阅读说明：本技术 基于tcv的同时沉默2个内源基因的vigs载体 (TCV-based VIGS vector capable of simultaneously silencing 2 endogenous genes ) 是由 张秀春 吴坤鑫 刘志昕 于 2019-11-06 设计创作，主要内容包括：本申请属于植物功能基因组研究技术领域,具体涉一种基于TCV的同时沉默2个内源基因的VIGS载体的专利申请。所述2个基因,其中一个为内源基因PDS,另一个为内源基因“X基因”,该VIGS载体用于植物中基因沉默。构建步骤包括：用人工合成片段构建基于TCV高效沉默PDS基因的病毒诱导沉默载体CPB1B、构建基于TCV高效沉默目标基因“X基因”和PDS的VIGS载体CPB1B-X等步骤。由于利用该载体对拟南芥内源靶标基因进行功能鉴定时,具有直观、快速、高通量、易操作等明显优势,因此具有较好的实用价值和推广应用意义。同时该构建方法还为其他同时沉默两个、多个功能基因的VIGS载体提供了较好借鉴和参考。(The application belongs to the technical field of plant functional genome research, and particularly relates to a TCV-based VIGS vector capable of silencing 2 endogenous genes simultaneously. One of the 2 genes is an endogenous gene PDS, and the other is an endogenous gene 'X gene', and the VIGS vector is used for gene silencing in plants. The construction steps comprise: the virus induction silencing vector CPB1B based on the TCV high-efficiency silencing PDS gene is constructed by using the artificially synthesized fragment, the VIGS vector CPB1B-X based on the TCV high-efficiency silencing target gene X gene and PDS is constructed, and the like. When the vector is used for carrying out function identification on an arabidopsis endogenous target gene, the vector has the obvious advantages of intuition, rapidness, high flux, easiness in operation and the like, so that the vector has better practical value and popularization and application significance. Meanwhile, the construction method also provides better reference and reference for other VIGS vectors capable of silencing two or more functional genes simultaneously.)

1. A TCV-based VIGS vector for simultaneously silencing 2 endogenous genes, wherein one of the 2 genes is endogenous gene PDS and the other is endogenous gene "X gene", the VIGS vector being for gene silencing in plants; the VIGS vector is constructed by the following steps:

construction of virus-induced silencing vector CPB1B based on TCV high-efficiency silencing PDS gene by utilizing artificially synthesized fragment

(1) Gene fragment design and synthesis of target PDS

Firstly, the sequence of the generated siRNA is determined to be shown in SEQ ID NO.1, and specifically: 5'-AAGATGGTTTATCAGTCAAAG-3', respectively;

then, a PDS gene fragment PDS1-F of 44 bp containing the sequence was artificially synthesized, and a reverse complementary sequence PDS1-R (TAC) to which a base "TAC" was added at the 3' end was additionally synthesized;

the final synthetic sequences were:

PDS1-F：5’-CAAGATGGTTTATCAGTCAAAGAATGGATGGAAAAGCAGGGAGTAC-3’，

PDS1-R(TAC)：5’-TCCCTGCTTTTCCATCCATTCTTTGACTGATAAACCATCTTGGTAC-3’；

(2) construction of Virus-induced silencing vector CPB1B

Firstly, equally mixing artificially synthesized fragments PDS1-F and PDS1-R (TAC), and then carrying out denaturation and annealing treatment;

secondly, carrying out Kpn I single enzyme digestion on the TCV viral vector CPB-CC with the weakened virus silencing function inhibitor function, recovering a linearization product from gel, and carrying out dephosphorylation treatment;

thirdly, connecting the recovered phosphorylation products with a mixture of denatured and annealed artificially synthesized fragments PDS1-F and PDS1-R (TAC);

fourthly, after the ligation products are transferred into competent cells, Amps are randomly selected⁺Carrying out plasmid extraction on a colony with a normal shape on the LB plate, carrying out sequencing identification on a positive clone identified by enzyme digestion to ensure correct recombination, and finally obtaining a VIGS vector CPB1B only reserving one Kpn I enzyme digestion site for later use;

