CN106520828B - Whole corn TRV vector mediated virus induced gene silencing method - Google Patents

Whole corn TRV vector mediated virus induced gene silencing method Download PDF

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CN106520828B
CN106520828B CN201611082938.8A CN201611082938A CN106520828B CN 106520828 B CN106520828 B CN 106520828B CN 201611082938 A CN201611082938 A CN 201611082938A CN 106520828 B CN106520828 B CN 106520828B
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corn
slpds
trv1
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CN106520828A (en
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李成伟
张菊
于德水
王健
陈璨
刘坤
徐克东
张怡
谭光轩
张福丽
廖立冰
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Zhoukou Normal University
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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)
    • C12N15/8201—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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    • 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 belongs to the technical field of plant genetic engineering, and particularly relates to a method for inducing gene silencing by a TRV vector mediated virus of a whole corn plant. The method comprises the following specific steps: taking 50 corn seeds, and cutting off seed coats above the embryos by using a blade; disinfecting seeds with a sodium hypochlorite disinfectant with 1% of available chlorine for later use; connecting the amplified tomato PDS gene segment to a TRV virus-induced gene silencing vector pTRV2 by using a genetic engineering method; agrobacterium transformation and culture; preparing a transformed bacterium liquid; and (4) corn seed infection. The corn genome is complex, mutants are not easy to obtain, genetic transformation is difficult and is limited by genotypes, the corn PDS gene silencing is realized by initially utilizing TRV and adopting tomato PDS virus genes to infect corn seeds with virus vectors, and the method is simple, convenient, fast, efficient, low in cost and free of gene transformation, is full-plant silencing, has high efficiency and is convenient for researching the functions of target genes in various tissues.

Description

Whole corn TRV vector mediated virus induced gene silencing method
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for inducing gene silencing by a TRV vector mediated virus of a whole corn plant.
Background introduction
Plant mutants are difficult to obtain, mutation of some genes can be lethal to plants, gene silencing is relatively easy to obtain, Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing technology, and is widely applied to research of plant genetic engineering as an effective reverse genetics technology, and the action principle of the technology is that plants can initiate an RNA silencing (RNA silencing) mechanism when resisting Virus infection. Dicer-like protein4 in plants recognizes and cleaves dsRNA produced during viral replication into 21-24nt siRNAs (small interfering RNAs), one strand of which binds to proteins such as agnataue (ago) to form an RNA-induced silencing complex (RISC), and the specific binding to a homologous target mRNA ultimately leads to degradation of the target mRNA, i.e., viral-induced gene silencing. Similarly, if a fragment of the host plant gene is cloned into the isease VIGS vector, the corresponding host gene will also be silenced when the host plant elicits a defense response by RNA silencing.
A series of engineered RNA or DNA viral vectors were used for VIGS (Becker and Lange, 2010). Tobacco Rattle Virus (TRV) is a virus vector which has wide application and good efficiency and durability, and can mediate gene silencing without virus-induced symptoms. The modified virus can promote the insertion of non-virus sequences and the subsequent infection of plants, and can also identify the genes of the growing points of host plants, so that the TRV has wide application in the identification of plant gene functions.
Corn (scientific name: Zea mays), also known as maize, corn and corn cobs. Yue is called maize and Minnan is called Fanmi. Is an annual gramineous herbaceous plant, is an important food crop and an important feed source, and is also the food crop with the highest overall yield all over the world. Corn is also an important food crop in China, and the preliminary completion of corn genome sequencing provides important reference for the research of corn functional genomics (Schnable et al, 2009). However, the corn genome is complex, mutants are not easy to obtain, genetic transformation is difficult, and the research on the corn gene function is limited by genotype and the like, which brings great inconvenience.
Disclosure of Invention
In order to solve the problems, the invention provides a rapid and convenient method for silencing the TRV vector-mediated virus-induced gene of the whole corn plant and application thereof, and can rapidly and efficiently silence a target gene in the whole corn plant.
