CN113652447A - VIGS-based efficient peach leaf gene silencing method - Google Patents
VIGS-based efficient peach leaf gene silencing method Download PDFInfo
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Abstract
The invention relates to a VIGS-based efficient peach leaf root gene silencing method, which comprises the following steps of: 1. constructing pCaRNA 3-target gene recombinant plasmid, and transferring to Agrobacterium tumefaciens GV3101 to obtain positive Agrobacterium transferred to the recombinant plasmid; 2. preparing VIGS transformed bacterial liquid by using the positive agrobacterium transferred with the recombinant plasmid, and injecting the VIGS transformed bacterial liquid into all fully-unfolded leaves of the fully-unfolded leaf peach seedlings with the seedling age of 5-6; 3. culturing the injected peach seedlings in weak light for 2 days, placing the peach seedlings in a plant growth chamber under the conditions of (22 +/-1) DEG C, 16h of light/8 h of dark, and continuously culturing, and irrigating a Hoagland nutrient solution during the peach seedling culture period. The invention optimizes the practical application of the VIGS gene silencing technology in peaches, establishes a set of efficient peach leaf gene silencing system, and has high silencing efficiency and good stability.
Description
Technical Field
The invention relates to the field of peach gene silencing methods, in particular to a VIGS-based efficient peach leaf gene silencing method.
Background
VIGS (Virus-induced gene silencing) is a Virus-induced post-transcriptional gene silencing technique. According to the technology, a recombinant virus vector with a target gene is introduced into a host plant by using a virus, and the endogenous gene of the plant is silenced after the virus is successfully infected, so that the phenotype of the host is changed, and the functional analysis of the target gene is further realized. The VIGS technology has the advantages of short time, quick response, simple operation and the like, can obtain gene silencing plants without genetic transformation, and becomes one of powerful technical means for researching gene functions. VIGS technology has been successfully applied to gene silencing in many plants. However, at present, a set of stably mature genetic transformation system has not been established for peach (Prunus persica), which seriously hinders the research on the gene function and the related mechanism of the species.
Common VIGS vectors are TRV vectors (TRV1 and TRV2) transformed by tobacco rattle virus, the vectors have higher silencing efficiency in tobacco, arabidopsis thaliana, petunia, tomato, wheat and other species, and are mostly applied to fruit gene silencing on peaches. In 2017, Cui et al constructed a set of virus-mediated gene silencing vectors (pCaRNA1&2 and pCaRNA3) suitable for peaches by utilizing plum necrotic ringspot virus (PNRSV), and successfully induced silencing of peach endogenous genes. However, in the practical application process of the two sets of vectors, the application aspects of using peach leaves as materials and utilizing agrobacterium tumefaciens to mediate recombinant plasmid transfection and the like have the problems of low efficiency, instability and the like, and a system for efficiently inducing plant endogenous gene silencing in peach seedlings by utilizing the VIGS technology is urgently needed to be established.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for efficiently silencing peach leaf slices based on VIGS.
The VIGS-based efficient peach leaf gene silencing method comprises the following steps:
and 3, culturing the injected peach seedlings in a low light environment for 2 days, placing the peach seedlings in a plant growth chamber under the conditions of (22 +/-1) DEG C, 16h of light/8 h of dark environment for continuous culture, and irrigating 1 time of Hoagland nutrient solution during the culture of the peach seedlings to obtain the peach plants with the silent target genes.
Further, the temperature for continuing the culture in said step 3 is 22 ℃.
Further, the peach seedlings in the step 2 are 5-6 fully unfolded leaves.
