGGTTTGTCT
the growth of peppers before and after 15d inoculation by injection is shown in figure 1.
The results show that: after 15d inoculation by injection, the pepper plants showed photo-bleaching.
The relative expression level of PDS gene of capsicum was analyzed by RT-PCR using the capsicum reverse transcription of the extracted RNA of capsicum leaf, which had undergone gene silencing to cause albino, as shown in fig. 2.
The results show that: compared with the control wild material, the relative expression level of the PDS gene of the capsicum in the albino capsicum material is very low, which indicates that the silencing system can carry out gene silencing on the corresponding target gene in the capsicum.
Example 2 Effect of post-infection growth temperature on silencing efficacy in Capsici fructus VIGS System
Peppers belong to warm-loving vegetable crops, whereby the growth temperature obtained after inoculation infestation may have an impact on the silencing efficiency of pepper VIGS. This example sets a different post-infection growth temperature, and the other methods are the same as in example 1. The effect of growth temperature on silencing efficiency after different infestations is shown in figure 3.
The results show that: when the temperature of the capsicum is 20 ℃, the infection silencing efficiency is 11%; when the growth temperature after infection is 24 ℃, the infection silencing efficiency is 40%; when the growth temperature after infection is 28 ℃, the silencing efficiency of the capsicum gene is 13%. The result shows that the growth temperature after infection by inoculation has a great influence on the gene silencing efficiency of the capsicum VIGS, and the optimal growth temperature after infection and inoculation is 24 ℃.
Example 3 Effect of Agrobacterium seed concentration on Capsici fructus VIGS silencing efficiency
The agrobacterium-mediated virus infection and genetic transformation are not the same in tolerance degree for different crops, so that the agrobacterium infection concentration of the optimal VIGS of the peppers is explored, and the different inoculation concentrations of the agrobacterium for improving the VIGS silencing efficiency of the peppers and the tolerance degree of the crop for the agrobacterium have a significant effect on the VIGS silencing plant rate of the tomatoes. In this example, different concentrations of Agrobacterium infection were set, and the other methods were the same as in example 1. The effect of different Agrobacterium infection concentrations on silencing efficiency is shown in FIG. 4.
The results show that: when the agrobacterium tumefaciens bacteria solution OD600 = 0.01, the silencing proportion of the PDS gene is the highest and is 15%; when the agrobacterium tumefaciens bacteria solution od600=0.005, the silencing proportion of PDS gene is 13%; while the silencing ratio at the time of the agrobacteria liquid OD600 = 0.001 and 0.5 is only 5% and 3%. From this, it was shown that the agrobacterium infection concentration also has a great influence on the gene silencing efficiency of the pepper VIGS, and that the optimal agrobacterium infection concentration is od600=0.01.
Example 4 Effect of infection methods in Pepper VIGS Gene silencing on silencing efficiency
At present, the living inoculation of agrobacterium mainly comprises a vacuum infiltration method and an injection method, and simultaneously, the superposition of the two methods is used for improving the infection efficiency of the agrobacterium. In this example, the effect of vacuum infiltration and injection on silencing efficiency was studied, and the results are shown in FIG. 5 for the other methods as in example 1.
The results show that: for pepper seedlings after about 15d sowing, the efficiency of PDS gene silencing of the peppers by using a vacuum infiltration method and an injection method is 8% and 21%, respectively, and when the two methods are overlapped, the gene silencing efficiency of the peppers is reduced to 6%, probably because the damage to the peppers is overlarge under the condition of weaker vacuum infiltration during the early growth of the pepper seedlings, the method is not suitable for VIGS verification of the peppers.