A method for mediating virus-induced gene silencing by a TRV vector of a whole corn plant comprises the following specific steps:
step (1): taking 50 corn seeds, and cutting off seed coats above the embryos by using a blade; using hypochlorous acid with 1% available chlorine
Disinfecting seeds with sodium disinfectant for later use;
step (2): connecting the amplified gene segment of tomato phytoene dehydrogenase (hereinafter referred to as tomato PDS) to a TRV virus-induced gene silencing vector pTRV2 by using a genetic engineering method to obtain pTRV-SlPDS;
the method comprises the following specific steps:
and (3) cDNA synthesis: extracting total RNA of the tomato according to the operation instruction of a TaKaRa extraction kit, synthesizing a first chain of cDNA by taking 1 mu g of total RNA as a template according to the operation instruction of a TaKaRa cDNA synthesis kit, and then diluting by 10 times for later use;
PDS gene amplification: designing primers, namely an upstream primer 5'-CGGGGTACCGGCACTCAACTTTATAAACC-3' and a downstream primer 5'-CGGGGATCCTTCAGTTTTCTGTCAAACC-3', and amplifying by using a 50 mu L system of KAPA HiFiPCR Kits by using a first strand of cDNA of tomato as a template; the amplification procedure was pre-denaturation at 95 ℃ for 3 min; 30 cycles of denaturation at 98 ℃ for 20s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 30 s; extending for 5min at 72 deg.C, and storing at 4 deg.C; detecting the PCR product as a single band by electrophoresis, and purifying for later use; the product of the tomato cDNA amplified by PCR is 409-bp tomato PDS gene segment;
construction of pTRV-SlPDS plasmid: carrying out double enzyme digestion on the purified PCR gene fragment with KpnI-BamHI, connecting the PCR gene fragment with pTRV2 plasmid subjected to double enzyme digestion with KpnI-BamHI by using T4-DNA ligase, transforming a connecting product into escherichia coli DH5 alpha competent cells, coating the competent cells on an LB plate culture medium containing 50mg/L Kan antibiotic, inverting the competent cells at 37 ℃ for overnight culture to grow a monoclonal, selecting the monoclonal to be a liquid LB culture medium containing 50mg/Lkan antibiotic, carrying out shake culture at 37 ℃ at 250 rpm for 12 hours, extracting the plasmid, and verifying the correctness for later use;
and (3) agrobacterium transformation and culture:
3.1) correctly verified pTRV-SlPDS, pTRV2 and pTRV1 plasmids are respectively transformed into agrobacterium GV3101 competent cells by a liquid nitrogen freeze-thaw method, wherein the liquid nitrogen freeze-thaw method specifically comprises the following steps: taking agrobacterium GV3101 competent cells, melting on ice, adding 200ng plasmid to be transferred, ice-bathing for 30min, liquid nitrogen quick-freezing for 2min, water bathing for 2min in a water bath kettle at 37 ℃, ice-bathing for 2min, adding 800 microliter of nonreactive LB liquid culture medium, shaking and culturing for 3h at 28 ℃ and 180 rpm, coating the solution on a solid LB culture medium containing 50mg/L Kna and 50mg/L Rif antibiotics, inverting at 28 ℃ and culturing in a dark place for 3 days to grow a monoclonal; selecting a single clone to be cultured in a liquid LB culture medium containing 50mg/L Kan and 50mg/L Rif antibiotics in a shaking way at the temperature of 28 ℃ and at 200 rpm for 24 hours, and obtaining agrobacterium GV3101 positive strains containing plasmids pTRV1, pTRV2 and pTRV-SlPDS after being qualified by PCR identification of bacteria liquid, wherein the positive strains are marked as TRV1, TRV2 and TRV-SlPDS and are reserved for later use;
3.2) streaking the agrobacterium GV3101 bacterial liquid containing TRV1, TRV2 and TRV-SlPDS plasmids on LB plate culture medium containing antibiotics respectively, and performing inversion and light-proof culture at 28 ℃ for 3 days to grow a single clone;
3.3) respectively picking the monoclonals in the step 3.2) and placing the monoclonals in an LB liquid culture medium containing antibiotics, and performing shake culture for 24 hours by using a centrifugal tube under the conditions of 28 ℃ and 200 r/min to realize the proliferation of the monoclonals;