Further, in the VIGS transformed bacterial liquid obtained in the step 2, the concentration of the bacterial liquid is OD600＝1.0。
Further, the step 1 specifically includes the following steps:
step 1.1, extracting total RNA of peach leaves, and carrying out reverse transcription to obtain cDNA;
step 1.2, searching an mRNA sequence of a target gene in a peach genome, designing a primer by taking the sequence as a template, amplifying a CDS full-length sequence of the gene, connecting the CDS full-length sequence to a pEasy blunt zero vector, transferring the CDS full-length sequence into escherichia coli, and selecting monoclonal positive detection to obtain a recombinant plasmid connected with the CDS sequence of the target gene;
step 1.3, amplifying primers by using target gene specific fragments, adding sequences of enzyme cutting sites at two ends of the primers, and amplifying to obtain target gene fragments by using the recombinant plasmids in the step 1.2 as templates; and constructing a recombinant plasmid pCaRNA 3-target gene by T4 ligase (Takara, Beijing), selecting a monoclonal positive detection sequence to be correct, extracting the plasmid and transferring the plasmid into agrobacterium tumefaciens by a heat shock method.
Further, the step 2 specifically includes the following steps:
step 2.1, inoculating the positive agrobacterium tumefaciens single colony transferred with the recombinant plasmid to 1mL of the single colony containing 50 mg.L-1Kanamycin and 20 mg. L-1In LB liquid culture medium of rifampicin, at 28 deg.C, 220 r.min-1Carrying out shake culture for 12h under the condition;
step 2.2, transferring the bacterial liquid finally obtained in the last step to 50mL of bacterial liquid containing 50 mg.L according to the volume ratio of 1:50-1KanamycinAnd 20 mg. L-1In LB liquid culture medium of rifampicin, at 28 deg.C, 220 r.min-1Carrying out shake culture for 16h under the condition;
step 2.3, centrifuging 5000g of the bacterial liquid finally obtained in the last step for 5min at the temperature of 4 ℃, discarding the supernatant, suspending the supernatant by using 5mL of MMA, centrifuging again, discarding the supernatant, suspending the supernatant by using 5mL of MMA, measuring the OD value, and adjusting the bacterial liquid to the target concentration by using MMA to obtain a first bacterial liquid with the target concentration;
step 2.4, preparing positive agrobacterium transformed into pCaRNA1&2, and preparing a second bacterial liquid with the same concentration as the first bacterial liquid by using the positive agrobacterium transformed into pCaRNA1&2 and adopting the method of the step 2.1-step 2.3;
step 2.5, mixing the first bacterial liquid and the second bacterial liquid according to a volume ratio of 1: 1, uniformly mixing, and standing at room temperature in a dark place for 2 hours to prepare VIGS transformed bacterial liquid;
and 2.6, injecting the VIGS transformed bacterial liquid into the peach leaves from the back sides of the peach seedling leaves until the whole peach leaves are wetted, and injecting all the fully-unfolded leaves of the peach seedlings.
The technical scheme for solving the technical problems is as follows:
the invention has the beneficial effects that: compared with conventional TRV vectors (TRV1 and TRV2) in peach seedling leaves for the first time, the invention discovers that the vectors (pCaRNA1&2 and pCaRNA3) constructed by PNRSV have good effect, optimizes technical details, and has high silencing efficiency and good stability.
Drawings
FIG. 1 is an electrophoresis chart showing the result of PCR amplification of CDS fragment of PpPDS gene;
FIG. 2 is the electrophoresis diagram of the target band detection of the pCaRNA3-PpPDS recombinant vector;
FIG. 3 is a phenotype diagram of peach seedling plants infected by a leaf injection method for 25 days;
FIG. 4 is a chart of different site patterns after the peach seedling leaves are infected by a leaf injection method;
FIG. 5 shows silencing efficiency of genes at different temperatures and at different ages;
FIG. 6 shows the silencing efficiency of pCaRNA3-PpPDS at different bacterial fluid concentrations;
FIG. 7 shows the relative expression levels of different parts of peach seedlings under the treatment of pTRV 2-PpPDS;
FIG. 8 shows the relative expression levels of different parts of peach seedlings under the treatment of pCaRNA 3-PpPDS;
FIG. 9 shows the relative expression level of PpERF98 gene under PNRSV-VIGS treatment of peach seedlings.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
Example 1
The silencing vectors pTRV2-PpPDS and pCaRNA3-PpPDS are constructed by taking a Phytoene Dehydrogenase (PDS) gene as a target gene, and the influence of temperature, the inoculation seedling age and the concentration of an inoculation bacterial liquid on the silencing efficiency of the peach TRV-VIGS and PNRSV-VIGS is explored by taking peach leaves as injection materials by adopting an agrobacterium tumefaciens mediated method.
