CN113652447B - High-efficiency peach leaf gene silencing method based on VIGS - Google Patents
High-efficiency peach leaf gene silencing method based on VIGS Download PDFInfo
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Abstract
The invention relates to a high-efficiency peach leaf gene silencing method based on VIGS, which comprises the following steps: 1. constructing pCaRNA 3-target gene recombinant plasmid, and transferring the recombinant plasmid into agrobacterium tumefaciens GV3101 to obtain positive agrobacterium tumefaciens transferred into the recombinant plasmid; 2. preparing a VIGS conversion bacterial liquid by utilizing positive agrobacterium transferred into a recombinant plasmid, and injecting the VIGS conversion bacterial liquid into all fully-unfolded blades of which the seedling age is 5-6 and fully-unfolded She Taomiao; 3. culturing the injected peach seedlings with weak light for 2 days, placing the peach seedlings in a plant growth room at the temperature of (22+/-1) ℃ under the condition of 16h illumination/8 h darkness for continuous culture, and irrigating 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 high-efficiency 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 high-efficiency peach leaf gene silencing method based on VIGS.
Background
VIGS (Virus-induced gene silencing) is a post-transcriptional gene silencing technique induced by viruses. The technology utilizes viruses to introduce recombinant viral vectors with target genes into host plants, and induces the endogenous genes of the plants to silence after the viruses are successfully infected, so that the phenotype of the host is changed, and the functional analysis of the target genes is further realized. The VIGS technology has the advantages of short time, quick response, simple operation and the like, and can obtain the gene silencing plant without genetic transformation, thus becoming one of powerful technical means for researching gene functions. VIGS technology has been successfully applied to the gene silencing of many plants. However, at present, a stable mature genetic transformation system of peach (Prunus persica) has not been established, and the research on the gene functions and related mechanisms of the species is seriously hindered.
The common VIGS vector is a TRV vector (TRV 1 and TRV 2) modified by tobacco brittle fracture virus, has high silencing efficiency in tobacco, arabidopsis thaliana, petunia, tomato, wheat and other species, and is mostly applied to fruit gene silencing on peaches. In 2017, cui et al constructed a set of virus-mediated gene silencing vectors (pCaRNA 1&2 and pCaRNA 3) for peach using Plum Necrosis Ring Spot Virus (PNRSV) and successfully induced silencing of the peach endogenous gene. However, in the practical application process of the two sets of vectors, the problems of low efficiency, instability and the like exist in the application aspects of taking peach leaves as materials and utilizing agrobacterium tumefaciens to mediate recombinant plasmid transfection and the like, and a system for efficiently inducing plant endogenous gene silencing in peach seedlings by utilizing the VIGS technology is needed to be established.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for high-efficiency peach leaf gene silencing based on VIGS.
The high-efficiency peach leaf gene silencing method based on the VIGS comprises the following steps:
step 1, constructing pCaRNA 3-target gene recombinant plasmid, and transferring into agrobacterium tumefaciens to obtain positive agrobacterium transferred into the recombinant plasmid;
step 2, preparing a VIGS conversion bacterial liquid by utilizing positive agrobacterium transferred into a recombinant plasmid, and injecting the VIGS conversion bacterial liquid into peach leaves by utilizing a syringe with a needle removed;
and 3, culturing the injected peach seedlings for 2 days under weak light, and then placing the peach seedlings in a plant growth room under the conditions of (22+/-1) DEG C, 16h illumination/8 h darkness for continuous culture, and irrigating 1 time of Hoagland nutrient solution during the peach seedling culture period to obtain the peach plants with the silent target genes.