3.4) mixing the TRV1 obtained in 3.3) with a TRV2 monoclonal culture solution in a ratio of 1:1, mixing the TRV1 and the TRV-SlPDS monoclonal culture solution in a ratio of 1:1, transferring the mixture to the same LB culture medium in a ratio of 1:100 respectively, and continuously culturing the mixture in a triangular flask until the O.D of a bacterial liquid.600＝0.04-1.And 5, adjusting the O.D of the bacterial liquid in the two triangular flasks.600The values are the same;
preparing a transformed bacterium solution:
adding Acetosyringone (AS) into the bacterial liquid obtained in the step (3) to the final concentration of 19.62 mg.L-1And cysteine (Cys) at a final concentration of 400 mg.L-1And Tween 20(Tween 20) at a final concentration of 5 ml. L-1Uniformly mixing to obtain a transformed bacterium liquid containing TRV1 and TRV2 and a transformed bacterium liquid containing TRV1 and TRV-SlPDS, and placing the transformed bacterium liquid in an ultra-clean workbench for later use;
and (5) corn seed infection: respectively putting the same amount of treated corns into 10ml glass bottles with rubber plugs, marking as glass bottles 1 and 2, adding 5ml of transformed bacterium liquid containing TRV1 and TRV2 into the glass bottle 1, and adding 5ml of transformed bacterium liquid containing TRV1 and TRV-SlPDS into the glass bottle 2, so that seeds are completely soaked in the transformed bacterium liquid; vacuumizing the glass bottle by using a 20ml syringe, keeping for 15s each time, and operating for 1-4 times; finally, respectively pouring the corn seeds and the transforming bacteria liquid in the glass bottle 1 and the glass bottle 2 into the corresponding residual transforming bacteria liquid in the step (4), and culturing at 28 ℃ and 200 rpm overnight for 0-25 hours;
after the culture is finished, the culture medium is cleaned by sterile water in an ultra-clean bench, and the culture medium is subjected to TRV1 and TRV2 in a ratio of 1:1 mixed infested maize and TRV1 mixed with TRV-SlPDS at a ratio of 1:1, cleaning the mixed infected corns, and respectively transferring the corns to the same solid MS culture medium under the culture conditions that: 19-22 deg.C, and light intensity of 150 μmol/m-2·s-1The photoperiod is 16 hours of illumination/8 hours of darkness, the relative humidity is 55%, the cultivation is carried out until leaves grow out, the leaves are transplanted to soil and continue to grow for two weeks, the phenotype is observed, DNA is extracted, the amplification condition of tomato PDS virus is detected by adopting TRV coat protein gene, RNA is extracted, and the silencing effect of PDS gene transcript in the silenced corn is detected by using a semi-quantitative RT-PCR method.
Further, in the step (3), OD of the bacterial liquid cultured in a 100ml Erlenmeyer flask600The optimum value of (b) is 0.3.
Further, in the step (4), the mixed solution of acetosyringone, cysteine and tween 20 is adopted for the best culture effect, and the final concentration of the acetosyringone in the mixed solution is 19.62mg·L-1(ii) a Cysteine at a final concentration of 400 mg.L-1B, carrying out the following steps of; tween 20 with final concentration of 5 ml. L-1。
Further, in the step (5), 5ml of transforming bacterium liquid is added into a 10ml glass bottle with a rubber plug, and the seeds are completely soaked in the transforming bacterium liquid; the vacuum is pumped by a 20ml syringe, the operation is carried out for 4 times each time for 15 seconds, and the effect is optimal.
Further, in the step (5), the corn seeds and the transforming bacteria liquid in the glass bottle 1 and the glass bottle 2 are respectively poured into the residual transforming bacteria liquid corresponding to the step (4), and are cultured overnight for 15 hours at the temperature of 28 ℃ and at the speed of 200 rpm, so that the infection effect is optimal.
Compared with the prior art, the invention has the following technical innovation points:
(1) the corn genome is complex, mutants are not easy to obtain, genetic transformation is difficult and is limited by genotypes, the corn PDS gene silencing is realized by initially utilizing TRV and adopting tomato PDS virus genes to infect corn seeds with virus vectors, and the method is simple, convenient, fast, efficient, low in cost and free of gene transformation, is full-plant silencing, has high efficiency and is convenient for researching the functions of target genes in various tissues.
(2) The method for carrying out negative pressure assisted infection on the corn seeds is initiated, and is convenient and quick and good in infection effect.