1. Construction of silencing vectors pTRV2-PpPDS and pCaRNA3-PpPDS
(1) Taking wild peach leaves as a material, extracting total RNA by using an EASY spin Plus plant RNA rapid extraction kit (Aidlab, Beijing), detecting the concentration and quality of RNA by using Nanodrop one (Thermo, USA) and gel electrophoresis, and then using a reverse transcription kit
Performing reverse transcription by using an RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) to obtain cDNA;
(2) search for the mRNA sequence of PpPDS in the peach genome and use this sequence as a template to design primers (primer 1 and primer 2) using primer5, using Novonoprazan Hi-Fi enzyme
Amplifying the CDS full-length sequence of the gene by a Super-Fidelity DNA Polymerase (Vazyme, Nanjing) kit, connecting the CDS full-length sequence to a pEasy blunt zero vector, transferring the vector into escherichia coli, and selecting single clone for positive detection to obtain a recombinant plasmid connected with the CDS sequence of PpPDS;
(3) primer5 was used to design PpPDS specific fragment amplification primers (primer 3 and primer 4) based on the restriction sites of the different vectors (enzyme TRV2)Cutting sites are BamH I and Sma I; xba I) is added into the pCaRNA3, a sequence with the restriction enzyme site is added, the recombinant plasmid in the step (2) is used as a template, and the Novozam high-fidelity enzyme
Amplifying a Super-Fidelity DNA Polymerase (Vazyme, Nanjing) kit to obtain a PpPDS gene 100bp fragment; recombinant plasmids pTRV2-PpPDS and pCaRNA3-PpPDS are constructed by T4 ligase (Takara, Beijing), and after a single clone positive detection sequence is picked to be correct, plasmids are extracted and are transformed into agrobacterium tumefaciens GV3101 by a heat shock method.
2. Preparation and transfection of agrobacterium tumefaciens bacterial liquid
(1) The plasmids (pTRV1, pTRV2, pTRV2-PpPDS and pCaRNA 1) were individually selected and transferred to the target plasmid&2. pCaRNA3 and pCaRNA3-PpPDS) were inoculated to 1mL of a single colony containing 50 mg. multidot.L-1Kanamycin (Kan) and 20 mg.L-1Rifampicin (Rif) in LB liquid medium at 28 ℃ and 220 r.min-1Carrying out shake culture for 12 h;
(2) then transferring the bacterial liquid to 50mL containing 50 mg.L according to the ratio of 1:50(v: v)-1Kan and 20 mg.L-1Rif in LB liquid medium, 28 ℃,220 r.min-1Shaking and culturing for 16 h.
(3) The bacterial solution was centrifuged at 4 ℃ and 5000g for 5min, and 5mL MMA (10mM MgCl) was added after discarding the supernatant2150 mu M AS, 10mM MES, pH 5.6), centrifuging again, abandoning the supernatant, suspending with 5mL MMA, measuring the OD value, and adjusting the Agrobacterium tumefaciens bacterial liquid carrying different plasmids to the target concentration with MMA according to the test requirements;
(4) the bacterium solutions containing pTRV1 and pTRV2 and pTRV2-PpPDS were mixed in the ratio of 1: 1(v: v) and mixing; the same procedure was carried out for pCaRNA1&2 and pCaRNA3 and pCaRNA3-PpPDS, respectively, in the same manner as described in 1: 1(v: v), uniformly mixing, and standing at room temperature in a dark place for 2 hours to prepare a VIGS transformation bacterial liquid;
(5) injecting the bacterial liquid prepared in the step (4) into the peach seedlings from the backs of the peach seedlings by using a 1ml syringe with a needle removed until the whole peach seedlings are wetted, wherein the mature peach seedlings are taken as materials;
(6) and culturing the injected peach seedlings in weak light for 2 days, and then placing the peach seedlings in a plant growth chamber at the temperature of 22 ℃ under the condition of 16h of light/8 h of dark for continuous culture. Uniformly pouring 1-time Hoagland nutrient solution with the same amount as the required nutrient solution during the culture of the peach seedlings.
