CN112126646B - sgRNA, vector, host cell and method for resisting grape leaf curl virus-3 of plant - Google Patents
sgRNA, vector, host cell and method for resisting grape leaf curl virus-3 of plant Download PDFInfo
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- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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Abstract
The invention relates to the technical field of genetic engineering, in particular to sgRNA, a vector, a host cell and a method for resisting grape leaf curl virus-3 of plants. The upstream primer sequence of the sgRNA sequence is gattGGTGTTGATCAATTCCGTT shown in SEQ ID No. 2; the sequence of a downstream primer of the sgRNA sequence is cgagAACGGAATTGATCAACACC shown in SEQ ID No. 3. The sgRNA can be used for reducing the virus content of the grape leaf roll virus-3 in a plant body and inhibiting the replication of the grape leaf roll virus-3, so that the capability of the plant for resisting the grape leaf roll virus-3 is improved.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to sgRNA, a vector, a host cell and a method for resisting grape leaf curl virus-3 of plants.
Background
Grape leaf roll virus-3 (grape leaf-associated virus 3, GLRaV-3) can cause grape leaf roll disease (grape leaf roll disease), so that the growth vigor of grape plants is weakened, the root system is dysplastic, the stress resistance is weakened, and the grapes are easy to be frozen; the survival rate of branch and vine grafting is obviously reduced, and the rooting capability is poor.
Thus, there is a need for a strategy that would render plants resistant to grape leaf curl-3 virus.
Disclosure of Invention
The invention provides a sgRNA, a vector, a host cell and a method for resisting grape leaf roll virus-3 of a plant, which can be used for improving the resistance of grapes to the grape leaf roll virus-3.
In a first aspect, embodiments of the present invention provide sgrnas for plant resistance to grapevine leafroll virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGGTGTTGATCAATTCCGTT shown in SEQ ID No. 2; the sequence of a downstream primer of the sgRNA sequence is cgagAACGGAATTGATCAACACC shown in SEQ ID No. 3.
In a second aspect, embodiments of the present invention provide sgrnas for use in plants against grapevine leafroll virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGCTAAGGAATAGAGGGCTG shown in SEQ ID No. 5; the sequence of a downstream primer of the sgRNA sequence is cgagCAGCCCTCTATTCCTTAGC shown in SEQ ID No. 6.
In a third aspect, the embodiments of the present invention provide a sgRNA for plant resistance to grapevine leafroll virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGTCGTCTTCGTACTAGCAG shown in SEQ ID No. 8; the sequence of a downstream primer of the sgRNA sequence is cgagCTGCTAGTACGAAGACGAC shown in SEQ ID No. 9.
In a fourth aspect, embodiments of the invention provide sgrnas for plant resistance to grapevine leafroll virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGTCCTTAACGTATTTTAAG shown in SEQ ID No. 11; the sequence of a downstream primer of the sgRNA sequence is cgagCTTAAAATACGTTAAGGAC shown in SEQ ID No. 12.
In a fifth aspect, embodiments of the invention provide sgrnas for plant resistance to grapevine leafroll virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGGAGCTTTGCTGTAACCTC shown in SEQ ID No. 14; the sequence of a downstream primer of the sgRNA sequence is cgagGAGGTTACAGCAAAGCTCC shown in SEQ ID No. 15.
In any one of the first to fifth aspects, the plant is a grape. The grape can be Cabernet Sauvignon variety grape, and also can be other varieties of grape.
In a sixth aspect, the embodiments of the present invention provide a recombinant vector for resisting grape leaf roll virus-3 in a plant, including the sgRNA of any one of the first to fifth aspects.
In a seventh aspect, embodiments of the invention provide a host cell comprising the sgRNA of any one of the first to fifth aspects or the recombinant vector of the sixth aspect.
In an eighth aspect, an embodiment of the present invention provides a method for improving a plant resistance to grape leaf curl virus-3, including the following steps:
preparing a recombinant expression vector provided by the sixth aspect to transform agrobacterium;
transforming a plant of said plant with agrobacterium containing said recombinant expression vector to obtain a transfected plant with an increased resistance against GLRaV-3.
In a ninth aspect, the embodiments of the present invention provide the use of a recombinant vector comprising, but not limited to, the recombinant vector of the sixth aspect for breeding anti-grapevine leaf roll virus-3 plants.
In a tenth aspect, the embodiments of the present invention provide a method for improving the resistance of a plant to grape leaf curl virus-3, the method comprising the following steps:
immersing a plant to be transfected in a closed container by using agrobacterium liquid containing the recombinant vector provided by the sixth aspect;
and vacuumizing the closed container, maintaining the vacuum degree of the closed container at 0.085MPa for 20 minutes, then restoring to normal atmospheric pressure, and repeating for 3 times to obtain the transfected plants.