Drawings
FIG. 1 shows the comparison result of the similarity between corn PDS gene ZmPLDS and tomato PDS gene SlPDS fragments;
FIG. 2 TRV-mediated maize plant germination 16-day phenotype of tomato PDS gene silenced maize plants;
FIG. 3 is a diagram showing the semi-quantitative detection of PDS gene expression level in silent maize plants;
FIG. 4 TRV induces maize gene silencing effects under different parameters.
Detailed Description
The invention is further illustrated by the following examples, which are intended to better understand the present disclosure and not to limit the scope of the invention.
Example silencing maize PDS Gene
A method for inducing gene silencing by a whole-corn TRV vector mediated virus comprises the following steps:
the raw material sources are as follows: the corn variety is Zhengdan 958
KpnI-BamHI, purchased from NEB;
T4-DNA ligase from NEB;
the silencing vectors used in the VIGS experiments were binary expression vectors of RNA1 and RNA2cDNA of Tobacco Rattle Virus (TRV), TRV1 and TRV2, respectively, presented by professor David Baulcombe, university of cambridge and professor liuyule, university of qinghua. The pTRV1 vector contains genes encoding RNA-dependent RNA polymerase, motor protein and 16kD protein and is a helper viral vector for the VIGS system. The pTRV2 vector includes capsid protein (Cp) gene, Multiple Cloning Site (MCS), etc. for construction of a gene of interest.
The method comprises the following specific steps:
step (1): taking 50 corn seeds, and cutting off seed coats above the embryos by using a blade; disinfecting seeds with a sodium hypochlorite disinfectant with 1% of available chlorine for later use;
step (2): connecting the amplified gene segment of tomato phytoene dehydrogenase (hereinafter referred to as tomato PDS) to a TRV virus-induced gene silencing vector pTRV2 by using a genetic engineering method to obtain pTRV-SlPDS;
the method comprises the following specific steps:
and (3) cDNA synthesis: extracting total RNA of the tomato according to the operation instruction of a TaKaRa extraction kit, synthesizing a first chain of cDNA by taking 1 mu g of total RNA as a template according to the operation instruction of a TaKaRa cDNA synthesis kit, and then diluting by 10 times for later use;
PDS gene amplification: designing primers, namely an upstream primer 5'-CGGGGTACCGGCACTCAACTTTATAAACC-3' and a downstream primer 5'-CGGGGATCCTTCAGTTTTCTGTCAAACC-3', and amplifying by using a 50 mu L system of KAPA HiFiPCR Kits by using a first strand of cDNA of tomato as a template; the amplification procedure was pre-denaturation at 95 ℃ for 3 min; 30 cycles of denaturation at 98 ℃ for 20s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 30 s; extending for 5min at 72 deg.C, and storing at 4 deg.C; detecting the PCR product as a single band by electrophoresis, and purifying for later use; the product of tomato PDS after PCR amplification is 409-bp tomato PDS gene segment; the similarity between the tomato PDS gene segment obtained by PCR amplification of tomato PDS and the corn PDS gene ZmPLDS is 78.24%, and is shown in figure 1 in detail;
construction of pTRV-SlPDS plasmid: carrying out double enzyme digestion on the purified PCR gene fragment with KpnI-BamHI, connecting the PCR gene fragment with pTRV2 plasmid subjected to double enzyme digestion with KpnI-BamHI by using T4-DNA ligase, transforming a connecting product into escherichia coli DH5 alpha competent cells, coating the competent cells on an LB plate culture medium containing 50mg/L Kan antibiotic, inverting the competent cells at 37 ℃ for overnight culture to grow a monoclonal, selecting the monoclonal to be a liquid LB culture medium containing 50mg/Lkan antibiotic, carrying out shake culture at 37 ℃ at 250 rpm for 12 hours, extracting the plasmid, and verifying the correctness for later use;
and (3) agrobacterium transformation and culture:
3.1) correctly verified pTRV-SlPDS, pTRV2 and pTRV1 plasmids are respectively transformed into agrobacterium GV3101 competent cells by a liquid nitrogen freeze-thaw method, wherein the liquid nitrogen freeze-thaw method specifically comprises the following steps: taking agrobacterium GV3101 competent cells, melting on ice, adding 200ng plasmid to be transferred, ice-bathing for 30min, liquid nitrogen quick-freezing for 2min, water bathing for 2min in a water bath kettle at 37 ℃, ice-bathing for 2min, adding 800 microliter of nonreactive LB liquid culture medium, shaking and culturing for 3h at 28 ℃ and 180 rpm, coating the solution on a solid LB culture medium containing 50mg/L Kna and 50mg/L Rif antibiotics, inverting at 28 ℃ and culturing in a dark place for 3 days to grow a monoclonal; selecting a single clone to be cultured in a liquid LB culture medium containing 50mg/L Kan and 50mg/L Rif antibiotics in a shaking way at the temperature of 28 ℃ and at 200 rpm for 24 hours, and obtaining agrobacterium GV3101 positive strains containing plasmids pTRV1, pTRV2 and pTRV-SlPDS after being qualified by PCR identification of bacteria liquid, wherein the positive strains are marked as TRV1, TRV2 and TRV-SlPDS and are reserved for later use;
