CN114634947B - Method for inducing cotton PR5 gene silencing by TRV virus and application thereof - Google Patents

Abstract

The invention discloses a method for inducing cotton PR5 gene silencing by TRV virus and application thereof, wherein the method for inducing gene silencing by TRV is used for carrying out experiments, PR5 genes are cloned to a plasmid pTRV2 for expressing TRV RNA2, then co-injection is carried out with a plasmid pTRV1 for expressing TRV RNA1, agrobacterium carrying pTRV1 and pTRV2 is co-injected to cotton cotyledon, newly grown top leaf and root materials are taken for extracting RNA after 2 weeks, and qRT-PCR is used for detecting the expression condition of PR5. The invention uses a more direct and efficient cotton root inoculation method, reduces the background expression level of the target gene PR5 of cotton, and is more effective for preventing soil-borne diseases such as verticillium wilt of cotton.

Description

Method for inducing cotton PR5 gene silencing by TRV virus and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for inducing cotton PR5 gene silencing by TRV virus and application thereof.

Background

The verticillium dahliae of cotton in China is mainly caused by verticillium dahliae (Verticillium dahliae), the semi-living nutritional soil-borne pathogenic fungi exist in soil and hosts in the whole life cycle, and chemical agents are difficult to exert effects on the soil and hosts. The microsclerotium of verticillium dahliae can survive in soil for more than 10 years, colonize from the root and infect 600 or more natural, non-natural host plants. The verticillium dahliae is difficult to effectively prevent and treat due to the specificity of the infection mode of the verticillium dahliae, the persistence of the survival of pathogenic matters and the wide spread of the transmission. In the face of the severe situation, advanced gene editing technology is used for improving and screening high-quality disease-resistant cotton seeds, and key and fundamental guarantees are provided for agricultural production.

In the interaction and long evolution of plants with pathogenic microorganisms, some genes of the plants become targets for attack by the pathogenic microorganisms, and pathogenic bacteria secrete some effector factors to interact with the targets so as to inhibit plant immune response. These plant target gene expression levels are too high, which can aggravate the disease and make it more susceptible.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for inducing cotton PR5 gene silencing by TRV virus and application thereof, which aims to reduce the background expression level of target genes by a TRV virus (tobacco embrittlement virus) -mediated plant gene silencing system so as to enable plants to be more disease-resistant. In specific operation, the invention uses a more direct and efficient root inoculation method, reduces the background expression level of a target gene (PR 5, coding plant disease course related protein, thaumatin family member) of cotton roots, and is more effective in preventing soil-borne diseases such as cotton verticillium wilt.

The invention is realized by the following technical scheme:

a method for inducing cotton PR5 gene silencing by TRV virus, comprising the steps of:

step 1) connecting cDNA of cotton PR5 gene to plasmid pTRV2 virus vector expressing TRV RNA2, preparing recombinant vector, transforming competent cells of escherichia coli, extracting plasmid pTRV2 with correct sequence for standby; wherein the nucleotide sequence of the cotton PR5 gene is shown as SEQ ID NO. 1;

PR5 and plasmid pTRV1 expressing TRV RNA1 are respectively transformed into agrobacterium, LB plate culture medium containing kanamycin and rifampicin is coated, dark culture is carried out at 28 ℃ for 2d, single colony is selected for identification, and then seed preservation is carried out;

step 3), transferring the agrobacterium of step 2) into 100mL of liquid LB culture medium for shake culture, and adding kanamycin, rifampicin, MES and AS;

step 4) centrifugation at 4000rpm for 10min to collect the cells of Agrobacterium of step 3) with 10mM MgCl ₂ OD (optical density) adjustment ₆₀₀ =0.6, adding AS with a final concentration of 200. Mu.M, mixing two agrobacteria transformed by pTRV 2:PR 5 and pTRV1 in equal volume, and placing in the dark for 36h, preparing agrobacterium liquid for later use;

step 5) preparing cotton seedlings growing for 9 days, and respectively performing the following operations:

step 5-1), pricking a non-permeable small hole on the left and right sides of a cotton cotyledon by using a needle head, uniformly injecting the agrobacterium tumefaciens bacterial liquid in the step 4) into the cotyledon by using a 1mL injector, and performing dark culture in water for 1d, wherein the temperature is controlled within the range of 22-25 ℃;

step 5-2), immersing the part below the cotton hypocotyl in the agrobacterium tumefaciens liquid in step 4), vacuumizing for 3 times, and performing dark culture in water for 1d, wherein the temperature is controlled within the range of 22-25 ℃;

and step 6), taking the cotton top leaf material in the step 5-1) and the cotton root material in the step 5-2) after two weeks, and performing qRT-PCR to detect whether the cotton gene is silenced.