Further, the temperature of the continuous culture in the 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 in the step 2, the bacterial liquid is concentratedDegree of OD 600 ＝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 mRNA sequence of a target gene in a peach genome, designing a primer by taking the sequence as a template, amplifying CDS full-length sequence of the gene, connecting the sequence to a pEasy batch zero vector, transferring the vector into escherichia coli, and then picking up monoclonal positive detection to obtain recombinant plasmid connected with the CDS sequence of the target gene;
step 1.3, amplifying a primer by utilizing the target gene specific fragment, adding sequences of enzyme cutting sites at two ends of the primer, and amplifying by taking the recombinant plasmid in the step 1.2 as a template to obtain a target gene fragment; and constructing a recombinant plasmid pCaRNA 3-target gene by T4 ligase (Takara, beijing), picking up the correct monoclonal positive detection sequence, 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 together the recombinant plasmid-transferred positive Agrobacterium single colony to 1mL containing 50mg.L -1 Kanamycin and 20 mg.L -1 Rifampicin LB liquid medium at 28 ℃ and 220 r.min -1 Shake culturing for 12h under the condition;
step 2.2, transferring the bacterial liquid obtained in the last step to 50mL containing 50 mg.L according to the volume ratio of 1:50 -1 Kanamycin and 20 mg.L -1 Rifampicin LB liquid medium at 28 ℃ and 220 r.min -1 Shake culturing for 16h under the condition;
step 2.3, centrifuging 5000g of the bacterial liquid finally obtained in the previous step for 5min at the temperature of 4 ℃, suspending the supernatant with 5mL of MMA, centrifuging again, suspending the supernatant with 5mL of MMA, measuring an OD value, and regulating the bacterial liquid with MMA to a target concentration to obtain a first bacterial liquid with the target concentration;
step 2.4, preparing positive agrobacterium transferred into pCaRNA1&2, and preparing a second bacterial liquid with the same concentration as the first bacterial liquid by using the positive agrobacterium transferred into pCaRNA1&2 and adopting the method of step 2.1-step 2.3;
step 2.5, the first bacterial liquid and the second bacterial liquid are mixed according to the volume ratio of 1:1, uniformly mixing, and standing at room temperature in a dark place for 2 hours to prepare a VIGS-transformed bacterial solution;
and 2.6, injecting the VIGS-transformed bacterial liquid into the peach leaves from the back of the peach leaves until the whole 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 beneficial effects of the invention are as follows: compared with conventional TRV vectors (TRV 1 and TRV 2) in peach seedling leaves for the first time, the invention discovers that the vectors (pCaRNA 1&2 and pCaRNA 3) constructed by PNRSV have good effect, optimize technical details, and have high silencing efficiency and good stability.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification result of CDS fragment of PpPDS gene;
FIG. 2 is a diagram showing the detection electrophoresis of the target band of the pCaRNA3-PpPDS recombinant vector;
FIG. 3 is a phenotype diagram of peach seedlings plants infected with leaf injection for 25 days;
FIG. 4 is a phenotype chart of different parts of the leaf of the peach seedling infected by the leaf injection method;
FIG. 5 shows the silencing efficiency of different temperature, shoot age size genes;
FIG. 6 shows silencing efficiency of pCaRNA3-PpPDS at different bacterial fluid concentrations;
FIG. 7 shows the relative expression levels of different parts of peach seedlings under pTRV2-PpPDS treatment;
FIG. 8 shows the relative expression levels of different parts of peach seedlings under pCaRNA3-PpPDS treatment;
FIG. 9 shows the relative expression level of the PpERF98 gene of peach seedlings under PNRSV-VIGS treatment.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended 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, adopting an agrobacterium tumefaciens-mediated method, taking peach leaves as injection materials, and exploring the influence of temperature, inoculation seedling age and inoculation bacterial liquid concentration on the silencing efficiency of the peach TRV-VIGS and PNRSV-VIGS.
1. Construction of silencing vectors pTRV2-PpPDS and pCaRNA3-PpPDS
(1) Taking wild peach leaf as material, extracting total RNA with EASY spin Plus plant RNA rapid extraction kit (Aidlab, beijing), detecting RNA concentration and quality by Nanodrop one (Thermo, USA) and gel electrophoresis, and reverse transcription kitRT Reagent Kit with gDNA Eraser (TaKaRa, dalia, china) to obtain cDNA;
(2) Searching mRNA sequence of PpPDS in peach genome, designing primers (primer 1 and primer 2) by using the sequence as template and using Novozan high-fidelity enzymeAmplifying the CDS full-length sequence of the gene by using a Super-Fidelity DNA Polymerase (Vazyme, nanjin) kit, connecting the amplified CDS full-length sequence to a pEasy blast zero vector, transferring the amplified product into escherichia coli, and carrying out monoclonal positive detection to obtain a recombinant plasmid connected with the CDS sequence of PpPDS;
(3) Primer5 is used for designing a PpPDS specific fragment amplification primer (primer 3 and primer 4), and a sequence with the enzyme cutting site is added according to the enzyme cutting sites of different vectors (the enzyme cutting site of TRV2 is BamHI and SmaI; the enzyme cutting site of pCaRNA3 is XbaI), and the recombinant plasmid in (2) is used as a template, so that the Norvirjoy high-fidelity enzyme is obtainedThe 100bp fragment of the PpPDS gene is obtained by amplification of a Super-Fidelity DNA Polymerase (Vazyme, nanjing) kit; and constructing recombinant plasmids pTRV2-PpPDS and pCaRNA3-PpPDS by T4 ligase (Takara, beijing), picking up the correct monoclonal positive detection sequence, extracting the plasmids and transferring the plasmids into agrobacterium tumefaciens GV3101 by a heat shock method.
2. Preparation and transfection of agrobacterium tumefaciens bacterial liquid
(1) Respectively picking up and transferring target plasmids (pTRV 1, pTRV2-PpPDS and pCaRNA 1)&2. pCaRNA3 and pCaRNA 3-PpPDS) were inoculated into 1mL of a single colony of positive Agrobacterium containing 50 mg.L -1 Kanamycin (Kan) and 20mg.L -1 Rifampicin (Rif) in LB liquid medium at 28deg.C, 220r.min -1 Shake culturing for 12h;
(2) Then the bacterial liquid is transferred to 50mL containing 50 mg.L according to the ratio of 1:50 (v: v) -1 Kan and 20 mg.L -1 In LB liquid medium of Rif, 28 ℃,220 r.min -1 Shaking culture for 16h.