3.2) streaking the agrobacterium GV3101 bacterial liquid containing TRV1, TRV2 and TRV-SlPDS plasmids on LB plate culture medium containing antibiotics respectively, and performing inversion and light-proof culture at 28 ℃ for 3 days to grow a single clone;
3.3) respectively picking the monoclonals in the step 3.2) and placing the monoclonals in an LB liquid culture medium containing antibiotics, and performing shake culture for 24 hours by using a centrifugal tube under the conditions of 28 ℃ and 200 r/min to realize the proliferation of the monoclonals;
3.4) mixing the TRV1 obtained in 3.3) with a TRV2 monoclonal culture solution in a ratio of 1:1, mixing the TRV1 and the TRV-SlPDS monoclonal culture solution in a ratio of 1:1, transferring the mixture to the same LB culture medium in a ratio of 1:100 respectively, and continuously culturing the mixture in a triangular flask until the O.D of a bacterial liquid.600＝0.04-1.5(OD600The optimum value was 0.3, see fig. 4(C)), and the o.d. of the inoculum solution in both flasks was adjusted.600The values are the same;
preparing a transformed bacterium solution:
adding Acetosyringone (AS) into the bacterial liquid obtained in the step (3) to the final concentration of 19.62 mg.L-1And cysteine (Cys) at a final concentration of 400 mg.L-1And Tween 20(Tween 20) at a final concentration of 5 ml. L-1(the best culture effect is obtained by using a mixed solution of acetosyringone, cysteine and Tween 20, the TRV induced gene silencing effect is shown in figure 4 under different parameters, the influence of different components of an infection solution on the gene silencing efficiency is shown in figure 4A, the result shows that the best culture effect is obtained by using the mixed solution of acetosyringone, cysteine and Tween 20, and the final concentration of the acetosyringone in the mixed solution is 19.62 mg.L-1(ii) a Cysteine at a final concentration of 400 mg.L-1(ii) a Tween 20 with final concentration of 5 ml. L-1) Uniformly mixing to obtain a transformed bacterium liquid containing TRV1 and TRV2 and a transformed bacterium liquid containing TRV1 and TRV-SlPDS, and placing the transformed bacterium liquid in an ultra-clean workbench for later use;
and (5) corn seed infection:
respectively putting the same amount of treated corns into 10ml glass bottles with rubber plugs, marking as glass bottles 1 and 2, adding 5ml of transformed bacterium liquid containing TRV1 and TRV2 into the glass bottle 1, and adding 5ml of transformed bacterium liquid containing TRV1 and TRV-SlPDS into the glass bottle 2, so that seeds are completely soaked in the transformed bacterium liquid; vacuumizing a glass bottle by using a 20ml syringe, keeping the vacuumizing for 15s each time, operating for 1-4 times (the vacuumizing of the syringe can be used for effectively assisting infection, and the vacuum treatment is carried out for 15s each time, namely the silencing efficiency is highest for four times, namely the total time is 60 s) (see a figure 4 (B));
after the culture is finished, the culture medium is cultured,cleaning the clean water in an ultra-clean bench, and mixing the water treated by TRV1 and TRV2 at a ratio of 1:1 mixed infested maize and TRV1 mixed with TRV-SlPDS at a ratio of 1:1, cleaning the mixed infected corns, and respectively transferring the corns to the same solid MS culture medium under the culture conditions that: 19-22 deg.C, and light intensity of 150 μmol/m-2·s-1The photoperiod is 16 hours of illumination/8 hours of darkness, the relative humidity is 55%, the cultivation is carried out until leaves grow out, the leaves are transplanted to soil and continue to grow for two weeks, the phenotype is observed, DNA is extracted, the amplification condition of tomato PDS virus is detected by adopting TRV coat protein gene, RNA is extracted, and the silencing effect of PDS gene transcript in the silenced corn is detected by using a semi-quantitative RT-PCR method.
