CN114606261A - Method for establishing radix codonopsis gene transient silencing system based on VIGS technology - Google Patents
Method for establishing radix codonopsis gene transient silencing system based on VIGS technology Download PDFInfo
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Abstract
The invention belongs to the technical field of plant genetic engineering, and discloses a construction method of a radix codonopsis gene transient silencing system based on a VIGS technology, which comprises the following steps: (1) cloning the gene to obtain CpPDS gene; (2) constructing a pTRV2 virus silencing expression vector containing CpPDS gene specific fragments; (3) transforming agrobacterium by a recombinant vector freeze-thawing method; (4) infecting the root system of the codonopsis pilosula by an agrobacterium infection liquid root irrigation method; (5) culturing the infected codonopsis pilosula under certain conditions; (6) and observing the change of the codonopsis pilosula leaves by a photobleaching method. The method establishes a radix codonopsitis gene transient silencing system based on VIGS technology, and can rapidly identify gene functions.
Description
Technical Field
The invention relates to the field of plant genetic engineering, in particular to a method for establishing a radix codonopsitis gene transient silencing system based on a VIGS technology.
Background
Radix Codonopsis (Codonopsis pilosula (Franch.) Nannf.) is a perennial herb of Codonopsis of Campanulaceae, and radix Codonopsis produced in Shanxi Shandangshen area is used as the top grade product, and has effects of invigorating spleen and replenishing qi, invigorating spleen and benefiting lung. However, the mechanism of quality formation is not clear. At present, a plurality of genes are cloned from codonopsis pilosula, but because a transformation system is immature, the functional analysis of the genes is not carried out. The currently established genetic transformation systems of codonopsis pilosula comprise hairy root genetic transformation systems and callus genetic transformation systems, but have the defects of complicated transformation procedures, lack of plant morphology observation and the like.
The main mechanism of action of virus-induced gene silencing (VIGS) is: after infecting host plant with the virus vector containing target gene segment, the virus vector can be replicated in plant tissue in large quantity, and then ds RNA is synthesized under the action of RdRp. When ds RNA accumulates to a certain level, it is cleaved by a Dicer endonuclease to si RNA of about 23nt in size. The combination of specific RNA protein and si RNA in host body forms RISC, and this complex can specifically recognize single-stranded RNA in target gene segment, so that mRNA of target gene is degraded, and the gene is silenced at RNA level. Among them, gene silencing (TRV-VIGS) induced by Tobacco Rattle Virus (TRV) is the most widely used viral vector at present due to its advantages of light virus symptoms, long duration, high silencing efficiency, wide host range, and capability of infecting plant meristems. Gene silencing caused by VIGS technology can cause the phenotypical change of plants or the change of indexes such as physiology, biochemistry and the like, thereby carrying out gene function analysis. On the other hand, the biggest advantage of VIGS is that the gene function identification is completed in a short time, and the plant can be symptomatic after 4-6 weeks after infection without a lengthy plant regeneration process.
The phytoene dehydrogenase gene (PDS), is an enzyme required in the carotenoid synthesis pathway. Successful silencing of the PDS gene in tobacco resulted in significant photobleaching symptoms in the plants (Ruiz et al 1998). Because the phenotype after the gene is silenced is remarkably changed and is easy to observe, the PDS gene becomes a reporter gene for evaluating whether the VIGS system causes effective silencing in different crops. The radix codonopsitis VIGS system is constructed by taking radix codonopsitis phytoene dehydrogenase base CpPDS as a positive report gene.
Disclosure of Invention
The invention aims to provide a method for establishing a codonopsis pilosula gene transient silencing system based on a VIGS technology by using codonopsis pilosula phytoene dehydrogenase base CpPDS as a positive report gene. At present, some silencing systems are realized by gene knockout or interference RNA operation, and many plants are injected into plant leaves or other tissues when the silencing systems are introduced. Because the codonopsis pilosula has no silencing system, the invention creatively adopts a virus infection mode to introduce the silencing system and adopts a direct soil injection mode to carry out transformation in the research process, the transformation time is short, and the operation is simple.
The invention is realized by the following technical scheme: the application of the radix codonopsis CpPDS gene in constructing a transient silencing vector of the radix codonopsis gene;
the nucleotide sequence of the radix codonopsis CpPDS gene is shown in SEQ ID NO. 1.