(II) constructing VIGS vector CPB1B-X based on TCV high-efficiency silencing target gene X gene and PDS

(1) Gene silencing sequence selection and primer design of target gene

Firstly, analyzing the sequence of an endogenous target gene X gene, and determining a sequence for generating siRNA as a target sequence;

secondly, designing forward and reverse primers for amplifying the target sequence; when designing a primer, respectively adding a partial sequence ' 5'-TTGACTGATAAACCATCTTGGTAC-3' ' of a sequence on both sides of an enzyme cutting site Kpn I of a virus vector CPB1B and a partial sequence ' 5'-CTAAGATGAGAAGACTACACTATG-3' ' of a 5 ' end in forward and reverse primer design;

(2) amplification of target gene sequences

Preparing a template for PCR amplification, and performing PCR amplification by using the primer designed in the step (1); after the PCR product is identified by electrophoresis, the target fragment is purified and recovered;

(3) construction of VIGS Virus vector CPB1B-X for silencing 2 endogenous genes simultaneously

Firstly, carrying out single enzyme digestion on the virus vector CPB1B constructed in the step (I) by using Kpn I, and recovering a linearized enzyme digestion product;

secondly, connecting the recovered linearized enzyme digestion product of CPB1B with the endogenous target gene sequence amplified in the step (2);

and finally, after the ligation product is transferred into a competent cell, screening and identifying to construct and obtain a VIGS virus vector CPB1B-X capable of silencing 2 genes simultaneously.

2. The TCV-based VIGS vector for simultaneous silencing of 2 endogenous genes of claim 1, wherein the endogenous gene "X gene" is the DRB2 gene.

3. The TCV-based VIGS vector for simultaneous silencing of 2 endogenous genes according to claim 2, wherein the sequence for generating siRNA is determined to be DRB2A sequence or DRB2B sequence in step (1) of step (ii) for the endogenous gene DRB2 gene;

the sequence of DRB2A is shown in SEQ ID NO.2, and specifically comprises:

TTTCCAAGGCTGCTGCCATAATTCTGTCAATACCGGAAGTTGCTGCTAAGGATGATCTGCTGCTTCTGGAAGGTTGTTTTGGGCAAATAAAGGGTAAGATTC；

the sequence of DRB2B is shown in SEQ ID NO.3, and specifically comprises:

GTTGTTTTGGGCAAATAAAGGGTAAGATTCTCGATGTGGTTGCAGGAGAAGGCTGTGGACTTGTTGGTCTAAGGTTCTGCATAAAGAACTTCTTTGCAAA。

4. the TCV-based VIGS vector for simultaneous silencing of 2 endogenous genes according to claim 3, wherein the primer sequences in step (1) of step (two) are designed for DRB2A and DRB2B sequences:

G-CPB1B-DRB2AF：

5’-TTGACTGATAAACCATCTTGGTACTTTCCAAGGCTGCTGCCATAATTC-3’，

G-CPB1B-DRB2AR：

5’-CTAAGATGAGAAGACTACACTATGGAATCTTACCCTTTATTTGCCCA-3’；

G-CPB1B-DRB2BF：

5’-TTGACTGATAAACCATCTTGGTACGTTGTTTTGGGCAAATAAAGGGT-3’，

G-CPB1B-DRB2BR：

5’-CTAAGATGAGAAGACTACACTATGTTTGCAAAGAAGTTCTTTATGCAG-3’。

5. the TCV-based VIGS vector for simultaneous silencing of 2 endogenous genes according to claim 1, wherein the VIGS vector is used for silencing of 2 endogenous genes in arabidopsis thaliana.

Technical Field

The application belongs to the technical field of plant functional genome research, and particularly relates to a TCV-based VIGS vector capable of silencing 2 endogenous genes simultaneously.