(3) Centrifuging the bacterial liquid at 4deg.C for 5min at 5000g, discarding supernatant, and adding 5mL MMA (10 mM MgCl) 2 150 μm AS,10mm mes, ph=5.6), and centrifuging again, discarding the supernatant, suspending with 5mL MMA, determining the OD value and adjusting the agrobacterium tumefaciens bacteria carrying the different plasmids with MMA to the target concentration AS required by the experiment;
(4) The bacterial solutions containing pTRV1 and pTRV2-PpPDS were prepared according to a ratio of 1:1 (v: v) mixing uniformly; pCaRNA1&2 was prepared in the same manner as the bacterial solutions of pCaRNA3 and pCaRNA3-PpPDS according to 1:1 (v: v) mixing uniformly, and standing at room temperature in a dark place for 2 hours to prepare a bacterial solution for VIGS conversion;
(5) Taking mature peach seedling leaves as materials of the bacterial liquid prepared in the step (4), and injecting the bacterial liquid into the peach leaves from the back surfaces of the peach seedling leaves by using a 1ml syringe without a needle head until the whole leaves are wetted;
(6) Culturing the injected peach seedlings for 2 days under weak light, and then placing the peach seedlings in a plant growth chamber under the conditions of 22 ℃ and 16h light/8 h darkness for continuous culture. Uniformly pouring an equal amount of 1-time liquid Hoagland nutrient solution according to requirements during the peach seedling culture period.
3. Concentration, temperature and age treatment of inoculated bacteria
Temperature: dividing the peach seedlings subjected to the agrobacterium tumefaciens injection into two groups, and respectively placing the two groups of peach seedlings at 22 ℃ and 28 ℃ for 16h illumination/8 h dark condition for culturing;
seedling age: dividing peach seedlings into 5-6 fully-developed leaves, 7-8 fully-developed leaves and 10-11 fully-developed leaves, and carrying out injection agrobacterium tumefaciens bacteria liquid treatment on 3 batches of seedling ages;
concentration of inoculum: the agrobacteria liquid in (3) in 2.3 was adjusted to OD about 0.6, 1.0 and 1.4 with MMA, respectively, and then tested according to steps (4) (5) and (6) in 2.3.
The test timing was used for phenotypic observation, and comparison of gene silencing effects was performed from the quantitative efficiency of the silencing plants, the phenotype of the silencing plants, and the relative expression levels of PpPDS. The relative expression level of the PpPDS gene is analyzed by adopting a qRT-PCR method, and the steps are as follows: selecting tissues of each part of pTRV2-PpPDS and pCaRNA3-PpPDS silent plants to extract RNA, reversely transcribing the RNA into cDNA, and calculating outside the PpPDS gene silent fragments to obtain a primer5 and a primer 6; ppTEF2 (Translation enlongation factor 2) (Tong et al 2009) was used as the reference gene (amplification primers: primer 7 and primer 8, 2) -ΔΔCt The method calculates to obtain the relative expression quantity of the PpPDS gene.
The sequences related to the genes are as follows:
CDS full length 1722bp, numbering: prupe.1G174100