The invention also provides a radix codonopsitis gene transient silencing vector, which comprises a pTRV2-CpPDS vector, wherein the nucleotide sequence of the pTRV2-CpPDS vector is shown as SEQ ID NO. 2.
The invention further provides a method for establishing a radix codonopsitis gene transient silencing system based on VIGS technology, which introduces a transient silencing vector containing radix codonopsitis CpPDS gene into root cells of radix codonopsitis plants by an agrobacterium infection root irrigation method, and silences the gene of the radix codonopsitis CpPDS gene in a target plant through culture.
The invention also provides a method for establishing the radix codonopsitis gene transient silencing system of the VIGS technology, which comprises the following steps:
(a) inserting a target fragment containing the Codonopsis pilosula CpPDS gene into a virus vector to obtain a recombinant virus vector; (b) preparing an agrobacterium infection solution, wherein the agrobacterium infection solution comprises: a recombinant viral vector;
(c) introducing the agrobacterium infection solution into root cells of codonopsis pilosula plants by adopting a root irrigation method so as to generate infected plant tissues;
(d) culturing, and inducing the gene silencing of the Codonopsis pilosula CpPDS gene in infected Codonopsis pilosula plants.
As a further improvement of the technical scheme of the establishing method, the viral vector is pTRV2 vector.
As a further improvement of the technical scheme of the establishing method, the agrobacterium infection solution also comprises a pTRV1 strain.
As a further improvement of the technical scheme of the establishing method, when the agrobacterium infection solution is prepared, the recombinant virus vector is transformed into agrobacterium by a freeze-thaw method.
The codonopsis pilosula VIGS silencing system is constructed for the first time, and the virus-induced gene silencing system constructed by the technical scheme of the invention has the advantages of short transformation time, quick symptom occurrence of plants, capability of quickly identifying gene functions, easiness in operation and implementation and the like, and provides a basis for large-scale development of codonopsis pilosula gene function research.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the PCR amplification results for CpPDS target fragments; in the figure: lane 1 (M in the figure) represents DL500marker, and lanes 2-3 (1, 2 in the figure) represent CpPDS.
FIG. 2 shows the results of double-restriction electrophoresis of recombinant vector pTRV 2-CpPDS; in the figure: lane 1 (M in the figure) represents DL5000marker, and lanes 2-4 (1, 2, 3 in the figure) represent the recombinant vector pTRV 2-CpPDS.
FIG. 3 shows the result of PCR identification of the plasmid of recombinant vector pTRV 2-CpPDS; in the figure: lane 1 (M in the figure) represents DL500marker, and lanes 2-3 (1, 2 in the figure) represent pTRV 2-CpPDS.
FIG. 4 shows the photo-bleaching results of the leaves of the young seedlings of Codonopsis pilosula.
Detailed Description
The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a specific embodiment of a method for establishing a radix codonopsitis gene transient silencing system based on VIGS technology, which comprises the following steps:
construction of silencing vector
1) Cloning of CpPDS gene-specific fragments: the nucleotide sequence of the gene is shown as a sequence shown in SEQ ID NO.1 (the gene sequence is selected from a subject group codonopsis pilosula transcriptome database, and CpPDS gene full length is obtained through RACE). Taking 200-and 300-bp fragments in CpPDS gene ORF as target sequences, carrying out enzyme cutting site analysis on the target sequences, designing specific primers with enzyme cutting sites on two sides after removing the enzyme cutting sites in the gene sequences, and adding protective bases, wherein the primer sequences are shown as follows.
CpPDS(BamHⅠ)F：CGCGGATCCACGAAGCGACTCAGAAA
CpPDS(SacⅠ)R：CGAGCTCAGATAGAACGGCACCCT
Amplifying by using radix codonopsis cDNA as a template to obtain CpPDS gene fragments: carrying out PCR amplification by using a recombinant plasmid pMD-19T-CpPDS with correct sequencing comparison as a template, wherein the amplification procedure is as follows: 95 ℃ for 5 min; 94 deg.C, 1min, 56 deg.C, 1min, 72 deg.C, 2min, 32 cycles; extension at 72 ℃ for 10 min. The PCR product was detected by 1.2% agarose gel electrophoresis.