Background

Virus Induced Gene Silencing (VIGS) is an RNA silencing technique developed based on the principle of plant resistance to virus invasion, and is a reverse genetic tool for gene function analysis. The action principle is as follows: RNA silencing is initiated when a plant is resistant to viral invasion, so that after a virus or a viral vector carrying cDNA infects the plant, double-stranded RNA (dsrna) is first formed in the host, the double-stranded RNA or a partially double-stranded hairpin structure is cleaved in the cell by a double-stranded RNA-specific endonuclease, Dicer or Dicer-like (DCL for short), to generate small interfering RNA (siRNA) of about 20 nt in length, and the siRNA is then bound by argonaute (ago) protein to form an RNA-induced silencing complex (RISC), which specifically recognizes and degrades homologous RNA in cytoplasm, thereby causing post-transcriptional gene silencing (PTGS). Compared with common biotechnology tools such as gene transformation, gene knockout, antisense suppression and the like, VIGS has the advantages of low cost, no need of stable genetic transformation, capability of transiently silencing gene expression, capability of allowing large-scale gene screening and the like. Therefore, this method has been widely used in many plant species for the study of disease resistance, stress resistance and metabolic regulation of genes and various plant developmental processes. At present, various viruses are used for constructing VIGS vectors, such as Tobacco Rattle Virus (TRV), Apple Latent Spherical Virus (ALSV) and the like which are commonly used, and the viruses are successfully applied to gene function researches in species such as Tobacco, arabidopsis thaliana, soybean, apple and the like.

Turnip rhyvirus (TCV) is a plant of the tomato bushy stunt virus familyTombusviridae) Musk Dianthus mottle virus genus (A)Carmovirus) Is a common virus. TCV primarily infects crucifers causing turnips (turnipBrassica rapa) Leaf shrinkage, mottle and slight distortion, sometimes with more severe plant deformity or low clumps. In the earlier research, the inventor inserts 92 bp Arabidopsis thaliana Phytoene Desaturase (PDS) sequence into TCV mutant CPB with weakened function of a silencing suppressor, constructs VIGS vector CPB-CC-PDS, and the vector can effectively induce wild type and Arabidopsis thalianadcl2、dcl4Single mutants were silenced by antiviral RNA, resulting in albinism (Zhang et al, Temperature-Dependent overview of Turnip Crinkle Virus-Infected Arabidopsis Plants Relies on an RNA Silencing-Based feed strategy DCL2, AGO2, and HEN1, 2012).

Due to practical research and application, the VIGS-based technology is mostly a silent monogene research. Because of the relevance of gene functions, the development and design of VIGS vectors which silence two or even more functional genes simultaneously has very important technical value.

Disclosure of Invention

Based on previous research work of the inventor, on the basis of the existing TCV mutant CPB, the invention aims to provide a method for simultaneously silencing 2 endogenous genes (one of which is an endogenous gene PDS, and the other is, for example, a DRB2 gene (Double strand RNA binding protein 2)) VIGS vector constructed by adopting a method of combining artificially synthesized fragments and seamless connection, and the vector can be used for functional research of target genes and can indicate whether plant leaves are whitened or not, so that whether virus-induced gene silencing occurs or not can be visually judged. Meanwhile, the construction method of the VIGS vector can provide certain reference and reference for developing and designing the VIGS vector capable of silencing two or even more functional genes simultaneously.

The technical solution adopted in the present application is detailed as follows.

A VIGS vector for simultaneous TCV-based silencing of 2 endogenous genes, one of the 2 genes being endogenous gene PDS and the other endogenous gene "X gene" being for example DRB2 gene (Double strand RNA binding protein 2), for gene silencing in plants (in particular for example arabidopsis thaliana); the VIGS vector is constructed by the following steps:

construction of virus-induced silencing vector CPB1B based on TCV high-efficiency silencing PDS gene by utilizing artificially synthesized fragment