ATGTCTCAGTGGGCTTGTGTCTCTGCTGCTAACTTGAGCTGTCAAGCTAGCATCATCAACACTCAAAAGCTACGAAACACTCCCAGATGCGATGCCTTTTCATTTAAAGGTAGTGAATTTATGGCTCAAAGCTGTAGATTTTTAAGCCCACAAACTATTTATGGAAGGCCGAGGAATGGTGCTTGCCCTTTGAAGGTGGTTTGCGTTGATTATCCAAGACCAGACCTTGACAATACTGCTAATTTCTTAGAAGCTGCATATTTCTCTTCCACTTTCCGAGCCTCTCCTCGTCCAGCTAAGCCGTTGAAGGTCGTGATTGCTGGTGCAGGTTTGGCTGGTCTGGCAACTGCAAAATATTTGGCTGATGCAGGTCATAAACCTATCTTACTGGAAGCAAGAGATGTTCTGGGCGGAAAGGTGGCAGCATGGAAAGATAAGGATGGAGACTGGTACGAAACAGGCCTACATATCTTCTTTGGGGCTTATCCGAATATTCAGAACCTGTTTGGTGAGCTTGGTATTGATGATCGATTGCAGTGGAAGGAGCATTCTATGATATTTGCAATGCCAAGCAAACCAGGAGAGTTCAGCCGGTTTGATTTCCCTGAAGTTTTACCAGCACCCTTAAATGGAATATGGGCCATATTGAAGAACAATGAGATGCTGACTTGGCCAGAGAAAATCAAGTTTGCAATTGGACTACTGCCAGCAATTCTTGGTGGGCAGGCTTATGTTGAAGCCCAAGATGGCTTGAGTGTAAAAGATTGGATGAGGAAACAGGGCATACCGGATCGAGTGACTACTGAGGTGTTTATTGCCATGTCAAAGGCCCTGAACTTTATTAACCCTGATGAACTTTCAATGCAATGCATATTGATTGCTTTGAACCGATTCCTTCAGGAGAAACACGGTTCCAAGATGGCTTTTTTGGATGGTAGTCCCCCTGAGAGACTCTGTGCACCAATTGTTGATCATATCCAGTCATTAGGCGGTGAAGTCCGAATTAATTCCCGAATACAGAGAATTGAGCTAAATAAAGATGGGACCGTGAAGAGTTTTGTACTAAATAATGGGAGCATGATTGAAGCAGATGCCTATGTATTCGCCACTCCAGTTGATATCCTAAAGCTTCTATTGCCTGATAACTGGAAAGAGATCCCATATTTCAAGAAATTGGAGAAACTGGTTGGCGTTCCAGTTATCAATGTTCACATATGGTTTGACAGAAAGCTGAAGAACACATATGATCATCTACTTTTTAGCAGAAGTCCTCTTTTAAGTGTCTATGCCGACATGTCCGTAACATGTAAGGAATATTACAATCCAAACCAGTCAATGCTGGAGTTGGTTTTTGCACCAGCAGAAGAATGGATATCATGCAGTGATTCAGAAATTATTGATGCTACACTCAAAGAACTTGCAAAACTCTTTCCAGATGAGATAGCTGTAGATCAAAGCAAAGCAAAGATTTTGAAGTACCATGTGGTGAAAACACCAAGGTCGGTTTACAAAACTGTACCAGGTTGTGAACCTTGCCGTCCCTTGCAAAGATCTCCCCTAGAGGGTTTCTATTTAGCTGGTGATTACACAAAACAAAAGTATTTAGCCTCAATGGAAGGTGCTGTTCTGTCAGGGAAACTTTGTGCACAAGCAATTGTACAGGATTACGAATTGCTTGTTGCTCGGGGACAAACAAGGGTGGCTGAGGCAAGCGTTCGGTGA
Primer 1:5'-ATGTCTCAGTGGGCTTGTGTCTCTG-3'
Primer 2:5'-TCACCGAACGCTTGCCTCAGCCACC-3'
2. Amplifying the PpPDS specific fragment, introducing forward and reverse primersXbaΙRestriction enzyme site (underlined; 100bp product)
Primer 3:5' -GCTCTAGAAGAAAGCTGAAGAACACAT-3’
Primer 4:5' -GCTCTAGAGATTGTAATATTCCTTACAT-3’
Primer for PpPDS quantification (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 FIG. 1, through agarose gel electrophoresis detection, FIG. 1 shows that the PCR amplification product of the PpPDS gene fragment has clear bands at about 1700bp, after recovery, sequencing verification is carried out, DNANAN software is used for comparison, and the sequence of the fragment is consistent with that of 1722bp of the PpPDS gene fragment, which shows that the cloning of the PpPDS gene fragment is successful.
As shown in FIG. 2, the pCaRNA3-PpPDS recombinant vector was transformed into Agrobacterium tumefaciens GV3101, and then the bacterial liquid plasmid of the positive clone was extracted. The pCaRNA3-PpPDS recombinant plasmid was amplified by PCR (primer 9 and primer 10). As detected by agarose gel electrophoresis, FIG. 2 shows that the band is about 375 and b p, which meets the expected aim, and proves that the pCaRNA3-PpPDS recombinant vector is successfully constructed. ( Primer 9:5'-GCTTCCCTAACGGGGCATCC-3'; primer 10:5'-AGGTCTTGGTTAGGGATTTG-3'. )
As shown in FIG. 3, the peach seedling plants are infected by adopting a leaf injection method, and the phenotype of pCaRNA3-PpPDS is obviously different from that of pTRV2-PpPDS after 25 days of infection. The phenotype of pCaRNA3-PpPDS presents large spots, and the new leaves always have albino phenotype, and the silent overall phenotype is obvious; the phenotype of pTRV2-PpPDS is a small thread, and only a few leaves are phenotyped, new leaves are not phenotyped any more, and the overall silencing effect is not obvious compared with pCaRNA 3-PpPDS.
As shown in fig. 4, leaves at different positions from the morphological lower end to the morphological upper end of peach seedlings have different phenotypes after leaf injection. In the figure, (1) injection leaf: represents injected leaf, (2) phenotypic leaf: leaves representing the appearance of phenotype, (3) phenotypically upper leaves: representing leaves above the phenotype where no phenotype appears. And (3) injection: the upper part of the pCaRNA3-PpPDS phenotypic leaves are phenotypic leaves, so that the phenotypic leaves are absent.