Preferably, the recombinant vector in step 1) is obtained by double cleavage of a cDNA fragment of the cotton PR5 gene with restriction endonucleases EcoRI and BamHI, and then ligating the double cleavage with restriction endonucleases EcoRI and BamHI into a TRV viral vector which is also digested with restriction endonucleases EcoRI and BamHI.

Preferably, the specific primers of the cotton PR5 gene in the step 1) are shown as SEQ ID NO.2 and SEQ ID NO. 3.

Preferably, the specific operation of step 5-2) of vacuum pumping 3 times is as follows: the air in the container containing the lower part of the cotton hypocotyl and the agrobacterium liquid is pumped out, then the sealing cover is quickly opened to allow the air to enter, and the agrobacterium liquid is hydraulically pressed into cotton root tissues, and the process is repeated for 3 times.

Application of TRV virus in cotton disease resistance is provided.

Preferably, the disease resistance is resistance to cotton verticillium wilt.

PR5 is preferably used to induce PR5 silencing by pTRV1 and pTRV2, which effectively inhibits significant upregulation of PR5 gene by Verticillium dahliae infection of cotton.

Preferably, the method of step 5-2) is used to induce PR5 silencing directly from the cotton root using pTRV1 and pTRV 2. PR5 is also described.

The beneficial effects of the invention are as follows:

the invention provides a more efficient and convenient cotton root gene silencing method; the target gene PR5 plays an important role in the verticillium wilt disease incidence process of cotton, and the expression level of the PR5 gene at the root of cotton is successfully reduced through a TRV virus-mediated plant gene silencing system, so that the verticillium wilt resistance of cotton is remarkably improved.

Drawings

FIG. 1 shows PR5 expression in the top leaf and root of the un-silenced cotton (US) and silenced cotton (S) 2 weeks after injection in test example 1;

FIG. 2 shows the expression of PR5 in cotton roots (R) and stems (S), respectively, after 2 weeks of growth in test example 1;

FIG. 3 shows qRT-PCR assay results for PR5 silenced cotton roots (2 weeks after Agrobacterium inoculation) in test example 2;

FIG. 4 shows the results of qRT-PCR detection of cotton root PR5 expression in test example 2: a is a comparison of PR5 silenced (S) and non-silenced (US) cotton root material 2 weeks after inoculation with agrobacterium; comparison of PR5 silenced (S) and non-silenced (US) cotton with no treatment (Mock) after 2 weeks of V592 infestation; c is a comparison of silenced cotton (S) before V592 infestation (S1) and after infestation (S1');

FIG. 5 is a graph showing the onset of PR5 silenced cotton in test 3 after V592 infestation (after 4 weeks): PR5 silenced cotton after V592 infection (S1, S11, S16, S7, -No. represents the number of bad cotton death in the corresponding group); b is the untonized cotton (US) after V592 infestation and the untreated cotton (Mock).

Detailed Description

The invention will be described in further detail with reference to the drawings and the specific embodiments.

V592 strain is a wild type strain of Verticillium dahliae with strong virulence selected from diseased cotton. V592 infection causes upregulation of PR5 expression dependent on VdSCP8, and the ΔVdSCP8 mutant greatly attenuated the pathogenicity of Verticillium dahliae. In order to examine the effect of reduced expression of PR5 on the pathogenicity of V592, the present invention performed the following experiment using a method for TRV to induce gene silencing.

The following examples relate to sources of biological material:

cotton was upland cotton (Gossypium hirsutum), purchased from a saline city seed station.

Agrobacterium is Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105, available from Invitrogen.