The results are shown in FIG. 1. In the figure: lane 1 (M in the figure) represents DL500marker, and lanes 2-3 (1, 2 in the figure) represent CpPDS. The results show that: the amplified band is single and bright, and the CpPDS gene is successfully amplified.
Wherein the sequence shown in SEQ ID NO.1 is:
ATGTCTGTATTTGGACATTTATCGGCTGTTAACTTTAGCGCGCAAAGCACTGCACTAAACCTTTCGAATGCAAATTCTACGTGGAGAAGCCGTTTCCATATCAGCTCAGTACAACCAAATGCTCTATCATTCAAAGGTACTCCGAGATTGCAATTTCCAGCTGCCCATCCTGTCCTACCAAGATCCAGGGGAAATGCAAGCCCATTAAAGGTGGTTTGCGTGGATTATCCAAGACCAGAGCTTGATAATACAGTTAATTATTTGGAAGCTGCGTACTTATCTTCTACATTTCGTACTTCCCCTCGTCCAGCTAAACCACTGAAGGTTGTAATTGCTGGTGCAGGCTTGGCTGGTTTATCTACTGCCAAATATTTGGCAGATGCAGGTCACAAGCCCATATTGTTGGAAGCAAGAGATGTTCTTGGTGGAAAGGTGGCAGCTTGGAAAGATGATGATGGAGACTGGTATGAGACTGGCTTACATATTTTCTTTGGGGCGTACCCCAATGTGCAAAACCTGTTTGGAGAACTAGGCATTAATGATAGATTGCAGTGGAAGGAGCATTCCATGATATTTGCAATGCCAAATAAGCCTGGAGAGTTCAGCCGGTTTGACTTCCCGGACGTTCTACCTGCTCCGTTGAATGGGATTTGGGCTATCTTGAAGAATAATGAAATGCTTACGTGGCCAGAGAAAGTCAAGTTTGCAATTGGATTATTGCCAGCAATGCTTGGTGGCCAGGCATATGTTGAGGCTCAAGATGGTTTGAGTGTACAAGATTGGATGAGAAAGCAAGGAATACCTGACCGAGTTACCACCGAGGTGTTTATTGCCATGTCAAAGGCATTAAATTTCATAAACCCGGATGAACTTTCAATGCAATGTATTTTGATTGCTTTGAATCGATTTCTTCAGGAGAAGCATGGTTCAAAAATGGCTTTCTTAGATGGTAATCCTCCCGAGAGGCTTTGCCAGCCAATTGTGGATCATATCGAGTCACTAGGTGGTGAAGTCAGGCTTAATTCTCGAATCCAAAAGATTGACTTGAATAAAGATGGAACTGTGAGAAACTTCTTATTGACTAATGGGGATATAATTGAAGGTGATGCTTACGTATCCGCTGCGCCAGTTGATATCCTGAAGCTTCTTTTGCCTGAAGAATGGAAAGAGATTCCATACTTCAGGAAATTGGACAAACTAGTTGGAGTTCCAGTTATAAATGTTCACATATGGTTTGACAGGAAATTGAAGAACACATACGATCATCTACTCTTCAGCAGAAGCCCTCTTCTCAGTGTGTATGCTGACATGTCTGTAACATGTAAGGAATATTACAATCCTAACCAGTCTATGTTGGAGTTGGTATTTGCACCTGCTGAAGAATGGATTTCACGAAGCGACTCAGAAATTATTGATGCTACAATGCATGAACTTGCAAAACTCTTTCCTGATGAAATTTCAGCTGATCAGAGTAAAGCGAAGATATTGAAGTACCATGTTGTTAAAACACCAAGGTCTGTTTATAAAACCGTACCAGACTGTGAACCTTGCCGTCCCTTGCAAAGATCTCCAATAGAGGGATTTTATCTAGCTGGCGACTACACCAAACAAAAGTATTTGGCTTCAATGGAGGGTGCCGTTCTATCTGGAAAATTTTGTGCACAAGCAATTGTAAAGGATAGTGAGTTGCTAGTTACCCGGGGTCAGAAAATGGTGGCTGAGGCGAGTCTTGTTTAA(SEQ ID NO.1)
2) and cutting the target fragment into gel, recovering the gel, and performing double enzyme digestion on the CpPDS and the pTRV2 vectors respectively, wherein the reaction system is as follows, the reaction temperature is 37 ℃, and the reaction time is 3 h.