As shown in FIG. 5, in order to verify the effect of temperature on silencing efficiency, peach seedlings were cultured at 22℃and 28℃respectively in this experiment. The results indicate that both pCaRNA3-PpPDS and pTRV2-PpPDS are affected by temperature. The gene silencing efficiencies of pCaRNA3-PpPDS and pTRV2-PpPDS are 85% and 64% at 22 ℃; however, at 28 ℃, the gene silencing efficiency was reduced to 53% and 29%, respectively. Higher temperatures significantly reduce VIGS silencing efficiency. Previous studies showed (Wang Hongzhi, 2005) that the silencing efficiency of genes was significantly affected by the age of the seedling, and in order to determine the optimum age of the seedling for maximum silencing efficiency, the test set 3 treatments for 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 affected by the age of seedlings, and is 64%, 24% and 0% respectively; the gene silencing efficiency of pCaRNA3-PpPDS was high at 3 treatments, 85%, 80% and 94%, respectively. Thus, the pCaRNA1&2 and pCaRNA3 vectors are more suitable than pTRV1 and pTRV2 vectors for silencing endogenous genes in peach seedlings using agrobacterium-mediated VIGS technology.
As shown in FIG. 6, to verify the effect of different bacterial concentrations on the silencing efficiency of pCaRNA3-PpPDS gene, the OD was set separately in this experiment 600 ＝0.6，OD 600 =1.0 and OD 600 Total 3 treatments =1.4. The results showed that the gene silencing efficiencies were 61%, 55% and 88%, respectively, 3 weeks after injection; the gene silencing efficiencies are 85%, 95% and 92% respectively 4 weeks after injection, and no significant difference exists between the three.
As shown in fig. 7, leaf injection method was used to infect peach leaves. After 25 days of infection, qRT-PCR analysis was performed on pTRV2 empty vector plants and pTRV2-PpPDS plant leaves, stems and roots with pTEF2 (Translation enlongation factor) as an internal reference gene and the gene expression was analyzed with 3 replicates per treatment (Tong et al 2009). The results show that compared with pTRV2 control, the expression quantity of the phenotypically upper leaf, the phenotypically lower leaf, the injected leaf, the stem and the root of the pTRV2-PpPDS plant is obviously reduced, and the albino phenotype of the pTRV2-PpPDS plant is caused by the silencing of the PpPDS gene and the silencing effect is obvious.
As shown in fig. 8, leaf injection method was used to infect peach leaves. After 25 days of infection, qRT-PCR analysis was performed on each part of pCaRNA3 empty vector plants and pCaRNA3-PpPDS plants using pCaRNA3 empty vector plants as a control, 3 replicates were taken per treatment, and the gene expression level was analyzed using PpTEF2 (Translation enlongation factor 2) (Tong et al 2009) as an internal reference gene. The results show that compared with pCaRNA3 control, the expression quantity of phenotype leaf, phenotype lower leaf, injection leaf, stem and root of pCaRNA3-PpPDS plant is obviously reduced, and the albino phenotype of pCaRNA3-PpPDS plant is proved to be caused by PpPDS gene silencing, and the silencing effect is obvious.
Example 2
The silencing effect was tested in the same manner as in example 1, using PpERF98 as the target gene.
As shown in fig. 9, leaf injection method was used to infect peach leaves. qRT-PCR analysis was performed on pCaRNA3 empty vector plants and pCaRNA3-PpERF98 plants, using pCaRNA3 empty vector plants as a control, 25 days after infection. The test set up 5 biological replicates, designated pCaRNA3-PpERF98#1, pCaRNA3-PpERF98#2, pCaRNA3-PpERF98#3, pCaRNA3-PpERF98#4 and pCaRNA3-PpERF98#5, respectively. qRT-PCR analysis was performed on the expression level of the target gene PpERF98 using PpTEF2 (Translationenlongation factor 2) (Tong et al 2009) as a reference gene. The results showed that the expression levels of pCaRNA3-PpERF98 plants were significantly reduced compared to the pCaRNA3 control.
The sequence listing related to the genes is as follows:
1. the CDS full length 492bp, numbered: prupe.1G037800
