CN114606261B - Method for establishing dangshen gene transient silencing system based on VIGS technology - Google Patents
Method for establishing dangshen 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 dangshen gene transient silencing system based on a VIGS technology, which comprises the following steps: (1) cloning genes to obtain CpPDS genes; (2) Constructing pTRV2 virus silencing expression vector containing CpPDS gene specific fragment; (3) the recombinant vector is transformed into agrobacterium by a freeze thawing method; (4) infecting root systems of radix codonopsis by an agrobacterium infection solution root irrigation method; (5) culturing the infected codonopsis pilosula under a certain condition; (6) observing the leaf changes of the codonopsis pilosula by a photobleaching method. The method establishes a dangshen gene transient silencing system based on the VIGS technology, and can rapidly identify the gene function.
Description
Technical Field
The invention relates to the field of plant genetic engineering, in particular to a method for establishing a dangshen gene transient silencing system based on a VIGS technology.
Background
Radix Codonopsis (Codonopsis pilosula (Franch.) Nannf.) is perennial herb of Codonopsis genus of Campanulaceae family, and has effects of invigorating spleen and lung, and invigorating spleen and replenishing qi. However, the quality formation mechanism is not clear. A plurality of genes have been cloned from Codonopsis pilosula, but many have not been subjected to gene function analysis due to the immaturity of the transformation system. The genetic transformation system of the dangshen established at present comprises a hairy root genetic transformation system and a callus genetic transformation system, but all have the defects of complicated transformation procedures, lack of plant morphology observation and the like.
The main mechanisms of action of virus-induced gene silencing (VIGS) are: after infection of a host plant with a viral vector containing the gene fragment of interest, a large number of copies are made in the tissues of the plant and ds RNA is then synthesized under the action of RdRp. When ds RNA accumulates to a certain level, it is cleaved by a Dicer endonuclease into siRNA of about 23 nt. The specific RNA protein in the host and the siRNA combine to form RISC, and the complex can specifically recognize single-stranded RNA in the target gene fragment, so that the mRNA of the target gene is degraded, and the gene is silenced at the RNA level. Among them, gene silencing (TRV-VIGS) induced by tobacco brittle virus (tobacco rattle virus, TRV) is the most currently used viral vector because of its advantages of light virus symptoms, long duration, high silencing efficiency, wide host range, and capability of infecting plant meristems. Gene silencing caused by the VIGS technology can cause plant re-phenotype or physiological, biochemical and other indexes to change, so that gene function analysis is performed. On the other hand, the biggest advantage of the VIGS is that the gene function identification is completed in a short time, and symptoms can appear in plants 4-6 weeks after infection without 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 plants show significant photobleaching symptoms (Ruiz et al 1998). Because the phenotype after the gene silencing changes obviously and is easy to observe, the PDS gene becomes a report gene for evaluating whether the VIGS system causes effective silencing in different crops. The invention constructs a codonopsis pilosula VIGS system by taking codonopsis pilosula phytoene dehydrogenase group CpPDS as a positive reporter gene.
Disclosure of Invention
The invention aims to provide a method for establishing a dangshen gene transient silencing system based on a VIGS technology by using dangshen phytoene dehydrogenase group CpPDS as a positive reporter gene. At present, many silencing systems are realized by gene knockout or RNA interference operation, and when many plants are introduced into the silencing systems, the silencing systems are injected into leaves or other tissues of the plants. Because the dangshen has no silencing system, the invention creatively adopts a virus infection mode to introduce the silencing system in the research process, adopts a direct soil injection mode to carry out transformation, has short transformation time and is simple to operate.
The invention is realized by the following technical scheme: application of target fragment of codonopsis pilosula CpPDS gene in construction of transient silencing vector of codonopsis pilosula gene;
the nucleotide sequence of the target fragment of the codonopsis pilosula CpPDS gene is shown as SEQ ID NO.2.
The invention also provides a dangshen gene transient silencing vector, which comprises a pTRV2-CpPDS vector, wherein the nucleotide sequence of a target fragment in the pTRV2-CpPDS vector is shown as SEQ ID NO.2.
The invention further provides a method for establishing a dangshen gene transient silencing system based on a VIGS technology, wherein an agrobacterium infection root irrigation method is used for introducing a transient silencing vector containing a dangshen CpPDS gene into root cells of dangshen plants, and the dangshen CpPDS gene in target plants is silenced by culturing.
The invention further provides a method for establishing a dangshen gene transient silencing system by the VIGS technology, which comprises the following steps:
(a) Inserting a target fragment containing the Codonopsis pilosula CpPDS gene into a viral vector to obtain a recombinant viral vector; (b) Preparing an agrobacterium infection solution, wherein the agrobacterium infection solution comprises the following components: a recombinant viral vector;
(c) Introducing agrobacterium infection liquid into root cells of codonopsis pilosula plants by adopting a root irrigation method to generate infected plant tissues;
(d) Culturing, inducing gene silencing of the CpPDS gene of Codonopsis pilosula in the infected Codonopsis pilosula plant.