TABLE 1 double digestion reaction System
3) After the double enzyme digestion is finished, 10 Xloading buffer is added to terminate the reaction, and the target fragment and the pTRV2 vector are respectively subjected to gel cutting and recovery.
4) The target fragment was ligated with pTRV2 vector in the following reaction system and ligated overnight at 16 ℃.
TABLE 2 ligation reaction System
5) The ligation product was introduced into E.coli DH 5. alpha. competent cells, spread on LB solid medium containing 50mg/L Kana, and cultured in an inverted state at 37 ℃ for 12-16 h.
6) And (3) picking positive clone, shaking bacteria, extracting plasmids, carrying out double enzyme digestion identification, sending the positive clone with correct identification to Beijing Hua DageneCo Ltd for sequencing and splicing to obtain pTRV2-CpPDS recombinant vector, wherein the result is shown in SEQ ID NO. 2.
The results are shown in FIG. 2. In the figure, lane 1 (M) represents DL10000 marker, and lanes 2-4 (1, 2, 3 in the figure) represent recombinant vector pTRV 2-CpPDS. The results show that: two bright bands, one is about 10kb and the other is about 250bp, which indicates that the target fragment is successfully connected with the vector.
The sequence shown in SEQ ID NO.2 is:
GATTTCACGAAGCGACTCAGAAATTATTGATGCTACAATGCATGAACTTGCAAAACTCTTTCCTGATGAAATTTCAGCTGATCAGAGTAAAGCGAAGATATTGAAGTACCATGTTGTTAAAACACCAAGGTCTGTTTATAAAACCGTACCAGACTGTGAACCTTGCCGTCCCTTGCAAAGATCTCCAATAGAGGGATTTTATCTAGCTGGCGACTACACCAAACAAAAGTATTTGGCTTCAATGGAGGGTGCCGTTCTATCTGGA(SEQ ID NO.2)
second, freeze thawing method for transforming agrobacterium GV3101
1) Thawing 100ul Agrobacterium GV3101 competent cells in ice bath;
2) adding 1ug of expression vector plasmid, mixing, and ice-cooling for 30 min;
3) quickly putting the centrifugal tube into liquid nitrogen for quick freezing for 90 s;
4) quickly taking out, and placing into water bath of 37 deg.C for heat shock for 5 min;
5) quickly taking out and placing on ice, and carrying out ice bath for 2 min;
6) adding 1ml LB culture medium heated to 28 deg.C in advance into each tube, culturing at 28 deg.C and 200rpm/min under shaking for 4-6 hr to recover plasmid;
7) the cell pellet was collected by centrifugation at 5000rpm/min for 5min at 4 ℃ and resuspended in 50uL LB, spread evenly on solid LB medium supplemented with 30ug/mL Kan, 50ug/mL Rif and 50ug/mL Gen, and cultured upside down at 28 ℃ for 36-48 h.
8) And carrying out colony PCR identification on the recombinant plasmid.
The results are shown in FIG. 3. In the figure: lane 1 (M in the figure) represents DL500marker, and lanes 2-3 (1, 2 in the figure) represent pTRV 2-CpPDS. The results show that: the recombinant vector was successfully introduced into Agrobacterium.
Third, preparation of agrobacteria infection liquid
1) The GV3101 strain containing the viral vectors pTRV1, pTRV2, pTRV2-CpPDS was monoclonally propagated in 5mL LB liquid medium containing 30ug/mLKan, 50ug/mL Rif and 50ug/mL Gen3 antibiotics at 28 ℃ to OD600The value reaches 0.8 to 1.0;
2) mixing the bacterial liquid according to the proportion of 1: diluted at 25, and cultured to OD 28 ℃ in induction medium containing 30ug/mL Kan, 50ug/mL Rif, 50ug/mLGen 10mM MES and 200. mu.M AS600The value reaches 0.5 to 0.8;
3) centrifuging at 5000rpm/min for 10min to collect thallus, and discarding supernatant;
4) the mycelia were infected with equal volume of VIGS buffer (10mM MgCl)210mM MES, 200. mu. MAS) and adjusting the OD600Standing at 28 deg.C for 3 hr or more until the temperature reaches 1.0-2.0;
5) pTRV1 strain was mixed with pTRV2 empty vector strain and pTRV2-CpPDS vector at a ratio of 1:1(V: V), respectively.