(1) Gene fragment design and synthesis of target PDS

Firstly, through the analysis of bioinformatics software (https:// www.genscript.com/tools/siRNA-target-finder), the analysis confirms that the siRNA sequence (the sequence is shown as SEQ ID NO. 1) possibly generated in the PDS fragment in the existing virus silencing vector CPB-CC-PDS is: 5'-AAGATGGTTTATCAGTCAAAG-3', respectively;

then, a PDS gene fragment PDS1-F of 44 bp containing the sequence was artificially synthesized, and a reverse complementary sequence PDS1-R (TAC) to which a base "TAC" was added at the 3' end was additionally synthesized;

the final synthetic sequences were:

PDS1-F：5’-CAAGATGGTTTATCAGTCAAAGAATGGATGGAAAAGCAGGGAGTAC-3’，

PDS1-R(TAC)：5’-TCCCTGCTTTTCCATCCATTCTTTGACTGATAAACCATCTTGGTAC-3’；

(2) construction of Virus-induced silencing vector CPB1B

Firstly, equally mixing artificially synthesized fragments PDS1-F and PDS1-R (TAC), and then carrying out denaturation and annealing treatment;

secondly, carrying out Kpn I single enzyme digestion on the TCV viral vector CPB-CC with the weakened virus silencing function inhibitor function, recovering a linearization product from gel, and carrying out dephosphorylation treatment;

thirdly, connecting the recovered phosphorylation products with a mixture of denatured and annealed artificially synthesized fragments PDS1-F and PDS1-R (TAC);

fourthly, after the ligation products are transferred into competent cells, Amps are randomly selected⁺Carrying out plasmid extraction on the colony with normal morphology on the LB plate, and carrying out sequencing identification on the positive clone identified by enzyme digestion to ensure correct recombination;

(3) detection and verification of silencing effect of CPB1B of VIGS viral vector

After the constructed VIGS vector CPB1B is linearized by Xba I single enzyme digestion and purified and recovered, in vitro transcription virus is carried out by using a transcription Aid T7 High Yield transcription Kit of Thermoscientific;

arabidopsis thaliana 4 weeks after transplantation by viral inoculation of transcriptiondcl4Performing phenotype observation and semi-quantitative RT-PCR detection on the mutant seedlings after virus inoculation so as to detect and evaluate the silencing effect;

it should be explained that in the above construction process, through the denaturation and annealing treatment of the artificially synthesized 2 fragments, a double-stranded DNA with Kpn I cohesive end is finally formed, so that the double-stranded DNA can be directly connected with the TCV mutant CPB cut by Kpn I, thereby constructing the VIGS vector CPB1B containing the artificially synthesized fragment and only reserving one Kpn I cutting site;

(II) constructing VIGS vector CPB1B-X based on TCV high-efficiency silencing target gene X gene and PDS

The "X gene" is specifically, for example, a DRB2 gene, and the specific process is as follows:

(1) gene silencing sequence selection and primer design of target gene

Firstly, analyzing the reverse complementary sequence of an endogenous target gene DRB2 gene, determining a target gene fragment of about 100bp of a sequence most likely to generate siRNA as a target sequence, and constructing a virus vector;

taking software analysis using website address https:// www.genscript.com/tools/siRNA-target-finder as an example, when the analysis is determined, a 100bp target gene fragment containing the first 5 most likely sequences to generate siRNA as far as possible in the software analysis is selected as a target sequence, so as to construct a viral vector;

in practice, as a reference control, simultaneously transfecting an exogenous GUS gene for transformation operation, and as a reference control, namely, selecting a targeting sequence aiming at the exogenous GUS gene, and constructing a virus vector inserted with exogenous GUS as a reference control;

the finally determined targeting sequences of the endogenous gene DRB2 and the exogenous GUS gene are respectively as follows:

DRB2A sequence (102 bp, shown as SEQ ID NO. 2):

TTTCCAAGGCTGCTGCCATAATTCTGTCAATACCGGAAGTTGCTGCTAAGGATGATCTGCTGCTTCTGGAAGGTTGTTTTGGGCAAATAAAGGGTAAGATTC

DRB2B sequence (100 bp, shown in SEQ ID NO. 3)

GTTGTTTTGGGCAAATAAAGGGTAAGATTCTCGATGTGGTTGCAGGAGAAGGCTGTGGACTTGTTGGTCTAAGGTTCTGCATAAAGAACTTCTTTGCAAA

GUS sequence (100 bp):