In some embodiments, the OD of the agrobacterium liquid600The value was 1, the As concentration was 200mM, and the pH was 5.8.
The sgRNA, the vector and the host cell designed by the invention can be used for specifically clearing virus RNA in a body in a plant-guided FnCas9 protein, and can effectively improve the capability of the plant in resisting grape leaf roll virus-3, thereby reducing or avoiding the influence of grape leaf roll disease on grape planting.
Drawings
FIG. 1 is a schematic diagram of the genome structure of grape leaf roll virus-3;
FIG. 2 is a diagram of the structure of pCambia1300-sgRNA-FnCas9 vector provided in the examples of the present specification;
FIG. 3a is a schematic diagram of expression level of FnCas9 in transfected plants;
FIG. 3b is a graph showing the amount of virus of grape leaf curl-3 in transfected plants;
FIG. 4 is an appearance of the transfected plants and the control group.
Detailed Description
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
A repetitive sequence in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome, which belongs to the immune system of bacteria, can eliminate viral DNA integrated into the bacterial genome or can cleave viral RNA in bacteria to inhibit viral replication. In the embodiments of the present disclosure, a sgRNA and a vector containing the sgRNA are provided, which can effectively cleave RNA of grape leaf roll virus-3 in a cell of a plant, thereby inhibiting replication of grape leaf roll virus-3 and reducing the content of grape leaf roll virus-3 in the plant.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BI OLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHOD IN ENZYMOLOGY, Vol.304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The experimental material sources used in the examples of this specification are as follows.
Healthy Cabernet Sauvignon 'grapes and GLRaV-3 infected Cabernet Sauvignon' grapes under the tissue culture system are from the gardening academy of northwest agriculture and forestry science university and the national key laboratory of crop biology in dry regions.
pCambia1300-FnCas9 vector-from southern China university of agriculture.
RNA extraction kit (R6827-01) -OMEGA.
Reverse transcription kit (AT311-02) -Transgen.
TransStart Green qPCR SuperMix (AQ101-02) -Transgen.
Example 1 target selection
The sequence length of the virus genome of the grape leaf curl virus-3 is 18026 bp. As shown in FIG. 1, the viral genome of grape leaf roll virus-3 has 12 open reading frames. Table 1 shows the function of these 12 open reading frames. Among them, five open reading frames, p5(5kDa protein), Hsp70h (reactive heat shock protein 70), Hsp90h (reactive heat shock protein 90), CP (coat protein), CPm (minor coat protein), correspond to the conserved domain of the virus. These five open reading frames were selected as targets.
TABLE 1
Example 2 sgRNA design
The sgRNA was designed according to the target sequence, and the specific sequence is shown in table 2. The design principle and method are as follows.
(1) And (3) selecting a target sequence: unlike SpCas9, sgRNA of FnCas9 does not need to look for NGG sequences;
(2) selecting an N18(18 bases) C sequence (N represents four bases of A, T, G and C), and taking a total of 19 base sequences as a designed sgRNA sequence;
(3) CGAGN18C was used as a reverse primer, and GATTGN18(GN18 is the reverse complement of the designed sgRNA) was used as a forward primer.
TABLE 2 target selection and sgRNA design
Example 3 transfection experiment
The transfection experiment included the following steps.
1. Mix 100. mu. mol forward and reverse primers 10. mu.L each, ddH2Fusing O80 μ L at 98 deg.C for 5 min;
2. the vector only contains one BsaI restriction enzyme cutting site, the vector pCambia1300-FnCas9 is subjected to enzyme cutting by using BsaI restriction enzyme, water bath is carried out at 37 ℃ for 20min, and the solution is recovered;
3. adding samples according to the sequence of 5 mu L of the treated sgRNA, 3 mu L of the vector after enzyme digestion, 1 mu L of T4 ligase and 1 mu L of T4Buffer, and fusing the sgRNA and the enzyme digestion vector;
4. coli (Top10 strain): adding the plasmid into 25 mu L of escherichia coli competence, blowing and mixing uniformly, carrying out ice bath for 30min, carrying out heat shock for 90s, carrying out ice bath for 5min, adding 200 mu L of LB solution into the solution, recovering for 30min by using a shaking table (180rpm) at 37 ℃, uniformly coating the bacterial solution on LB solid culture medium with 50mg/L Kan resistance in a sterile environment, and carrying out inverted culture in a 37 ℃ culture box. Picking single spots, extracting plasmids and finally obtaining pCambia1300-sgRNA-FnCas9 plasmids or pCambia1300-sgRNA-FnCas9 vectors; the structure of the pCambia1300-sgRNA-FnCas9 vector is shown in FIG. 2.