3. Treatment of concentration, temperature and seedling age of inoculated bacterial liquid
Temperature: dividing the peach seedlings subjected to agrobacterium tumefaciens injection into two groups, and respectively placing the two groups of peach seedlings at the temperature of 22 ℃, the temperature of 28 ℃, and the illumination for 16 h/dark for 8h for culture;
and (4) inoculating seedling age: dividing peach seedlings into 5-6 completely unfolded leaves, 7-8 completely unfolded leaves and 10-11 completely unfolded leaves, and injecting agrobacterium liquid for treatment in 3 batches of seedling ages;
the concentration of the inoculated bacterial liquid: the Agrobacterium culture solution of (3) in 2.3 was adjusted to OD of about 0.6, 1.0, 1.4 with MMA, and then the test was carried out according to the procedures (4), (5) and (6) in 2.3.
Phenotypic observations were made periodically in this experiment and the effect of gene silencing was compared from the number efficiency of silenced plants, phenotype of silenced plants and relative expression of PpPDS. The relative expression quantity of the PpPDS gene is analyzed by a qRT-PCR method, and the steps are as follows: selecting tissues of each part of a pTRV2-PpPDS silent plant and pCaRNA3-PpPDS to extract RNA, carrying out reverse transcription to obtain cDNA, and designing a primer5 and a primer 6 outside a PpPDS gene silent fragment; PpTEF2 (transformation activation factor 2) (Tong et al 2009) was used as an internal reference gene (amplification primers: primer 7 and primer 8, and 2 as-ΔΔCtThe method is used for calculating to obtain the relative expression quantity of the PpPDS gene.
The sequences of the genes involved are as follows:
CDS full length 1722bp of PpPDS, numbering: Prupe.1G174100
ATGTCTCAGTGGGCTTGTGTCTCTGCTGCTAACTTGAGCTGTCAAGCTAGCATCATCAACACTCAAAAGCTACGAAACACTCCCAGATGCGATGCCTTTTCATTTAAAGGTAGTGAATTTATGGCTCAAAGCTGTAGATTTTTAAGCCCACAAACTATTTATGGAAGGCCGAGGAATGGTGCTTGCCCTTTGAAGGTGGTTTGCGTTGATTATCCAAGACCAGACCTTGACAATACTGCTAATTTCTTAGAAGCTGCATATTTCTCTTCCACTTTCCGAGCCTCTCCTCGTCCAGCTAAGCCGTTGAAGGTCGTGATTGCTGGTGCAGGTTTGGCTGGTCTGGCAACTGCAAAATATTTGGCTGATGCAGGTCATAAACCTATCTTACTGGAAGCAAGAGATGTTCTGGGCGGAAAGGTGGCAGCATGGAAAGATAAGGATGGAGACTGGTACGAAACAGGCCTACATATCTTCTTTGGGGCTTATCCGAATATTCAGAACCTGTTTGGTGAGCTTGGTATTGATGATCGATTGCAGTGGAAGGAGCATTCTATGATATTTGCAATGCCAAGCAAACCAGGAGAGTTCAGCCGGTTTGATTTCCCTGAAGTTTTACCAGCACCCTTAAATGGAATATGGGCCATATTGAAGAACAATGAGATGCTGACTTGGCCAGAGAAAATCAAGTTTGCAATTGGACTACTGCCAGCAATTCTTGGTGGGCAGGCTTATGTTGAAGCCCAAGATGGCTTGAGTGTAAAAGATTGGATGAGGAAACAGGGCATACCGGATCGAGTGACTACTGAGGTGTTTATTGCCATGTCAAAGGCCCTGAACTTTATTAACCCTGATGAACTTTCAATGCAATGCATATTGATTGCTTTGAACCGATTCCTTCAGGAGAAACACGGTTCCAAGATGGCTTTTTTGGATGGTAGTCCCCCTGAGAGACTCTGTGCACCAATTGTTGATCATATCCAGTCATTAGGCGGTGAAGTCCGAATTAATTCCCGAATACAGAGAATTGAGCTAAATAAAGATGGGACCGTGAAGAGTTTTGTACTAAATAATGGGAGCATGATTGAAGCAGATGCCTATGTATTCGCCACTCCAGTTGATATCCTAAAGCTTCTATTGCCTGATAACTGGAAAGAGATCCCATATTTCAAGAAATTGGAGAAACTGGTTGGCGTTCCAGTTATCAATGTTCACATATGGTTTGACAGAAAGCTGAAGAACACATATGATCATCTACTTTTTAGCAGAAGTCCTCTTTTAAGTGTCTATGCCGACATGTCCGTAACATGTAAGGAATATTACAATCCAAACCAGTCAATGCTGGAGTTGGTTTTTGCACCAGCAGAAGAATGGATATCATGCAGTGATTCAGAAATTATTGATGCTACACTCAAAGAACTTGCAAAACTCTTTCCAGATGAGATAGCTGTAGATCAAAGCAAAGCAAAGATTTTGAAGTACCATGTGGTGAAAACACCAAGGTCGGTTTACAAAACTGTACCAGGTTGTGAACCTTGCCGTCCCTTGCAAAGATCTCCCCTAGAGGGTTTCTATTTAGCTGGTGATTACACAAAACAAAAGTATTTAGCCTCAATGGAAGGTGCTGTTCTGTCAGGGAAACTTTGTGCACAAGCAATTGTACAGGATTACGAATTGCTTGTTGCTCGGGGACAAACAAGGGTGGCTGAGGCAAGCGTTCGGTGA
Primer 1: 5'-ATGTCTCAGTGGGCTTGTGTCTCTG-3'
Primer 2: 5'-TCACCGAACGCTTGCCTCAGCCACC-3'
2. Amplifying PpPDS specific segment, introducing positive and negative primersXbaΙRestriction sites (underlined; product 100bp)
Primer 3: 5' -GCTCTAGAAGAAAGCTGAAGAACACAT-3’
Primer 4: 5' -GCTCTAGAGATTGTAATATTCCTTACAT-3’
Primer for PpPDS quantitation (145 bp product)
Primer 5: 5'-CCTTGCAAAGATCTCCCCTA-3'
Primer 6: 5'-CGAGCAACAAGCAATTCGTA-3'
Quantitative primer of PpTEF2 (147 bp product)
Primer 7: 5'-AGCAAGTCACCCAACAAGCATA-3'
Primer 8: 5'-CCAACCAAACTCTTCAGCCAAT-3'
As shown in figure 1, agarose gel electrophoresis detection shows that figure 1 shows that the PCR amplification product of the PpPDS gene segment has a clear band at about 1700bp, and after recovery, sequencing verification is carried out, and the PpPDS gene segment is compared with the sequence of the 1722bp segment of the PpPDS gene with an expected result by using DNANN software, so that the cloning success of the PpPDS gene segment is shown.
As shown in FIG. 2, after Agrobacterium tumefaciens GV3101 was transformed with the pCaRNA3-PpPDS recombinant vector, a plasmid of a bacterial solution of a positive clone was extracted. The pCaRNA3-PpPDS recombinant plasmid was amplified by PCR (primer 9 and primer 10). Through agarose gel electrophoresis detection, FIG. 2 shows that the band is about 375b p, which is in line with the expected target, and the construction of the pCaRNA3-PpPDS recombinant vector is proved to be successful. (primer 9: 5'-GCTTCCCTAACGGGGCATCC-3'; primer 10: 5'-AGGTCTTGGTTAGGGATTTG-3'.)