ATGGAAGACCCTCGTAGAGGAAAGGATCTACAGAAGCAAGGAATGGAAGAGAAGGGAAGCGAAGAAGTGCGTTATCGGGGAGTCCGAAGGAGGCCATGGGGGAAGTTTGCTTCTGAGATAAGAGACCCTTCAAGGCAAGGTGCTCGTCTGTGGCTTGGCACATTTGATACGGCTGAAGAAGCTGCAAGGGCTTATGACAGAGCTGCTTTTAATTTAAGGGGTCATCTTGCCATACTCAACTTTCCAAATGAGTATTATTCTCAAGTTATGGGCTCTCCTCCTCATCCTCCTAGATTCTCATCATCATTTTCTTCTTCTTCTTCTTCTTCATCTTCTTCTTCTTCTTCTTCTTCTGCTGCTGCTGCTGCTGCTGCTCCTGGTGGAAGTTCATCTACTGGACAAGGAAGGCAAGTCTTTGAGATTGAGTATTTGGATGACCATGTGTTGGAGGACCTTCTTGATTCTCAAGAGGAGAAAAACAAACGGAAGTGA
2. VIGS interference fragment amplification (product 100 bp)
PpERF98-F：5’-GCTCTAGAATGGAAGACCCTCGTAGAGG-3’
PpERF98-R：5’-GCTCTAGACCCATGGCCTCCTTCGGA-3’
3. Quantitative primer: (product 115 bp)
PpERF98-F1：5’-AAATGTGCCAAGCCACAGAC-3’
PpERF98-R1：5’-GAAGGGAAGCGAAGAAGTGC-3’
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> university of agriculture in China
<120> efficient peach leaf gene silencing method based on VIGS
<140> 2021109064174
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1722
<212> DNA
<213> peach (Prunus persica)
<400> 1
atgtctcagt gggcttgtgt ctctgctgct aacttgagct gtcaagctag catcatcaac 60
actcaaaagc tacgaaacac tcccagatgc gatgcctttt catttaaagg tagtgaattt 120
atggctcaaa gctgtagatt tttaagccca caaactattt atggaaggcc gaggaatggt 180
gcttgccctt tgaaggtggt ttgcgttgat tatccaagac cagaccttga caatactgct 240
aatttcttag aagctgcata tttctcttcc actttccgag cctctcctcg tccagctaag 300
ccgttgaagg tcgtgattgc tggtgcaggt ttggctggtc tggcaactgc aaaatatttg 360
gctgatgcag gtcataaacc tatcttactg gaagcaagag atgttctggg cggaaaggtg 420
gcagcatgga aagataagga tggagactgg tacgaaacag gcctacatat cttctttggg 480
gcttatccga atattcagaa cctgtttggt gagcttggta ttgatgatcg attgcagtgg 540
aaggagcatt ctatgatatt tgcaatgcca agcaaaccag gagagttcag ccggtttgat 600
ttccctgaag ttttaccagc acccttaaat ggaatatggg ccatattgaa gaacaatgag 660
atgctgactt ggccagagaa aatcaagttt gcaattggac tactgccagc aattcttggt 720
gggcaggctt atgttgaagc ccaagatggc ttgagtgtaa aagattggat gaggaaacag 780
ggcataccgg atcgagtgac tactgaggtg tttattgcca tgtcaaaggc cctgaacttt 840
attaaccctg atgaactttc aatgcaatgc atattgattg ctttgaaccg attccttcag 900
gagaaacacg gttccaagat ggcttttttg gatggtagtc cccctgagag actctgtgca 960
ccaattgttg atcatatcca gtcattaggc ggtgaagtcc gaattaattc ccgaatacag 1020
agaattgagc taaataaaga tgggaccgtg aagagttttg tactaaataa tgggagcatg 1080
attgaagcag atgcctatgt attcgccact ccagttgata tcctaaagct tctattgcct 1140
gataactgga aagagatccc atatttcaag aaattggaga aactggttgg cgttccagtt 1200
atcaatgttc acatatggtt tgacagaaag ctgaagaaca catatgatca tctacttttt 1260
agcagaagtc ctcttttaag tgtctatgcc gacatgtccg taacatgtaa ggaatattac 1320
aatccaaacc agtcaatgct ggagttggtt tttgcaccag cagaagaatg gatatcatgc 1380
agtgattcag aaattattga tgctacactc aaagaacttg caaaactctt tccagatgag 1440
atagctgtag atcaaagcaa agcaaagatt ttgaagtacc atgtggtgaa aacaccaagg 1500
tcggtttaca aaactgtacc aggttgtgaa ccttgccgtc ccttgcaaag atctccccta 1560
gagggtttct atttagctgg tgattacaca aaacaaaagt atttagcctc aatggaaggt 1620
gctgttctgt cagggaaact ttgtgcacaa gcaattgtac aggattacga attgcttgtt 1680
gctcggggac aaacaagggt ggctgaggca agcgttcggt ga 1722
<210> 2
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atgtctcagt gggcttgtgt ctctg 25
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tcaccgaacg cttgcctcag ccacc 25
<210> 4
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
gctctagaag aaagctgaag aacacat 27
<210> 5
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gctctagaga ttgtaatatt ccttacat 28
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ccttgcaaag atctccccta 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
cgagcaacaa gcaattcgta 20
<210> 8
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
agcaagtcac ccaacaagca ta 22
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
ccaaccaaac tcttcagcca at 22
<210> 10
<211> 492
<212> DNA
<213> peach (Prunus persica)
<400> 10
atggaagacc ctcgtagagg aaaggatcta cagaagcaag gaatggaaga gaagggaagc 60
gaagaagtgc gttatcgggg agtccgaagg aggccatggg ggaagtttgc ttctgagata 120
agagaccctt caaggcaagg tgctcgtctg tggcttggca catttgatac ggctgaagaa 180