5. Agrobacterium (GV3101 strain) transformation: transformation of the plasmid into Agrobacterium: and (3) taking the plasmid on ice, adding 25 mu L of competent cells, blowing and beating the competent cells uniformly, carrying out ice bath for 30min, freezing the competent cells for 1min by liquid nitrogen, carrying out water bath at 37 ℃ for 5min, adding 200 mu L of LB solution, recovering the competent cells for 3h by using a shaking table at 28 ℃, uniformly coating the bacterial solution on LB plates with 50mg/L Kan resistance, Rif resistance and Gent resistance in a sterile environment, and carrying out inverted culture in an incubator at 28 ℃. Selecting a single spot, adding 5mL of LB solution, placing on a shaking table at 28 ℃, shaking at 180rpm for 16-18h, sucking bacterial liquid and 30% glycerol, respectively weighing 800 mu L, mixing to obtain bacterial liquid for experiments, and placing in a refrigerator at-80 ℃ for storage;
6. the Empty vector pCambia1300-FnCas9 (Empty) and the Agrobacterium strain solution 100uL of the five constructed vectors pCambia1300-sgRNA-FnCas9 (designated by sgRNA-ID shown in Table 2: A, B, C, D, E) were aspirated, 5mL of LB solution (with Kan, Gent, Rif resistance) were added, and the mixture was shaken at 180rpm on a shaker at 28 ℃ for 16-18h to complete the primary activation. Taking 100-200 mu L of activated bacteria liquid, adding the activated bacteria liquid into a liquid LB culture medium containing the same antibiotics again, and shaking the bacteria for 16-18h to complete secondary activation of the bacteria liquid; wherein, the constructed pCambia1300-sgRNA-FnCas9 can be abbreviated as FnCas 9;
7.5000rpm for 10min, discard the supernatant, add the resuspension solution (2.130g/L MES +2.033g/L MgCl)2+200mM As 4mL/L, pH 5.8), and repeating the step;
8. dilute resuspension bacterial solution to OD600Standing for 3-4h under the condition of keeping out of the sun for activating the bacterial liquid, wherein the weight is 1.0;
9. selecting healthy Cabernet Sauvignon 'tissue culture seedlings cultured for 6 weeks and GLRaV-3 infected Cabernet Sauvignon' tissue culture seedlings (cultured on 1/2MS culture medium), and performing the following operations under aseptic conditions;
10. immersing the infected plants with the treated bacterial liquid, vacuumizing, maintaining the vacuum degree of 0.085MPa for 20min, recovering (i.e. removing the vacuumizing and restoring to normal atmospheric pressure) for 5min, repeating the step for 3 times, and pouring out the bacterial liquid;
11. culturing in dark place for 12h, cutting single bud stem, eluting with eluent (MS 2.215g/L + sucrose 20g/L +450mg/L Cef +250mg/L Carb) for 2-3 times (3 min each time), and gently washing with sterile water for 2-3 times (3 min each time). The leaf and stem sections were adsorbed with sterile filter paper, air dried slightly, and cultured on 4% PEG solid medium (MS 2.215g/L + sucrose 30g/L +7g/L agar +40g/L PEG8000+200mg/L Carb).
The transformation scheme provided in example 3 is a high-efficiency whole grape living body transient transformation system. The transient transformation system can ensure that the vector can be continuously and stably expressed in the plant for 2 weeks to 1 month, thereby realizing the elimination of the grape leaf curl virus-3 in the plant body.
Example 4 detection of the expression level of FnCas 9and the viral content of grape leaf curl virus-3 in plants
Comprises the following steps.
1. Samples were taken 5d after the incubation treatment in step 11 of example 3 for RT-qPCR fluorescence quantification using the primers shown in Table 3.
TABLE 3 RT-qPCR primers
2. Grape leaf RNA was extracted using an RNA extraction kit (R6827-01) from OMEGA according to the instruction manual. The concentration of RNA was measured using Nandrop 2000. The integrity of RNA was checked by electrophoresis using 0.5% agarose gel, and the qualified grape leaf RNA was detected by reverse transcription and cDNA synthesis using the instructions of the reverse transcription kit (AT311-02) from Transgen. The concentration of the cDNA was measured using Nandrop 2000.
3. Real-time quantitative PCR analysis was performed with reference to the instructions of TransStart Green qPCR SuperMix (AQ101-02) from Transgen. The reaction system is shown in Table 4.