As a further improvement of the technical scheme of the establishment method, the viral vector is pTRV2 vector.
As a further improvement of the technical scheme of the establishment method, the agrobacterium tumefaciens invasion solution also comprises pTRV1 strain.
As a further improvement of the technical scheme of the establishment method, when preparing the agrobacterium infection solution, the recombinant viral vector converts agrobacterium by a freeze thawing method.
The invention constructs the Codonopsis pilosula VIGS silencing system 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, rapid appearance of symptoms of plants, rapid identification of gene functions, easy operation and realization, and the like, and provides basis for large-scale research on the gene functions of the Codonopsis pilosula.
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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 that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of PCR amplification of CpPDS target fragment; in the figure: lanes 1 (M in the figure) represent DL500 marker and lanes 2-3 (1, 2 in the figure) represent CpPDS.
FIG. 2 shows the results of double digestion electrophoresis of recombinant vector pTRV 2-CpPDS; in the figure: lanes 1 (M in the figure) represent DL5000 markers and lanes 2-4 (1, 2, 3 in the figure) represent the recombinant vector pTRV2-CpPDS.
FIG. 3 shows the PCR identification result of the recombinant vector pTRV2-CpPDS plasmid; in the figure: lanes 1 (M in the figure) represent DL500 marker and lanes 2-3 (1, 2 in the figure) represent pTRV2-CpPDS.
FIG. 4 shows photobleaching results of leaves of silent Codonopsis pilosula seedlings.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a concrete embodiment of a method for establishing a dangshen gene transient silencing system based on a VIGS technology, which comprises the following steps:
1. construction of silencing vector
1) Cloning of CpPDS Gene-specific fragments: the nucleotide sequence of the gene is shown as SEQ ID NO.1 (the gene sequence is selected from the topic group codonopsis transcriptome database, and the full length of the CpPDS gene is obtained by RACE). A200-300 bp fragment in the ORF of the CpPDS gene is taken as a target sequence, enzyme digestion site analysis is carried out on the target sequence, specific primers with enzyme digestion sites are designed on two sides after enzyme digestion sites in the gene sequence are eliminated, and protective bases are added, wherein the primer sequences are shown as follows.
CpPDS(BamHⅠ)F：CGCGGATCCACGAAGCGACTCAGAAA
CpPDS(SacⅠ)R：CGAGCTCAGATAGAACGGCACCCT
Amplifying the codonopsis pilosula root cDNA as a template to obtain a CpPDS gene fragment: the PCR amplification is carried out by taking the recombinant plasmid pMD-19T-CpPDS with correct sequencing comparison as a template, and the amplification procedure is as follows: 95 ℃ for 5min;94 ℃, 1min,56 ℃, 1min,72 ℃, 2min,32 cycles; extending at 72℃for 10min. The PCR products were detected by 1.2% agarose gel electrophoresis.
The results are shown in FIG. 1. In the figure: lanes 1 (M in the figure) represent DL500 marker and lanes 2-3 (1, 2 in the figure) represent CpPDS. The results show that: the amplified band was single and bright, and was successfully amplified to the CpPDS gene.
Wherein the sequence shown in SEQ ID NO.1 is:
ATGTCTGTATTTGGACATTTATCGGCTGTTAACTTTAGCGCGCAAAGCACTGCACTAAACCTTTCGAATGCAAATTCTACGTGGAGAAGCCGTTTCCATATCAGCTCAGTACAACCAAATGCTCTATCATTCAAAGGTACTCCGAGATTGCAATTTCCAGCTGCCCATCCTGTCCTACCAAGATCCAGGGGAAATGCAAGCCCATTAAAGGTGGTTTGCGTGGATTATCCAAGACCAGAGCTTGATAATACAGTTAATTATTTGGAAGCTGCGTACTTATCTTCTACATTTCGTACTTCCCCTCGTCCAGCTAAACCACTGAAGGTTGTAATTGCTGGTGCAGGCTTGGCTGGTTTATCTACTGCCAAATATTTGGCAGATGCAGGTCACAAGCCCATATTGTTGGAAGCAAGAGATGTTCTTGGTGGAAAGGTGGCAGCTTGGAAAGATGATGATGGAGACTGGTATGAGACTGGCTTACATATTTTCTTTGGGGCGTACCCCAATGTGCAAAACCTGTTTGGAGAACTAGGCATTAATGATAGATTGCAGTGGAAGGAGCATTCCATGATATTTGCAATGCCAAATAAGCCTGGAGAGTTCAGCCGGTTTGACTTCCCGGACGTTCTACCTGCTCCGTTGAATGGGATTTGGGCTATCTTGAAGAATAATGAAATGCTTACGTGGCCAGAGAAAGTCAAGTTTGCAATTGGATTATTGCCAGCAATGCTTGGTGGCCAGGCATATGTTGAGGCTCAAGATGGTTTGAGTGTACAAGATTGGATGAGAAAGCAAGGAATACCTGACCGAGTTACCACCGAGGTGTTTATTGCCATGTCAAAGGCATTAAATTTCATAAACCCGGATGAACTTTCAATGCAATGTATTTTGATTGCTTTGAATCGATTTCTTCAGGAGAAGCATGGTTCAAAAATGGCTTTCTTAGATGGTAATCCTCCCGAGAGGCTTTGCCAGCCAATTGTGGATCATATCGAGTCACTAGGTGGTGAAGTCAGGCTTAATTCTCGAATCCAAAAGATTGACTTGAATAAAGATGGAACTGTGAGAAACTTCTTATTGACTAATGGGGATATAATTGAAGGTGATGCTTACGTATCCGCTGCGCCAGTTGATATCCTGAAGCTTCTTTTGCCTGAAGAATGGAAAGAGATTCCATACTTCAGGAAATTGGACAAACTAGTTGGAGTTCCAGTTATAAATGTTCACATATGGTTTGACAGGAAATTGAAGAACACATACGATCATCTACTCTTCAGCAGAAGCCCTCTTCTCAGTGTGTATGCTGACATGTCTGTAACATGTAAGGAATATTACAATCCTAACCAGTCTATGTTGGAGTTGGTATTTGCACCTGCTGAAGAATGGATTTCACGAAGCGACTCAGAAATTATTGATGCTACAATGCATGAACTTGCAAAAC TCTTTCCTGATGAAATTTCAGCTGATCAGAGTAAAGCGAAGATATTGAAGTACCATGTTGTTAAAACACCAAGGTCTGTTTATAAAACCGTACCAGACTGTGAACCTTGCCGTCCCTTGCAAAGATCTCCAATAGAGGGATTTTATCTAGCTGGCGACTACACCAAACAAAAGTATTTGGCTTCAATGGAGGGTGCCGTTCTATCTGGAAAATTTTGTGCACAAGCAATTGTAAAGGATAGTGAGTTGCTAGTTACCCGGGGTCAGAAAATGGTGGCTGAGGCGAGTCTTGTTTAA(SEQ ID NO.1)
2) And (3) performing gel cutting recovery on the target fragment, and performing double enzyme cutting on the CpPDS and pTRV2 vector respectively, wherein the reaction system is as follows, and the reaction temperature is 37 ℃ and the reaction time is 3 hours.
Table 1 double cleavage reaction System
3) After the double enzyme digestion is completed, 10×loading buffer is added to terminate the reaction, and the target fragment and pTRV2 vector are respectively subjected to gel digestion recovery.
4) The target fragment was ligated with pTRV2 vector according to the following reaction system at 16℃overnight.
TABLE 2 ligation reaction System
5) The above-mentioned ligation product was introduced into E.coli DH 5. Alpha. Competent cells, spread on LB solid medium containing 50mg/L Kana, and cultured upside down at 37℃for 12-16h.
6) And (3) picking positive clones, shaking, extracting plasmids, carrying out double enzyme digestion identification, and sending the positive clones with correct identification to Beijing Hua big gene limited company for sequencing and splicing to obtain the pTRV2-CpPDS recombinant vector, wherein the nucleotide sequence of the target fragment is shown as SEQ ID NO.2.
The results are shown in FIG. 2. Lanes 1 (M in the figure) represent DL10000 marker, and lanes 2-4 (1, 2, 3 in the figure) represent recombinant vector pTRV2-CpPDS. The results show that: two bright bands, one about 10kb and one about 250bp, indicate successful ligation of the target fragment to the vector.
The sequence shown in SEQ ID NO.2 is:
GATTTCACGAAGCGACTCAGAAATTATTGATGCTACAATGCATGAACTTGCAAAACTCTTTCCTGATGAAATTTCAGCTGATCAGAGTAAAGCGAAGATATTGAAGTACCATGTTGTTAAAACACCAAGGTCTGTTTATAAAACCGTACCAGACTGTGAACCTTGCCGTCCCTTGCAAAGATCTCCAATAGAGGGATTTTATCTAGCTGGCGACTACACCAAACAAAAGTATTTGGCTTCAATGGAGGGTGCCGTTCTATCTGGA(SEQ ID NO.2)
2. freeze thawing process of transforming agrobacterium GV3101
1) Melting 100ul of Agrobacterium GV3101 competent cells in ice bath;
2) Adding 1ug expression vector plasmid, mixing thoroughly, and ice-bathing for 30min;
3) Quickly freezing the centrifuge tube in liquid nitrogen for 90s;
4) Rapidly taking out, and placing into a 37 ℃ water bath for heat shock for 5min;
5) Rapidly taking out and placing on ice, and ice-bathing for 2min;
6) 1ml of LB culture medium which is heated to 28 ℃ in advance is added into each tube, and shaking culture is carried out for 4-6 hours at the temperature of 28 ℃ and the rpm/min of 200, so that plasmids are recovered;
7) Centrifugation was performed at 5000rpm/min for 5min at 4℃to collect cell pellet, and the cells were 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 36-48h.