gctgcaaggg cttatgacag agctgctttt aatttaaggg gtcatcttgc catactcaac 240
tttccaaatg agtattattc tcaagttatg ggctctcctc ctcatcctcc tagattctca 300
tcatcatttt cttcttcttc ttcttcttca tcttcttctt cttcttcttc ttctgctgct 360
gctgctgctg ctgctcctgg tggaagttca tctactggac aaggaaggca agtctttgag 420
attgagtatt tggatgacca tgtgttggag gaccttcttg attctcaaga ggagaaaaac 480
aaacggaagt ga 492
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
aaatgtgcca agccacagac 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gaagggaagc gaagaagtgc 20
Claims (3)
1. The high-efficiency peach leaf gene silencing method based on the VIGS is characterized by comprising the following steps of:
step 1, constructing pCaRNA 3-target gene recombinant plasmid, and transferring into agrobacterium tumefaciens to obtain positive agrobacterium transferred into the recombinant plasmid, wherein the target gene is PpPDS gene or PpERF98 gene;
the nucleotide sequence of the PpPDS gene is as follows:
ATGTCTCAGTGGGCTTGTGTCTCTGCTGCTAACTTGAGCTGTCAAGCTAGCATCATCAACACTCAAAAGCTACGAAACACTCCCAGATGCGATGCCTTTTCATTTAAAGGTAGTGAATTTATGGCTCAAAGCTGTAGATTTTTAAGCCCACAAACTATTTATGGAAGGCCGAGGAATGGTGCTTGCCCTTTGAAGGTGGTTTGCGTTGATTATCCAAGACCAGACCTTGACAATACTGCTAATTTCTTAGAAGCTGCATATTTCTCTTCCACTTTCCGAGCCTCTCCTCGTCCAGCTAAGCCGTTGAAGGTCGTGATTGCTGGTGCAGGTTTGGCTGGTCTGGCAACTGCAAAATATTTGGCTGATGCAGGTCATAAACCTATCTTACTGGAAGCAAGAGATGTTCTGGGCGGAAAGGTGGCAGCATGGAAAGATAAGGATGGAGACTGGTACGAAACAGGCCTACATATCTTCTTTGGGGCTTATCCGAATATTCAGAACCTGTTTGGTGAGCTTGGTATTGATGATCGATTGCAGTGGAAGGAGCATTCTATGATATTTGCAATGCCAAGCAAACCAGGAGAGTTCAGCCGGTTTGATTTCCCTGAAGTTTTACCAGCACCCTTAAATGGAATATGGGCCATATTGAAGAACAATGAGATGCTGACTTGGCCAGAGAAAATCAAGTTTGCAATTGGACTACTGCCAGCAATTCTTGGTGGGCAGGCTTATGTTGAAGCCCAAGATGGCTTGAGTGTAAAAGATTGGATGAGGAAACAGGGCATACCGGATCGAGTGACTACTGAGGTGTTTATTGCCATGTCAAAGGCCCTGAACTTTATTAACCCTGATGAACTTTCAATGCAATGCATATTGATTGCTTTGAACCGATTCCTTCAGGAGAAACACGGTTCCAAGATGGCTTTTTTGGATGGTAGTCCCCCTGAGAGACTCTGTGCACCAATTGTTGATCATATCCAGTCATTAGGCGGTGAAGTCCGAATTAATTCCCGAATACAGAGAATTGAGCTAAATAAAGATGGGACCGTGAAGAGTTTTGTACTAAATAATGGGAGCATGATTGAAGCAGATGCCTATGTATTCGCCACTCCAGTTGATATCCTAAAGCTTCTATTGCCTGATAACTGGAAAGAGATCCCATATTTCAAGAAATTGGAGAAACTGGTTGGCGTTCCAGTTATCAATGTTCACATATGGTTTGACAGAAAGCTGAAGAACACATATGATCATCTACTTTTTAGCAGAAGTCCTCTTTTAAGTGTCTATGCCGACATGTCCGTAACATGTAAGGAATATTACAATCCAAACCAGTCAATGCTGGAGTTGGTTTTTGCACCAGCAGAAGAATGGATATCATGCAGTGATTCAGAAATTATTGATGCTACACTCAAAGAACTTGCAAAACTCTTTCCAGATGAGATAGCTGTAGATCAAAGCAAAGCAAAGATTTTGAAGTACCATGTGGTGAAAACACCAAGGTCGGTTTACAAAACTGTACCAGGTTGTGAACCTTGCCGTCCCTTGCAAAGATCTCCCCTAGAGGGTTTCTATTTAGCTGGTGATTACACAAAACAAAAGTATTTAGCCTCAATGGAAGGTGCTGTTCTGTCAGGGAAACTTTGTGCACAAGCAATTGTACAGGATTACGAATTGCTTGTTGCTCGGGGACAAACAAGGGTGGCTGAGGCAAGCGTTCGGTGA；
the nucleotide sequence of the ppenf 98 gene is:
ATGGAAGACCCTCGTAGAGGAAAGGATCTACAGAAGCAAGGAATGGAAGAGAAGGGAAGCGAAGAAGTGCGTTATCGGGGAGTCCGAAGGAGGCCATGGGGGAAGTTTGCTTCTGAGATAAGAGACCCTTCAAGGCAAGGTGCTCGTCTGTGGCTTGGCACATTTGATACGGCTGAAGAAGCTGCAAGGGCTTATGACAGAGCTGCTTTTAATTTAAGGGGTCATCTTGCCATACTCAACTTTCCAAATGAGTATTATTCTCAAGTTATGGGCTCTCCTCCTCATCCTCCTAGATTCTCATCATCATTTTCTTCTTCTTCTTCTTCTTCATCTTCTTCTTCTTCTTCTTCTTCTGCTGCTGCTGCTGCTGCTGCTCCTGGTGGAAGTTCATCTACTGGACAAGGAAGGCAAGTCTTTGAGATTGAGTATTTGGATGACCATGTGTTGGAGGACCTTCTTGATTCTCAAGAGGAGAAAAACAAACGGAAGTGA；
step 2, preparing a VIGS transformed bacterial solution by using positive agrobacterium transferred into a recombinant plasmid, specifically preparing a first bacterial solution by using positive agrobacterium transferred into the recombinant plasmid, preparing a second bacterial solution by using positive agrobacterium transferred into pCaRNA1&2, adjusting the concentration of the first bacterial solution to be consistent with that of the second bacterial solution, and mixing the first bacterial solution and the second bacterial solution according to a volume ratio of 1:1, uniformly mixing, and standing for 2 hours at room temperature in a dark place to prepare a VIGS-transformed bacterial solution;
injecting the VIGS-transformed bacterial liquid into the wild peach leaves by using a syringe without a needle; the peach plants in the step 2 have 5-6 fully-unfolded leaves; the concentration of the VIGS transformed bacteria solution is OD 600 = 1.0;