Example 1

A method for inducing cotton PR5 gene silencing by TRV virus comprises the following specific steps:

(1) Preparation of the target gene: the cDNA of the cotton PR5 gene is obtained by reverse transcription of total RNA of cotton, the nucleotide sequence of the cotton PR5 gene is shown as SEQ ID NO.1, and the specific steps are as follows:

double-stranded cDNA was reverse transcribed using Box1 in the Dualsystems Biotech company EasyClone normalized cDNA library construction package kit. The minimum amount of total RNA or mRNA used as template is 250ng and 100ng, respectively, and the recommended amount of total RNA or mRNA is 1 to 1.5. Mu.g and 0.5. Mu.g, respectively. Before synthesizing the first strand, the RNA sample is heated at 65℃for 1-2 min to prevent RNA aggregation.

(i) Add 3. Mu.L RNA sample, 1. Mu.L CDS-3M adapter, 1. Mu.L Plugoligo-3M adapter to PCR tube, gently mix and throw, place into PCR instrument and run the procedure shown in Table 1 below:

TABLE 1 PCR procedure

(ii) RT Master mix was prepared simultaneously during the reaction of step (i) above, and the formulation is shown in Table 2 below:

TABLE 2 RT Master mix formulation

The first strand of cDNA can be used directly for double-stranded cDNA synthesis or stored at-20℃for up to 3 months.

(iii) In the process of synthesizing double-stranded cDNA by PCR, the number of cycles is critical, more non-specific PCR products are generated, the yield of the double-stranded cDNA is reduced, and the downstream cloning efficiency is affected, so that the optimal number of cycles is required to be searched for by pre-experiment. The optimal number of cycles used in this experiment was 19. If the first cDNA strand is taken from-20deg.C, it is preheated at 65deg.C for 1min, and double-stranded cDNA is synthesized according to the following formula shown in Table 3 and the procedure shown in Table 4:

table 3 formulation

TABLE 4 PCR amplification procedure

4. Mu.L of the PCR product was taken and subjected to electrophoresis on a 1.2% agarose gel to examine the synthesis of double-stranded cDNA, using a DNA Marker as a reference. The double-stranded cDNA may be stored at-20℃for up to 6 months.

(iv) Homogenization of double-stranded cDNA (Normalization of ds cDNA). Since the double-stranded cDNA synthesized in this example was homogeneous by electrophoresis, this step was not performed.

(v) The double-stranded cDNA was purified with CHROMA SPINTM TE-400 Columb for subsequent experiments.

(vi) Performing PCR reaction according to the reaction system of Table 5 and the reaction conditions of Table 6 by using the double-stranded cDNA obtained in the step (v) as a template, and amplifying the cDNA of the cotton PR5 gene by using a specific primer of the cotton PR5 gene.

Specific primers for the cotton PR5 gene were as follows:

the upstream primer is shown as SEQ ID NO.2, and concretely comprises:

5’-CGGAATTCATGACCATTTCCCAAATCTCCTCCCTCCTCTTC-3’。

the downstream primer is shown as SEQ ID NO.3, and concretely comprises:

5’-CGGGATCCTTATTTTTCTTGGCTCTTACTTCCGACCAT-3’。

TABLE 5 PCR reaction System

TABLE 6 PCR reaction conditions

(2) The cDNA of the cotton PR5 gene is connected to a plasmid pTRV2 virus vector expressing TRV (tobacco brittle virus) RNA2 to prepare a recombinant vector, competent cells of escherichia coli are transformed, and plasmid pTRV2 with correct sequencing is extracted for standby.

The recombinant vector is a TRV virus vector obtained by double cleavage of a cDNA fragment of the cotton PR5 gene with restriction enzymes EcoRI and BamHI and then ligation of the same into the TRV virus vector.

(3) PR5 and the plasmid pTRV1 expressing TRV RNA1 are transformed into agrobacterium respectively, LB plate culture medium containing kanamycin and rifampicin is coated, dark culture is carried out at 28 ℃ for 2d, and single colony is selected for identification and then seed preservation is carried out.