TABLE 4
At 95 deg.C for 2 min; 95 ℃ for 10s, 60 ℃ for 10s,72 ℃ for 30s, 40 cycles. And calculating the relative expression quantity of the target gene by using Actin as an internal reference and adopting 2-delta-Delta Ct.
The result of detecting the expression level of FnCas9 is shown in FIG. 3a, and the result of detecting the virus level of grape leaf curl virus-3 is shown in FIG. 3 b.
Wherein, health: healthy plants;
mock: plants infected with GLRaV-3;
FnCas 9-ns: infecting GLRaV-3 plants after being treated by non-specific pCambia1300-FnCas9 bacterial liquid;
FnCas 9-A: infecting GLRaV-3 plants treated by pCambia1300-FnCas9 bacterial liquid with a target spot of p 5;
FnCas 9-B: infecting GLRaV-3 plants after being treated by pCambia1300-FnCas9 bacterial liquid with a target spot of Hsp70 h;
FnCas 9-C: infecting GLRaV-3 plants after being treated by pCambia1300-FnCas9 bacterial liquid with a target spot of Hsp90 h;
FnCas 9-D: infected GLRaV-3 plant treated by pCambia1300-FnCas9 bacterial liquid with CP target point
FnCas 9-E: infecting a GLRaV-3 plant after being treated by pCambia1300-FnCas9 bacterial liquid with a target spot of CPm;
fig. 3a and 3b show that the FnCas9 gene expression of different vectors is significantly different, but can be stably expressed. Compared with the control, the GLRaV-3 content is remarkably reduced by the treatment except the FnCas9-C treatment, wherein the FnCas9-D (Target-CP) treatment efficiency is the highest, and the FnCas9-E (Target-CPm) inhibits the virus less efficiently.
Example 5 appearance of grape plants
1. After 15d of the culture treatment in step 11 of example 3, a significant difference in symptoms appeared. Specifically, as shown in FIG. 4, the stem segments of healthy plants appeared green and showed no red leaf symptoms. The areas of redness in the leaves were small or even absent in the FnCas9-D (Target-CP) and FnCas9-E (Target-CPm) compared to the susceptible controls Mock and FnCas 9-ns. The sgRNA, the vector and the host cell provided by the embodiment of the specification are shown to effectively inhibit the content of the virus, and greatly relieve the red leaf symptom of the grape leaf curl virus-3 on the cabernet sauvignon'.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
<120> sgRNA, vector, host cell and method for plant to resist grape leaf curl virus-3
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Claims (9)
1. An sgRNA for grape anti-grape leaf roll virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGGTGTTGATCAATTCCGTT shown in SEQ ID No. 2; the sequence of a downstream primer of the sgRNA sequence is cgagAACGGAATTGATCAACACC shown in SEQ ID No. 3.
2. A sgRNA for grape anti-grape leaf curl virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGCTAAGGAATAGAGGGCTG shown in SEQ ID No. 5; the sequence of a downstream primer of the sgRNA sequence is cgagCAGCCCTCTATTCCTTAGC shown in SEQ ID No. 6.
3. A sgRNA for grape anti-grape leaf curl virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGTCGTCTTCGTACTAGCAG shown in SEQ ID No. 8; the sequence of a downstream primer of the sgRNA sequence is cgagCTGCTAGTACGAAGACGAC shown in SEQ ID No. 9.
4. An sgRNA for grape anti-grape leaf roll virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGTCCTTAACGTATTTTAAG shown in SEQ ID No. 11; the sequence of a downstream primer of the sgRNA sequence is cgagCTTAAAATACGTTAAGGAC shown in SEQ ID No. 12.
5. A sgRNA for grape anti-grape leaf curl virus-3; wherein the upstream primer sequence of the sgRNA sequence is gattGGAGCTTTGCTGTAACCTC shown in SEQ ID No. 14; the sequence of a downstream primer of the sgRNA sequence is cgagGAGGTTACAGCAAAGCTCC shown in SEQ ID No. 15.
6. A recombinant vector for grape anti-grape leaf curl virus-3, comprising the sgRNA of any one of claims 1-5.
7. A host cell containing the sgRNA of any one of claims 1 to 5 or the recombinant vector of claim 6.
8. A method for improving the resistance of grapes to grape leaf curl virus-3, which is characterized by comprising the following steps:
preparing an agrobacterium solution containing the recombinant vector of claim 6;
and transforming the grape plant by using agrobacterium liquid containing the recombinant expression vector to obtain a transfected plant, and obtaining the transfected plant with improved anti-GLRaV-3 capability from the transfected plant.
9. The method of claim 8, wherein the OD of the Agrobacterium solution600The value was 1, the As concentration was 200mM, and the pH was 5.8.
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