and 3, culturing the injected peach seedlings for 2 days under weak light, and then placing the peach seedlings in a plant growth room under the conditions of (22+/-1) DEG C and 16h illumination/8 h darkness for continuous culture, and irrigating 1 time of Hoagland nutrient solution during the peach seedling culture period to obtain wild peach plants for silencing target genes, wherein the temperature of the continuous culture in the step 3 is 22 ℃.
2. The VIGS-based high-efficiency peach leaf gene silencing method according to claim 1, wherein the step 1 specifically comprises the steps of:
step 1.1, extracting total RNA of peach leaves, and carrying out reverse transcription to obtain cDNA;
step 1.2, searching mRNA sequence of a target gene in a peach genome, designing a primer by taking the sequence as a template, amplifying a nucleotide sequence of the gene, connecting the amplified nucleotide sequence to a pEasy batch zero vector, transferring the amplified nucleotide sequence into escherichia coli, and selecting a monoclonal positive test to obtain a recombinant plasmid connected with the nucleotide sequence of the target gene;
step 1.3, amplifying a primer by utilizing the target gene specific fragment, adding sequences of enzyme cutting sites at two ends of the primer, and amplifying by taking the recombinant plasmid in the step 1.2 as a template to obtain a target gene fragment; and constructing a recombinant plasmid pCaRNA 3-target gene by using T4 ligase, picking up the correct single clone positive detection sequence, extracting the plasmid and transferring the plasmid into agrobacterium tumefaciens by a heat shock method.
3. The VIGS-based high-efficiency peach leaf gene silencing method according to claim 1, wherein the step 2 specifically comprises the steps of:
step 2.1, inoculating together the recombinant plasmid-transferred positive Agrobacterium single colony to 1mL containing 50mg.L -1 Kanamycin and 20 mg.L -1 Rifampicin LB liquid medium at 28 ℃ and 220 r.min -1 Shake culturing for 12h under the condition;
step 2.2, transferring the bacterial liquid obtained in the last step to 50mL containing 50 mg.L according to the volume ratio of 1:50 -1 Kanamycin and 20 mg.L -1 Rifampicin LB liquid medium at 28 ℃ and 220 r.min -1 Shake culturing for 16h under the condition;
step 2.3, centrifuging 5000g of the bacterial liquid obtained in the last step at 4 ℃ for 5min, discarding the supernatant, and then using 5 mM MA containing 10mM MgCl 2 150 μM AS and 10mm mes, ph=5.6; centrifuging again after suspending, discarding supernatant, suspending with 5mL MMA, measuring OD value, and regulating concentration of bacterial liquid with MMA to obtain first bacterial liquid;
step 2.4, preparing positive agrobacterium transferred into pCaRNA1&2, and preparing a second bacterial liquid with the same concentration as the first bacterial liquid by using the positive agrobacterium transferred into pCaRNA1&2 and adopting the method of step 2.1-step 2.3;
step 2.5, the first bacterial liquid and the second bacterial liquid are mixed according to the volume ratio of 1:1, uniformly mixing, and standing for 2 hours at room temperature in a dark place to prepare a VIGS-transformed bacterial solution;
and 2.6, injecting the VIGS-transformed bacterial liquid into the peach leaves from the back of the peach leaves until the whole leaves are wetted, and injecting all the fully-unfolded leaves of the peach seedlings.
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| CN116555321A (en) * | 2022-09-21 | 2023-08-08 | 华中农业大学 | Application of PpERF98 gene in regulation and control of gummosis resistance of peach |
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| An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene;Hongguang Cui等;Plant Biotechnology Journal;第344-356页 * |
| 扁桃AcCBF1基因VIGS载体构建与功能分析;宋恬;田嘉;李鹏;刘梦婕;张琦;郭长奎;李疆;;果树学报(第04期);第421-429页 * |
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