(4) The agrobacterium is transferred into 100mL of liquid LB culture medium for shake culture, and kanamycin, rifampin, MES (2-morpholinoethanesulfonic acid) and AS (acetosyringone) are added.

1M MES: 2.132g MES is weighed and dissolved in sterile water, the pH is regulated to 5.6 by KOH, the volume is fixed to 10mL, filtration and sterilization are carried out, and the mixture is packaged in a 1.5mL centrifuge tube and stored at the temperature of minus 20 ℃.

100mM AS: 0.1962g of AS was weighed and dissolved in 10mL of DMSO (dimethyl sulfoxide), and the mixture was packed into 1.5-mL centrifuge tubes and stored at-20 ℃.

(5) The cells of the above Agrobacterium were collected by centrifugation at 4000rpm for 10min and 10mM MgCl ₂ OD (optical density) adjustment ₆₀₀ About 0.6. Mu.M AS was added to the mixture to mix the two agrobacteria transformed by pTRV 2:PR 5 and pTRV1 in equal volume and then the mixture was left to standAnd preparing the agrobacterium liquid for standby after 3-6 hours in the dark.

(6) Preparing cotton seedlings for about 9 days, pricking a non-penetrating small hole on each of the left and right sides of cotton cotyledons by using a needle head, uniformly injecting the agrobacterium tumefaciens bacterial liquid into the leaves by using a 1mL syringe, and culturing in a dark place for 1d in water, wherein the temperature is controlled within the range of 22-25 ℃.

(7) The agrobacterium-mediated TRV virus vector replicates in plants, and after 2 weeks, cotton top leaf material is taken for qRT-PCR to detect whether cotton genes are silent.

Example 2

A method for inducing cotton PR5 gene silencing by TRV virus comprises the following specific steps:

(1) cDNA of cotton PR5 gene was prepared and ligated to plasmid pTRV2 virus vector expressing TRV RNA2 as shown in example 1 to prepare recombinant vector, competent cells of E.coli were transformed, and plasmid pTRV2:: PR5 with correct sequencing was extracted for use.

(2) PR5 and the plasmid pTRV1 expressing TRV RNA1 are transformed into agrobacterium respectively, LB plate culture medium containing kanamycin and rifampicin is coated, dark culture is carried out at 28 ℃ for 2d, and single colony is selected for identification and then seed preservation is carried out.

(3) The agrobacterium is transferred into 100mL of liquid LB culture medium for shake culture, and kanamycin, rifampin, MES and AS are added.

(4) The cells of the above Agrobacterium were collected by centrifugation at 4000rpm for 10min and 10mM MgCl ₂ OD (optical density) adjustment ₆₀₀ About 0.6, AS with a final concentration of 200 mu M is added, two agrobacteria transformed by pTRV 2:PR 5 and pTRV1 are mixed in equal volume and placed in the dark for 3-6 hours to prepare the agrobacteria liquid for standby.

(5) Preparing cotton seedlings for about 9 days, immersing the lower part of cotton hypocotyl in the agrobacterium liquid, vacuumizing for 3 times, and dark culturing in water for 1d at 22-25 ℃.

The specific operation of vacuum pumping 3 times is as follows: the air in the container containing the lower part of the cotton hypocotyl and the agrobacterium liquid is pumped out, then the sealing cover is quickly opened to allow the air to enter, and the agrobacterium liquid is hydraulically pressed into cotton root tissues, and the process is repeated for 3 times.

(6) The agrobacterium-mediated TRV virus vector replicates in plants, and after 2 weeks, cotton root material is taken for qRT-PCR to detect whether cotton genes are silent.

Test example 1

PR5 gene was cloned on a plasmid (pTRV 2) expressing TRV RNA2 and co-injected with a plasmid (pTRV 1) expressing TRV RNA 1. Agrobacteria carrying pTRV1 and pTRV2 are coinjected to the PR5 plasmid to obtain newly grown top leaf (example 1) and root (example 2) materials for RNA extraction after 2 weeks of cotyledons of cotton (cotton which is to induce PR5 gene silencing, hereinafter abbreviated as S), and qRT-PCR is used for detecting the expression condition of PR5, and the coinjected of agrobacteria carrying pTRV1 and pTRV2 empty vector plasmids to the cotton (namely cotton which cannot induce PR5 gene silencing, hereinafter abbreviated as US) is used as a control.