And (4) analyzing results:
1. for TRV1 and TRV2 at 1:1 mixed infested maize and TRV1 mixed with TRV-SlPDS at a ratio of 1:1, cleaning the mixed infected corns, and respectively transferring the corns to the same solid MS culture medium under the culture conditions that: 19-22 deg.C, and light intensity of 150 μmol/m-2·s-1The light period was 16 hours of light/8 hours of darkness, the relative humidity was 55%, the plant was cultured until leaves were grown, and the phenotype was observed after transplanting the plant into soil for two weeks, the results are shown in FIG. 2 (in the figure, A. shows the maize seeds used in the experiment, and the seed coat was cut with a scalpel; B shows that the maize seeds were infected with vacuum-assisted virus-carrying plasmids by Agrobacterium, C-E shows the phenotype after 16 days of germination of the maize seeds infected with TRV1 and TRV2 (as a control group), F-H shows the phenotype after 16 days of germination of the maize seeds infected with TRV1 and TRV-SlPDS at 1: 1; in the figure, 1.41 cm; (C, and F),1.27 cm; (D, E, G, and H),6.16 mm;): TRV1 and TRV2 at 1:1, the corn seeds infected by the mixture grow well after germinating for 16 days, and leaves are green or yellow-green, which indicates that genes for controlling chlorophyll synthesis are not silenced; through TRV1 and TRV-SlPDS 1:1, mixing infected corn seeds, wherein after the corn seeds germinate for 16 days, leaves are white, and a photobleaching phenomenon is generated to indicate that the PDS gene for controlling chlorophyll synthesis is silent; therefore, after the corn seeds are infected by the tomato PDS virus gene mediated by the TRV vector, the PDS gene silencing for controlling chlorophyll synthesis is controlled.
3. Extracting the mixture by using a TaKaRa extraction kit, namely extracting the mixture by using TRV1 and TRV 21: 1, mixing infected corn plants which germinate for 16 days and mixing the plants with TRV-SlPDS1 through TRV 1:1 Total RNA from plants that germinated 16 days after infection of maize. First strand cDNA was synthesized using 1. mu.g of the total RNA as a template according to the protocol of a cDNA Synthesis kit (TaKaRa), and then diluted 10-fold for use. And detecting the silencing effect of the PDS gene transcript in the silenced plant by adopting a semi-quantitative RT-PCR method. The maize Actin gene (accession number J01238) is an internal reference gene, and the maize PDS gene (accession number L39266). Semi-quantitative RT-PCR primer sequences (ZmActins: 5'-CAATGGCACTGGAATGGT-3' and 5'-ATCTTCAGGCGAAACACG-3', ZmPDS: 5'-GCTTACCTGGCCCGAGAAGGTGAAGTTT-3' and 5'-TTCAGTTTTCTGTCAAACC-3'). The reagents used for PCR were Taq enzyme premix (2 XTaq PCR StarMix with Loading Dye, available from Beijing Kangrun technology Co., Ltd.), 20. mu.l system, prepared according to the instructions, PCR conditions: pre-denaturation at 95 ℃ for 3 min; 28-36 cycles of denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30 s; extending for 5min at 72 deg.C, and storing at 4 deg.C; amplification was performed for 28, 30, 32, 34 and 36 different cycles as described above, and the PCR amplification products were detected by 1% agarose gel electrophoresis. The results are shown in FIG. 3: compared with the internal reference gene, the gene has the advantages that the gene is expressed by TRV1 and TRV-SlPDS 1:1 the expression level of the corn PDS gene is obviously reduced in the roots and leaves of plants which germinate for 16 days after mixed infection of the corns.