Fourthly, infecting codonopsis pilosula seedlings by agrobacterium
The root irrigation method is adopted for infecting codonopsis pilosula seedlings by agrobacterium. The pTRV1+ pTRV2 staining solution is used as a blank control, and the pTRV1+ pTRV2-CpPDS staining solution is used as an experimental group. Accurately sucking 2.5mL of infection liquid by using a 5mL syringe, inserting a needle into the soil of potted codonopsis pilosula, pouring the infection liquid near the roots of codonopsis pilosula, placing the codonopsis pilosula plants injected with infection into an artificial climate box for dark culture at 20 ℃ for 2 days, culturing at 25 ℃, 16h under illumination, 20 ℃ and 8h in dark, and watering regularly during the period.
Fifthly, observing the morphological change of plants by photobleaching method
The leaves of the transformed plants cultured for 4-6 weeks were observed by photobleaching, and the appearance of albino leaves as silent lines, as shown in FIG. 4, indicates that: the construction of the silencing system is successful.
The construction method of the codonopsis pilosula gene silencing system provided by the invention is simple and easy to operate, and the plants have quick symptom.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
<110> university of Shanxi medical science
<120> establishing method of radix codonopsis gene transient silencing system based on VIGS technology
<160>2
<210>1
<211>1737
<212>DNA
<213> Codonopsis pilosula (Franch.) Nannf.)
<220>
<223>CpPDS
<400>1
ATGTCTGTAT TTGGACATTT ATCGGCTGTT AACTTTAGCG CGCAAAGCAC 50
TGCACTAAAC CTTTCGAATG CAAATTCTAC GTGGAGAAGC CGTTTCCATA 100
TCAGCTCAGT ACAACCAAAT GCTCTATCAT TCAAAGGTAC TCCGAGATTG 150
CAATTTCCAG CTGCCCATCC TGTCCTACCA AGATCCAGGG GAAATGCAAG 200
CCCATTAAAG GTGGTTTGCG TGGATTATCC AAGACCAGAG CTTGATAATA 250
CAGTTAATTA TTTGGAAGCT GCGTACTTAT CTTCTACATT TCGTACTTCC 300
CCTCGTCCAG CTAAACCACT GAAGGTTGTA ATTGCTGGTG CAGGCTTGGC 350
TGGTTTATCT ACTGCCAAAT ATTTGGCAGA TGCAGGTCAC AAGCCCATAT 400
TGTTGGAAGC AAGAGATGTT CTTGGTGGAA AGGTGGCAGC TTGGAAAGAT 450
GATGATGGAG ACTGGTATGA GACTGGCTTA CATATTTTCT TTGGGGCGTA 500
CCCCAATGTG CAAAACCTGT TTGGAGAACT AGGCATTAAT GATAGATTGC 550
AGTGGAAGGA GCATTCCATG ATATTTGCAA TGCCAAATAA GCCTGGAGAG 600
TTCAGCCGGT TTGACTTCCC GGACGTTCTA CCTGCTCCGT TGAATGGGAT 650
TTGGGCTATC TTGAAGAATA ATGAAATGCT TACGTGGCCA GAGAAAGTCA 700
AGTTTGCAAT TGGATTATTG CCAGCAATGC TTGGTGGCCA GGCATATGTT 750
GAGGCTCAAG ATGGTTTGAG TGTACAAGAT TGGATGAGAA AGCAAGGAAT 800
ACCTGACCGA GTTACCACCG AGGTGTTTAT TGCCATGTCA AAGGCATTAA 850
ATTTCATAAA CCCGGATGAA CTTTCAATGC AATGTATTTT GATTGCTTTG 900
AATCGATTTC TTCAGGAGAA GCATGGTTCA AAAATGGCTT TCTTAGATGG 950
TAATCCTCCC GAGAGGCTTT GCCAGCCAAT TGTGGATCAT ATCGAGTCAC 1000
TAGGTGGTGA AGTCAGGCTT AATTCTCGAA TCCAAAAGAT TGACTTGAAT 1050
AAAGATGGAA CTGTGAGAAA CTTCTTATTG ACTAATGGGG ATATAATTGA 1100
AGGTGATGCT TACGTATCCG CTGCGCCAGT TGATATCCTG AAGCTTCTTT 1150
TGCCTGAAGA ATGGAAAGAG ATTCCATACT TCAGGAAATT GGACAAACTA 1200
GTTGGAGTTC CAGTTATAAA TGTTCACATA TGGTTTGACA GGAAATTGAA 1250
GAACACATAC GATCATCTAC TCTTCAGCAG AAGCCCTCTT CTCAGTGTGT 1300
ATGCTGACAT GTCTGTAACA TGTAAGGAAT ATTACAATCC TAACCAGTCT 1350