As shown in FIG. 3, when the peach seedling plants are infected by the leaf injection method, the phenotype of pCaRNA3-PpPDS is obviously different from that of pTRV2-PpPDS 25 days after infection. The phenotype of pCaRNA3-PpPDS shows large spots, and the new leaves always have albino phenotype, and the whole silent phenotype is obvious; the phenotype of pTRV2-PpPDS is small screw thread shape, only a few leaves show the phenotype, new leaves do not show the phenotype any more, and the overall effect of silencing is not obvious compared with pCaRNA 3-PpPDS.
As shown in fig. 4, the leaves from the lower morphological end to the upper morphological end of the peach seedling have different phenotypes after leaf injection. In the figure, the injection of leaves: representing injected leaves,. sup.: leaves representing the appearance of the phenotype, phenotypic upper leaves: representing leaves above phenotypic leaves where no phenotype is present. Note: the phenotypic leaves of pCaRNA3-PpPDS are all phenotypic leaves, so that the phenotypic leaves are not.
As shown in FIG. 5, in order to verify the effect of temperature on silencing efficiency, peach seedlings were cultured at 22 ℃ and 28 ℃ in this experiment. The results indicate that both pCaRNA3-PpPDS and pTRV2-PpPDS are affected by temperature. The silencing efficiency of pCaRNA3-PpPDS and pTRV2-PpPDS genes is 85 percent and 64 percent at 22 ℃; however, gene silencing efficiency decreased to 53% and 29% at 28 ℃ respectively. Higher temperatures significantly reduced VIGS silencing efficiency. Previous studies showed that (Wangmanghongzhi, 2005), the silencing efficiency of the gene was significantly affected by the size of the seedling age, and in order to determine the optimum size of the seedling age to obtain the maximum silencing efficiency, this experiment set the size of the seedling age to be 3 treatments of 5-6 fully expanded leaves, 7-8 fully expanded leaves, and 10-11 fully expanded leaves. The result shows that the gene silencing efficiency of pTRV2-PpPDS is obviously influenced by the seedling age and is respectively 64 percent, 24 percent and 0 percent; the gene silencing efficiency of pCaRNA3-PpPDS is high under 3 treatments, and is respectively 85%, 80% and 94%. Thus, the pCaRNA1&2 and pCaRNA3 vectors are more suitable than the pTRV1 and pTRV2 vectors for silencing endogenous genes in peach seedlings using agrobacterium-mediated VIGS technology.
As shown in FIG. 6, in order to verify the effect of different bacterial liquid concentrations on the silencing efficiency of pCaRNA3-PpPDS gene, OD was set in each experiment600＝0.6，OD6001.0 and OD600Total 3 treatments were 1.4. The results show that 3 weeks after injection, the gene silencing efficiency is 61%, 55% and 88% respectively; 4 weeks after injection, the gene silencing efficiency is 85%, 95% and 92%, and there is no significant difference among the three.
As shown in figure 7, the peach seedling leaves are infected by adopting a leaf injection method. After 25 days of infection, the leaf, stem and root of pTRV2 empty vector plant and pTRV2-PpPDS plant were subjected to qRT-PCR analysis using pTRV2 empty vector plant as a control, 3 replicates were taken per treatment, and gene expression levels were analyzed using PpTEF2 (transformation efficiency factor 2) (Tong et al 2009) as an internal reference gene. The results show that compared with pTRV2 control, the expression levels of phenotypic upper leaves, phenotypic lower leaves, injection leaves, stems and roots of pTRV2-PpPDS plants are obviously reduced, which indicates that the albino phenotype of the pTRV2-PpPDS plants is caused by PpPDS gene silencing and the silencing effect is obvious.