Result analysis 1, result analysis 2 and result analysis 3 prove that the method for inducing gene silencing by a whole-plant TRV vector mediated virus provided by the application can realize gene silencing of a specific gene from two aspects of plant phenotype and RNA level.

Claims (8)

1. A method for inducing tomato phytoene dehydrogenase PDS gene silencing by using a whole corn plant TRV vector mediated virus is characterized by comprising the following specific steps:
step (1): taking 50 corn seeds, and cutting off seed coats above the embryos by using a blade; disinfecting seeds with a sodium hypochlorite disinfectant with 1% of available chlorine for later use;
step (2): connecting the amplified tomato phytoene dehydrogenase PDS gene fragment to a TRV virus-induced gene silencing vector pTRV2 by using a genetic engineering method to obtain pTRV-SlPDS;
and (3) agrobacterium transformation and culture:
3.1) respectively transforming pTRV-SlPDS, pTRV2 and pTRV1 plasmids into agrobacterium GV3101 competent cells by a liquid nitrogen freeze thawing method, and obtaining agrobacterium GV3101 positive strains which respectively contain plasmids pTRV1, pTRV2 and pTRV-SlPDS and are marked as TRV1, TRV2 and TRV-SlPDS after the bacteria liquid PCR identification is correct, and preserving the strains for later use;
3.2) culturing the agrobacterium GV3101 bacterial solution containing TRV1, TRV2 and TRV-SlPDS plasmids on an LB plate culture medium containing antibiotics to grow a single clone;
3.3) respectively picking the monoclonals in the step 3.2) and putting the monoclonals in an LB liquid culture medium containing antibiotics for culture so as to realize the proliferation of the monoclonals;
3.4) mixing the TRV1 obtained in 3.3) with a TRV2 monoclonal culture solution in a ratio of 1:1, mixing the TRV1 and the TRV-SlPDS monoclonal culture solution in a ratio of 1:1, transferring the mixture to the same LB culture medium in a ratio of 1:100 respectively, and continuously culturing the mixture in a triangular flask until the O.D of a bacterial liquid.600O.d. of the inoculum solution in the two flasks was adjusted to 0.3.600The values are the same;
preparing a transformed bacterium solution:
adding acetosyringone, cysteine and Tween 20 into the bacterial liquid obtained in the step (3), uniformly mixing to obtain a transformed bacterial liquid containing TRV1 and TRV2 and a transformed bacterial liquid containing TRV1 and TRV-SlPDS, and placing the transformed bacterial liquids in an ultra-clean workbench for later use;
and (5) corn seed infection:
infecting the corn seeds treated in the step (1) with a transforming bacterium solution containing TRV1 and TRV2 and a transforming bacterium solution containing TRV1 and TRV-SlPDS, continuously culturing, observing phenotype, extracting RNA, and detecting the silencing effect of PDS gene transcripts in the silenced corn by using a semi-quantitative RT-PCR method.
2. The method for inducing tomato phytoene dehydrogenase PDS gene silencing by whole corn TRV vector mediated virus according to claim 1, wherein the detailed operation of step (2) is as follows:
and (3) cDNA synthesis: extracting total RNA of the tomato according to the operation instruction of a TaKaRa extraction kit, synthesizing a first chain of cDNA by taking 1 mu g of the total RNA as a template according to the operation instruction of a TaKaRa cDNA synthesis kit, and then diluting by 10 times for later use;
PDS gene amplification: using the first cDNA chain of tomato as template, using KAPA HiFi PCR kit, 50 uL system to make amplification; designing primers, an upstream primer 5'-CGGGGTACCGGCACTCAACTTTATAAACC-3' and a downstream primer 5'-CGGGGATCCTTCAGTTTTCTGTCAAACC-3'; detecting the PCR product as a single band by electrophoresis, and purifying for later use;
construction of pTRV-SlPDS gene silencing vector: the purified PCR gene fragment is subjected to double digestion by KpnI-BamHI, and then is connected with pTRV2 plasmid subjected to double digestion by KpnI-BamHI by T4DNA ligase, and the connection product is transformed into escherichia coli DH5 alpha competent cells to form the pTRV-SlPDS gene silencing vector.