ATGTTGGAGT TGGTATTTGC ACCTGCTGAA GAATGGATTT CACGAAGCGA 1400
CTCAGAAATT ATTGATGCTA CAATGCATGA ACTTGCAAAA CTCTTTCCTG 1450
ATGAAATTTC AGCTGATCAG AGTAAAGCGA AGATATTGAA GTACCATGTT 1500
GTTAAAACAC CAAGGTCTGT TTATAAAACC GTACCAGACT GTGAACCTTG 1550
CCGTCCCTTG CAAAGATCTC CAATAGAGGG ATTTTATCTA GCTGGCGACT 1600
ACACCAAACA AAAGTATTTG GCTTCAATGG AGGGTGCCGT TCTATCTGGA 1650
AAATTTTGTG CACAAGCAAT TGTAAAGGAT AGTGAGTTGC TAGTTACCCG 1700
GGGTCAGAAA ATGGTGGCTG AGGCGAGTCT TGTTTAA 1737
<210>2
<211>265
<212>DNA
<213> Artificial sequence
<220>
<223>pTRV2-CpPDS
<400>2
GATTTCACGA AGCGACTCAG AAATTATTGA TGCTACAATG CATGAACTTG 50
CAAAACTCTT TCCTGATGAA ATTTCAGCTG ATCAGAGTAA AGCGAAGATA 100
TTGAAGTACC ATGTTGTTAA AACACCAAGG TCTGTTTATA AAACCGTACC 150
AGACTGTGAA CCTTGCCGTC CCTTGCAAAG ATCTCCAATA GAGGGATTTT 200
ATCTAGCTGG CGACTACACC AAACAAAAGT ATTTGGCTTC AATGGAGGGT 250
GCCGTTCTAT CTGGA 265
Claims (7)
1. Codonopsis pilosulaCpPDSThe application of the gene in constructing a transient silencing vector of the codonopsis pilosula gene;
the radix CodonopsisCpPDSThe nucleotide sequence of the gene is shown in SEQ ID NO. 1.
2. A transient silencing vector for Codonopsis pilosula gene is characterized by comprising pTRV2-CpPDSVector, the pTRV2-CpPDSThe nucleotide sequence of the vector is shown as SEQ ID NO. 2.
3. A method for establishing radix Codonopsis gene transient silencing system based on VIGS technology is characterized in that radix Codonopsis is added by Agrobacterium tumefaciens infection root irrigation methodCpPDSThe transient silencing carrier of gene is introduced into root cell of dangshen plant and cultured to make dangshen inside the target plantCpPDSGene silencing of the gene.
4. A method for establishing a radix codonopsis gene transient silencing system of VIGS technology is characterized by comprising the following steps:
(a) will comprise radix CodonopsisCpPDSInserting the target segment of the gene into a virus vector to obtain a recombinant virus vector;
(b) preparing an agrobacterium infection solution, wherein the agrobacterium infection solution comprises: a recombinant viral vector;
(c) introducing the agrobacterium infection solution into root cells of codonopsis pilosula plants by adopting a root irrigation method to generate infected plant tissues;
(d) culturing, inducing radix Codonopsis in infected radix Codonopsis plantCpPDSGene silencing of the gene.
5. The method for establishing the transient silencing system of Codonopsis pilosula gene of VIGS technology as claimed in claim 4, wherein the viral vector is pTRV2 vector.
6. The method for establishing the transient silencing system of Codonopsis pilosula gene of VIGS technology as claimed in claim 4, wherein said Agrobacterium infection fluid further comprises pTRV1 strain.
7. The method for establishing the radix codonopsis gene transient silencing system according to the VIGS technology of claim 4, wherein the recombinant virus vector is used for transforming agrobacterium by a freeze-thaw method during preparation of an agrobacterium infection solution.
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