FIG. 1 shows PR5 expression in cotton top leaf and root after 2 weeks of injection, where US-L1 and US-L2 in FIG. 1 are the unslaked cotton (US) top leaf material and US-R1 and US-R2 are the unslaked cotton (US) root material; S-L1 and S-L2 are silenced cotton (S) apical leaf material, and S-R1 and S-R2 are silenced cotton (S) root material. As can be seen from FIG. 1, the leaf (US-L1, US-L2) of the first four control samples in normal growth was compared to the root (US-R1, US-R2) and the PR5 expression level in cotton leaves was approximately 6 times that in cotton roots.

As shown in FIG. 2, the expression of PR5 in the roots (R) and stems (S) of cotton respectively, and the expression level of PR5 in the roots (R) and stems (S) of cotton was compared with that of cotton material grown for 2 weeks, and the expression level in the stems was found to be similar to that of leaves and about 7 times that of the roots.

Experimental results show that TRV silencing (the last four samples of S-L1, S-R1, S-L2, and S-R2 in FIG. 1) did not provide good PR5 silencing in the first set of S-L1 cotton leaves; silencing in the second group of S-L2 leaves was better, with about a 5-fold reduction in PR5 levels compared to normal US control cotton leaves. Two experiments found that the silencing effect of PR5 in roots induced by TRV on leaves was not good, and the results of qRT-PCR showed that PR5 in roots was elevated compared to control samples (S-R1, S-R2). Since the infection of cotton with verticillium dahliae under simulated natural conditions was inoculated from the root, we have later tried to silence cotton PR5 directly from the root.

Test example 2

To silence PR5 in cotton roots and to examine the condition by inoculating Verticillium dahliae from the roots, agrobacterium expressing pTRV1 and pTRV2:: PR5 plasmids were introduced into cotton (S) roots by the method of root dipping and vacuuming (example 2). As a control, cotton (US) transformed with pTRV1 and pTRV2 empty vector plasmids was used.

FIG. 3 shows the qRT-PCR assay of PR5 silenced cotton roots (2 weeks after Agrobacterium inoculation). In FIG. 3, C1 is an unslaked cotton (US) material, and S1, S11, S16, and S7 are four sets of parallel silent cotton (S) materials, respectively.

FIG. 4 shows the results of qRT-PCR detection of cotton root PR5 expression. FIG. 4A is a comparison of PR5 silenced (S) and non-silenced (US) cotton root material 2 weeks after Agrobacterium inoculation; FIG. 4B is a comparison of PR5 silenced (S) and non-silenced (US) cotton with no treatment (Mock) after 2 weeks of V592 infestation; FIG. 4C is a comparison of silenced cotton (S) before V592 infestation (S1) and after infestation (S1').

From the results of the 3 rounds of repetition, PR5 silencing was less effective than silencing in cotton leaves by a factor of about 3, as shown in FIGS. 3 and 4A, because the background expression of PR5 was higher in cotton leaves than in cotton roots.

PR5 induced (S) and non-induced (US) cotton were again V592 infected with pTRV1 and pTRV2, and PR5 was found to have increased expression induced by the bacteria, approximately 20-fold in US roots of cotton with less PR5 induction than in S roots of healthy cotton without silencing and infection (Mock) (FIG. 4B). PR5 in S cotton roots was compared before and after infection with pathogenic fungi and it was found that PR5 was induced to increase approximately 3-fold after infection (as shown in fig. 4C).

The results of this example show that PR5 can be used to induce PR5 silencing directly from root and effectively inhibit the significant upregulation of PR5 gene caused by Verticillium dahliae infection in cotton by pTRV1 and pTRV 2.

Test example 3

PR 5-induced cotton (S), uninduced cotton (US), and healthy cotton without silencing and infection (Mock) were compared with V592 to pTRV1 and pTRV2, and after about 4 weeks of infection, the condition was examined and counted. As a result, it was found that the plant (S) with PR5 silencing at the cotton root exhibited stronger resistance to Verticillium dahliae than the cotton (US) without silencing, as shown in FIG. 5.