3. The method for inducing tomato phytoene dehydrogenase PDS gene silencing by whole corn TRV vector mediated virus according to claim 2, wherein in the step (2), the similarity between the tomato PDS gene segment obtained by PCR amplification of tomato PDS and the corn PDS gene ZmPLDS is 78.24%.
4. The method for inducing tomato phytoene dehydrogenase PDS gene silencing by whole-corn TRV vector mediated virus according to claim 1, wherein in the step (3), the liquid nitrogen freeze-thaw method comprises the specific steps of: taking agrobacterium GV3101 competent cells, melting on ice, adding 200ng plasmid to be transformed, carrying out ice bath for 30min, carrying out liquid nitrogen quick freezing for 2min, carrying out water bath in a water bath kettle at 37 ℃ for 2min, carrying out ice bath for 2min, adding into 800 mu L of non-resistant LB liquid culture medium, carrying out shake culture at 28 ℃ and 180 r/min for 3h, coating on a solid LB culture medium containing 50mg/L Kna and 50mg/L Rif antibiotic, and carrying out inversion and light-proof culture at 28 ℃ for 3 days to grow a monoclonal; selecting a single clone, culturing in a liquid LB culture medium containing 50mg/L Kan and 50mg/L Rif antibiotics at 28 ℃ for 24 hours with shaking at 200 r/min, and obtaining a positive strain containing plasmids pTRV1, pTRV2 and pTRV-SlPDS agrobacterium GV3101 after the PCR identification of bacterial liquid is correct.
5. The method for inducing tomato phytoene dehydrogenase PDS gene silencing by whole corn TRV vector mediated virus according to claim 1, wherein in step (3), O.D. of the bacterial liquid cultured in the triangular flask.6000.3; the final concentration of the acetosyringone in the step (4) is 19.62 mg.L-1The final concentration of cysteine was 400 mg.L-1And Tween 20 at a final concentration of 5 ml. L-1。
6. The method for inducing tomato phytoene dehydrogenase PDS gene silencing by whole corn TRV vector mediated virus according to claim 1, wherein in the step (5), the corn seed infection is specifically performed by the following steps:
respectively putting the same amount of treated corns into 10ml glass bottles with rubber plugs, marking as glass bottles 1 and 2, adding 5ml of transformed bacterium liquid containing TRV1 and TRV2 into the glass bottle 1, and adding 5ml of transformed bacterium liquid containing TRV1 and TRV-SlPDS into the glass bottle 2, so that seeds are completely soaked in the transformed bacterium liquid; vacuumizing the glass bottle by using a 20ml syringe, keeping for 15 seconds each time, and operating for 1-4 times; finally, respectively pouring the corn seeds and the transforming bacteria liquid in the glass bottle 1 and the glass bottle 2 into the corresponding residual transforming bacteria liquid in the step (4), and culturing at 28 ℃ and 200 r/min overnight for 0-25 hours;
at the end of the incubation, the cells were purified in a clean bench by TRV1 and TRV 21: 1, mixing the infected corn and the mixture of TRV1 and TRV-SlPDS 1:1, cleaning the mixed infected corns with sterile water, and respectively transferring the corns to the same solid MS culture medium, wherein the culture conditions are as follows: 19-22 deg.C, and light intensity of 150 μmol/m-2·s-1Culturing until leaves grow out, transplanting the leaves to soil for continuous growth for two weeks, observing phenotype, extracting RNA, and detecting the silencing effect of PDS gene transcripts in the silenced corn by using a semi-quantitative RT-PCR method.
7. The method for inducing tomato phytoene dehydrogenase PDS gene silencing by whole corn TRV vector-mediated virus according to claim 6, wherein in the step (5), 5ml of transforming bacteria solution is added into a 10ml glass bottle with a rubber plug, so that the seeds are completely soaked in the transforming bacteria solution; vacuum was applied using a 20ml syringe for 15 seconds each and the procedure was repeated 4 times.
8. The method for inducing tomato phytoene dehydrogenase PDS gene silencing by whole corn TRV vector-mediated virus according to claim 6, wherein in step (5), the corn seeds and the transforming bacteria liquid in glass bottle 1 and glass bottle 2 are poured into the remaining transforming bacteria liquid corresponding to step (4), and cultured overnight at 28 ℃ and 200 rpm for 15 h.
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