As shown in FIG. 8, the peach seedling leaves are infected by the leaf injection method. After infection for 25 days, each part of the pCaRNA3 empty vector plant and the pCaRNA3-PpPDS plant was subjected to qRT-PCR analysis using pCaRNA3 empty vector plant as a control, and 3 replicates were taken for each treatment, and gene expression levels were analyzed using PpTEF2 (transformation efficiency 2) (Tong et al 2009) as an internal reference gene. The results show that the expression levels of phenotype leaves, phenotype lower leaves, injection leaves, stems and roots of the pCaRNA3-PpPDS plant are obviously reduced compared with that of the pCaRNA3 control, and prove that the albino phenotype of the pCaRNA3-PpPDS plant is caused by PpPDS gene silencing and the silencing effect is obvious.
Example 2
The silencing effect test was performed in the same manner as in example 1 using PpERF98 as the target gene.
As shown in FIG. 9, the peach seedling leaves were infected by the leaf injection method. After 25 days of infection, the pCaRNA3 empty vector plant was used as a control, and qRT-PCR analysis was performed on the pCaRNA3 empty vector plant and the pCaRNA3-PpERF98 plant. In this experiment 5 biological replicates were set up and designated pCaRNA3-PpERF98# 1, pCaRNA3-PpERF98# 2, pCaRNA3-PpERF98# 3, pCaRNA3-PpERF98# 4 and pCaRNA3-PpERF98# 5, respectively. The expression level of the target gene PpERF98 was analyzed by qRT-PCR using PpTEF2 (transformation factor 2) (Tong et al 2009) as an internal reference gene. The results show that the expression level of the pCaRNA3-PpERF98 plants is obviously reduced compared with that of the pCaRNA3 control.
The sequence table of the related genes is as follows:
1. CDS full length 492bp of PpERF98, numbering: Prupe.1G037800
ATGGAAGACCCTCGTAGAGGAAAGGATCTACAGAAGCAAGGAATGGAAGAGAAGGGAAGCGAAGAAGTGCGTTATCGGGGAGTCCGAAGGAGGCCATGGGGGAAGTTTGCTTCTGAGATAAGAGACCCTTCAAGGCAAGGTGCTCGTCTGTGGCTTGGCACATTTGATACGGCTGAAGAAGCTGCAAGGGCTTATGACAGAGCTGCTTTTAATTTAAGGGGTCATCTTGCCATACTCAACTTTCCAAATGAGTATTATTCTCAAGTTATGGGCTCTCCTCCTCATCCTCCTAGATTCTCATCATCATTTTCTTCTTCTTCTTCTTCTTCATCTTCTTCTTCTTCTTCTTCTTCTGCTGCTGCTGCTGCTGCTGCTCCTGGTGGAAGTTCATCTACTGGACAAGGAAGGCAAGTCTTTGAGATTGAGTATTTGGATGACCATGTGTTGGAGGACCTTCTTGATTCTCAAGAGGAGAAAAACAAACGGAAGTGA
2. VIGS interference fragment amplification (product 100bp)
PpERF98-F：5’-GCTCTAGAATGGAAGACCCTCGTAGAGG-3’
PpERF98-R：5’-GCTCTAGACCCATGGCCTCCTTCGGA-3’
3. Quantitative primer: (product 115bp)
PpERF98-F1：5’-AAATGTGCCAAGCCACAGAC-3’
PpERF98-R1：5’-GAAGGGAAGCGAAGAAGTGC-3’
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. The VIGS-based efficient peach leaf gene silencing method is characterized by comprising the following steps of:
step 1, constructing pCaRNA 3-target gene recombinant plasmid, and transferring Agrobacterium tumefaciens to obtain positive Agrobacterium transformed with the recombinant plasmid;
step 2, preparing VIGS transformed bacteria liquid by using the positive agrobacterium transferred into the recombinant plasmid, and injecting the VIGS transformed bacteria liquid into the peach leaves by using an injector with a pinhead;
and 3, culturing the injected peach seedlings in a low light environment for 2 days, placing the peach seedlings in a plant growth chamber under the conditions of (22 +/-1) DEG C, 16h of light/8 h of dark environment for continuous culture, and irrigating 1 time of Hoagland nutrient solution during the culture of the peach seedlings to obtain the peach plants with the silent target genes.