FIG. 5 shows the onset of PR5 silenced cotton after V592 infestation (after 4 weeks). FIG. 5A is a graph of PR5 silenced cotton (S1, S11, S16, S7, -No. representing the number of cotton necrosed plants in the corresponding group) after V592 infestation; FIG. 5B is an unsmooth cotton (US) after V592 infestation and untreated cotton (Mock).

After PR5 silenced cotton (S) was infected with Verticillium dahliae, 4 replicates were immediately corresponding in disease extent to the results of qRT-PCR detection of PR5 expression (FIG. 3). Wherein, the S1 and S7 silencing is better, the infected plant has a slight degree of verticillium wilt, but none of the plants is completely necrotic, the S11 silencing effect is slightly worse, one of the plants is necrotic, and the S16 effect is worse, and two of the plants are necrotic (as shown in FIG. 5A). Essentially all of the PR5 silenced cotton (US) became necrotic after infection with Verticillium dahliae (as shown in FIG. 5B).

PR5 silenced cotton (S) and PR5 non-silenced cotton (US) were repeatedly infested with V592 for 3 rounds and the cotton disease statistics are shown in Table 7 below:

TABLE 7 statistics of conditions of cotton infested with Verticillium dahliae (3 rounds of repeated tests)

As can be seen from table 7, root PR5 silenced cotton exhibited stronger verticillium resistance than wild cotton (i.e., PR5 not silenced cotton).

Sequence listing

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<120> a method for inducing cotton PR5 gene silencing by TRV virus and application thereof

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Claims (3)

1. Use of a method for inducing cotton PR5 gene silencing by TRV virus for combating cotton verticillium, said method comprising the steps of:

step 1) connecting cDNA of cotton PR5 gene to plasmid pTRV2 virus vector expressing TRV RNA2, preparing recombinant vector, transforming competent cells of escherichia coli, extracting plasmid pTRV2 with correct sequence for standby; wherein the nucleotide sequence of the cotton PR5 gene is shown as SEQ ID NO. 1; PR5 and the plasmid pTRV1 expressing TRV RNA1 in the step 1) are respectively transformed into agrobacterium, LB plate culture medium containing kanamycin and rifampicin is coated, dark culture is carried out at 28 ℃ for 2d, and single colony is selected for identification and then seed preservation is carried out;

step 3), transferring the agrobacterium of step 2) into 100mL of liquid LB culture medium for shake culture, and adding kanamycin, rifampicin, MES and AS;

step 4) centrifugation at 4000rpm for 10min to collect the cells of Agrobacterium of step 3) with 10mM MgCl ₂ OD (optical density) adjustment ₆₀₀ Adding AS with a final concentration of 200 mu M into the mixture, mixing two agrobacterium transformed by PR5 and pTRV1 in equal volume, and standing in the dark for 3-6 h to prepare agrobacterium solution for later use;

step 5) preparing cotton seedlings growing for 9 days, and respectively performing the following operations:

step 5-1), pricking a non-permeable small hole on the left and right sides of a cotton cotyledon by using a needle head, uniformly injecting the agrobacterium tumefaciens liquid in the step 4) into the cotyledon by using a 1mL syringe, and performing dark culture in water for 1d, wherein the temperature is controlled within the range of 22-25 ℃;

step 5-2), immersing the part below the cotton hypocotyl in the agrobacterium tumefaciens liquid in step 4), vacuumizing for 3 times, and performing dark culture in water for 1d, wherein the temperature is controlled within the range of 22-25 ℃;

and step 6), taking the cotton top leaf material in the step 5-1) and the cotton root material in the step 5-2) after two weeks, and performing qRT-PCR to detect whether the cotton gene is silenced.

2. The method of claim 1, wherein PR5 is used to inhibit PR5 gene upregulation caused by infection of cotton with Verticillium dahliae by PR5 to induce PR5 silencing.

3. The method of claim 2, wherein PR5 is used to induce PR5 silencing directly from cotton roots using pTRV1 and pTRV 2. PR5 is used in the method of step 5-2).