8) Colony PCR identification was performed on the recombinant plasmid.
The results are shown in FIG. 3. In the figure: lanes 1 (M in the figure) represent DL500 marker and lanes 2-3 (1, 2 in the figure) represent pTRV2-CpPDS. The results show that: the recombinant vector was successfully introduced into Agrobacterium.
3. Preparation of agrobacteria invasion solution
1) The GV3101 strain containing the viral vectors pTRV1, pTRV2-CpPDS was propagated at 28℃to OD in 5mL of LB liquid medium containing 30ug/mLKan,50ug/mL Rif and 50ug/mL Gen3 antibiotics 600 The value reaches 0.8 to 1.0;
2) The bacterial liquid is prepared according to the following ratio of 1:25 ratio dilution, incubation at 28℃to OD in Induction Medium containing 30ug/mL Kan,50ug/mL Rif, 50ug/mL Gen 10mM MES and 200. Mu.M AS 600 The value reaches 0.5 to 0.8;
3) Centrifuging at 5000rpm/min for 10min to collect thallus, and discarding supernatant;
4) The cells were infected with an equal volume of VIGS-invasion buffer (10 mM MgCl 2 10mM MES, 200. Mu.M AS) and adjust the OD 600 Standing at 28deg.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 in a ratio of 1:1 (V: V), respectively.
4. Agrobacterium infects Codonopsis pilosula seedlings
The agrobacteria infects the codonopsis pilosula seedling by adopting a root irrigation method. pTRV1+pTRV2 is used as blank control and pTRV1+pTRV2-CpPDS is used as experimental group. Accurately sucking 2.5mL of dyeing liquor by using a 5mL syringe, inserting a needle into the soil of potted codonopsis pilosula, pouring the dyeing liquor near the roots of the codonopsis pilosula, placing the codonopsis pilosula plant after being injected and infected into a climatic chamber for dark culture for 2d at 20 ℃, culturing at 25 ℃ for 16h of illumination, 20 ℃ for 8h of darkness, and watering at fixed time.
5. Photo-bleaching method for observing plant morphology change
The leaves of the transformed plants cultured for 4-6 weeks were observed using the photobleaching method, and albino leaves appeared as silent lines, as shown in fig. 4, and the results showed that: the silencing system was constructed successfully.
The construction method of the codonopsis pilosula gene silencing system provided by the invention is simple and easy to operate, and the plant symptoms are fast.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
<110> university of mountain Western medicine
<120> method for establishing transient silencing system of codonopsis pilosula gene based on VIGS technology
<160>2
<210>1
<211>1737
<212>DNA
<213> dangshen (Codonopsis pilosula (frank.) 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 (4)
1. Radix codonopsis pilosulaeCpPDSApplication of gene in constructing transient silencing vector of codonopsis pilosula gene;
the radix codonopsis pilosulaeCpPDSThe nucleotide sequence of the target fragment of the gene is shown as SEQ ID NO.2.
2. A transient silencing vector for codonopsis pilosula gene is characterized by comprising pTRV2-CpPDSVector, pTRV2-CpPDSThe nucleotide sequence of the target fragment in the vector is shown as SEQ ID NO.2.
3. A method for establishing a pilose asiabell root gene transient silencing system by a VIGS technology, which is characterized by comprising the following steps:
(a) Radix CodonopsisCpPDSThe target fragment of the gene is connected with pTRV2 vector to obtain pTRV2-CpPDSA carrier; the radix codonopsis pilosulaeCpPDSThe nucleotide sequence of the target fragment of the gene is shown as SEQ ID NO. 2;
(b) pTRV1 vector and pTRV2-CpPDSThe vector was introduced into Agrobacterium, respectively, and pTRV1 strain and pTRV were introduced into the vector2-CpPDSMixing the strains, and preparing and obtaining agrobacterium tumefaciens infection solution;
(c) Introducing agrobacterium infection liquid into root cells of codonopsis pilosula plants by adopting a root irrigation method to generate infected plant tissues;
(d) Culturing, inducing Codonopsis pilosula in infected Codonopsis pilosula plantsCpPDSGene silencing of genes.
4. The method of claim 3, wherein the vector is introduced into the agrobacterium by freeze thawing method during the preparation of the agrobacterium infection solution.
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