2. The VIGS-based hypericum perforatum silencing method of claim 1, wherein the temperature for the continuous cultivation in step 3 is 22 ℃.
3. The VIGS-based high-efficiency peach leaf gene silencing method according to claim 1, wherein the size of the peach plant in the step 2 is 5-6 fully unfolded leaves.
4. The VIGS-based high-efficiency peach leaf base silencing method according to claim 1, wherein the concentration of the VIGS transformed bacterial liquid in the step 2 is OD600＝1.0。
5. The VIGS-based high-efficiency peach leaf gene silencing method according to claim 1, wherein the step 1 specifically comprises the following steps:
step 1.1, extracting total RNA of peach leaves, and carrying out reverse transcription to obtain cDNA;
step 1.2, searching an mRNA sequence of a target gene in a peach genome, designing a primer by taking the sequence as a template, amplifying a CDS full-length sequence of the gene, connecting the CDS full-length sequence to a pEasy blunt zero vector, transferring the CDS full-length sequence into escherichia coli, and selecting monoclonal positive detection to obtain a recombinant plasmid connected with the CDS sequence of the target gene;
step 1.3, amplifying primers by using target gene specific fragments, adding sequences of enzyme cutting sites at two ends of the primers, and amplifying to obtain target gene fragments by using the recombinant plasmids in the step 1.2 as templates; and constructing a recombinant plasmid pCaRNA 3-target gene by using T4 ligase, selecting a monoclonal positive detection sequence to be correct, extracting the plasmid, and transferring the plasmid into agrobacterium tumefaciens by a heat shock method.
6. The VIGS-based high-efficiency peach leaf gene silencing method according to claim 1, wherein the step 2 specifically comprises the following steps:
step 2.1, inoculating the positive agrobacterium tumefaciens single colony transferred with the recombinant plasmid to 1mL of the single colony containing 50 mg.L-1Kanamycin and 20 mg. L-1In LB liquid culture medium of rifampicin, at 28 deg.C, 220 r.min-1Carrying out shake culture for 12h under the condition;
step 2.2, transferring the bacterial liquid finally obtained in the last step to 50mL of bacterial liquid containing 50 mg.L according to the volume ratio of 1:50-1Kanamycin and 20 mg. L-1In LB liquid culture medium of rifampicin, at 28 deg.C, 220 r.min-1Carrying out shake culture for 16h under the condition;
step 2.3, the bacterial liquid finally obtained in the last step is centrifuged for 5min at the temperature of 4 ℃ at 5000g, 5mL of MMA containing 10mM MgCl is used after supernatant fluid is discarded2150 μ M AS and 10mM MES, pH 5.6; suspending, centrifuging again, discarding the supernatant, suspending with 5mL MMA, determining OD value, and adjusting the bacterial liquid to mesh with MMAMarking the concentration to obtain a first bacterial liquid with a target concentration;
step 2.4, preparing positive agrobacterium transformed into pCaRNA1&2, and preparing a second bacterial liquid with the same concentration as the first bacterial liquid by using the positive agrobacterium transformed into pCaRNA1&2 and adopting the method of the step 2.1-step 2.3;
step 2.5, mixing the first bacterial liquid and the second bacterial liquid according to a volume ratio of 1: 1, uniformly mixing, standing for 2 hours at room temperature in a dark place, and preparing VIGS transformed bacterial liquid;
and 2.6, injecting the VIGS transformed bacterial liquid into the peach leaves from the back sides of the peach seedling leaves until the whole peach leaves are wetted, and injecting all the fully-unfolded leaves of the peach seedlings.
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| CN108165579A (en) * | 2017-12-26 | 2018-06-15 | 北京农学院 | A kind of method of the VIGS silencing systems of the identification Chinese rose RhPDS genes of optimization |
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