In summary, in the types of capsicum towards the sky-pepper and the thin skin pepper, strict condition optimization is adopted to obtain the conclusion that the capsicum has obvious influence on the aspects of the growth temperature after the infection of agrobacterium, the infection concentration of agrobacterium, the inoculation mode of agrobacterium and the like, and research discovers that the optimal condition of a capsicum VIGS gene silencing system is that the growth temperature after the infection is 24 ℃, watering is carried out 1 day before injection, water is controlled 3 days after injection, the temperature is 18 ℃ after injection, the dark condition is carried out for 2 days, then the OD600 value of the inoculation concentration of agrobacterium is 0.01 under the conditions of 24 ℃/18 ℃ and 16h day/8h day, and the optimal inoculation method is an injection method.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The method for rapidly, efficiently and stably verifying the function of the capsicum gene is characterized by comprising the following steps of:
step one: construction of viral vector TRV:
designing a primer according to NCBI published or cloned capsicum gene sequences, connecting a cloned fragment with a viral vector, converting the fragment into escherichia coli, and sending the fragment to be detected after shaking PCR detection; detecting DNA sequences, shaking bacteria, extracting plasmid, and double enzyme cutting to obtain target gene segment; simultaneously converting the empty vector grains of pTRV2 into escherichia coli, performing PCR detection, shaking bacteria, extracting plasmids, performing double enzyme digestion, precipitating chloroform and ethanol, then running gel, and recovering gel to obtain digested pTRV2 vector fragments; connecting the target gene fragment with pTRV2 vector, transferring into escherichia coli, and performing PCR detection; shaking bacteria and extracting plasmid after sequencing without error; finally, transferring the extracted constructed viral vector plasmid into agrobacterium;
step two: and (3) preparation of an infection Buffer:
firstly, adding 95-100 ml of sterilized double distilled water into 95-100 ml of sterilized distilled water, then adding 0.5-1.5 ml of 1MMgcl 2 0.5 to 1.5ml of 1M MES and 180 to 220ul of 200mM acetosyringone, and finally adjusting the PH value to 5 to 6;
step three: preparation of an infectious microbe liquid:
performing a capsicum VIGS experiment by using an agrobacterium-mediated method, firstly transferring two plasmid vectors of TRV1 and TRV2+ target gene fragments into agrobacterium competent GV3101 respectively, and culturing for 1-3 d at 25-30 ℃; then selecting positive agrobacterium monoclonals in a liquid LB culture medium containing 45-55 mg/mL kanamycin and 8-12 mg/mL rifampicin; then centrifugally collecting thalli at 2500-3500 rpm, discarding supernatant, suspending bacteria by using an infection Buffer, and adjusting the concentration to OD < 600 > = 0.005-0.015; finally uniformly mixing TRV1+TRV2 and TRV1+ (TRV2+ target gene fragment) in equal volume, and standing for 2-4 hours at room temperature or shaking and culturing for 2-4 by a shaking table at 80-120 rpm at 25-30 ℃;
step four: growing and preparing capsicum:
about 20d after sowing the peppers, and inoculating after two true leaves grow out; and watering is needed before inoculation, and water is needed to be controlled after inoculation;
step five: control of the infestation conditions:
including the choice of the mode of inoculation, the use of the inoculation site, and the control of environmental conditions after injection.
2. The method according to claim 1, wherein the capsicum gene used in the first step is a capsicum PDS gene having a sequence shown in SEQ ID No. 1; the sequence of the obtained target gene fragment is shown as SEQ ID NO. 2.
3. The method according to claim 2, wherein the viral vector used in the first step is TRV, the cleavage sites are Xba I and Kpn I, the E.coli strain is Trans-5a, and the Agrobacterium strain is GV3101.
4. A method according to claim 3, wherein the specific process of the second step is: 98ml of sterilized double distilled water was added to 98ml of sterilized distilled water, followed by 1ml of 1M Mgcl 2 1ml of 1M MES and 200ul of 200mM acetosyringone, and finally the pH was adjusted to 5.6.
5. The method according to claim 4, wherein the specific process of the third step is: firstly, respectively transferring two plasmid vectors of TRV1 and TRV2+ target gene fragments into agrobacterium competent GV3101, and culturing for 2d at 28 ℃; then, positive agrobacterium is selected and monoclonal in a liquid LB culture medium containing 50mg/mL kanamycin and 10mg/mL rifampicin; then, the thalli are collected by centrifugation at 3000rpm, the supernatant is discarded, bacteria are suspended by using an infection Buffer, and the concentration is adjusted to be OD600 = 0.01; finally, mixing the mixture uniformly in equal volume, and placing the mixture for 3 hours at room temperature or shaking the mixture at a shaking table of 100rpm at 28 ℃ for 3 hours, wherein TRV1+ (TRV2+ target gene fragment).
6. The method according to claim 5, wherein the watering is performed 1d before inoculation and water is controlled 3d after inoculation in the step four.
7. The method of claim 6, wherein the inoculation mode selected in the fifth step is injection inoculation, and the inoculation position is the back of the true leaves.
8. The method according to claim 7, wherein the environmental conditions after injection in the fifth step are controlled by: after injection, the cells were grown for 2 days in the dark at 18℃and then cultured normally at 24℃at 18℃for 16h day/8h light.
9. The method of claim 8, further comprising the step of detecting the effect of gene silencing after infection is complete.
10. Use of the method according to any one of claims 1 to 9 for the functional detection of other pepper genes than the PDS gene of pepper.
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