GTCACGCGCTATCAGCTCTTTAATCGCCTGTAAGTGCGCTTGCTGAGTTTCCCCGTTGACTGCCTCTTCGCTGTACAGTTCTTTCGGCTTGTTGCCCGCT

secondly, designing forward and reverse primers for amplifying the target sequence (specifically, for example, designing a target sequence Primer by using software Primer premier 5.0); in designing primers, in order to facilitate subsequent fragment amplification and seamless connection with the viral vector CPB1B after single cleavage with Kpn I, a partial sequence "5'-TTGACTGATAAACCATCTTGGTAC-3'" of the sequence on both sides of the cleavage site Kpn I of the viral vector CPB1B and a partial sequence "5'-CTAAGATGAGAAGACTACACTATG-3'" of the 5 ' end are added in forward and reverse primer design, respectively, so that the final primer sequence is designed as follows:

G-CPB1B-DRB2AF：

5’-TTGACTGATAAACCATCTTGGTACTTTCCAAGGCTGCTGCCATAATTC-3’，

G-CPB1B-DRB2AR：

5’-CTAAGATGAGAAGACTACACTATGGAATCTTACCCTTTATTTGCCCA-3’；

G-CPB1B-DRB2BF：

5’-TTGACTGATAAACCATCTTGGTACGTTGTTTTGGGCAAATAAAGGGT-3’，

G-CPB1B-DRB2BR：

5’-CTAAGATGAGAAGACTACACTATGTTTGCAAAGAAGTTCTTTATGCAG-3;

G-CPB1B-GUSF：

5’-TTGACTGATAAACCATCTTGGTACGTCACGCGCTATCAGCTCTTTAATC-3’，

G-CPB1B-GUSR：5’-CTAAGATGAGAAGACTACACTATGAGCGGGCAACAAGCCGAAAGAACT-3’；’

(2) amplification of target gene sequences

Preparing a template for PCR amplification, and performing PCR amplification by using the primer designed in the step (1), wherein the DRB2 gene fragment takes Arabidopsis thaliana cDNA as the template, and the pCAMBIA1300 as the template during GUS gene fragment amplification;

after the PCR product is identified by 1.5 percent agarose gel electrophoresis, respectively purifying the target fragment according to the instruction of the kit and recovering the target fragment with the expected size;

(3) construction of VIGS viral vector CPB1B-X for silencing 2 endogenous genes simultaneously (named as CPB1B-DRB2 for target gene DRB2 gene and named as CPB1B-GUS for exogenous reference gene GUS gene)

Firstly, carrying out single enzyme digestion on the virus vector CPB1B constructed in the step (I) by using Kpn I, and recovering a linearized enzyme digestion product;

secondly, respectively connecting the recovered linearized enzyme digestion product of CPB1B with the endogenous target gene sequence amplified in the step (2) by adopting a Gibsob assembly kit;

finally, after the ligation products are transferred into competent cells, Amps are randomly selected⁺Carrying out plasmid extraction on a colony with normal morphology on the LB plate, carrying out sequencing identification on a positive clone identified by enzyme digestion, and ensuring that the recombination construction is correct, namely the VIGS virus vector CPB1B-X capable of silencing 2 genes simultaneously (namely, the endogenous gene DRB2 and PDS can be silenced simultaneously and used as a reference control, and after the exogenous gene GUS gene is transfected, the vector is equivalent to only silencing the PDS gene);

(4) detection of silencing effect of VIGS viral vector CPB1B-X

After the VIGS vector CPB1B-X (the VIGS virus vector CPB1B-DRB2 is taken as an example) is linearized by Xba I single enzyme digestion and purified and recovered, the virus is transcribed in vitro by using a TranscriptAID T7 High Yield transcription Kit of Thermo Scientific;

inoculation of Arabidopsis thaliana 4 weeks after transplantation with 10ng/μ l of transcription Virusdcl2drb4And (3) inoculating 3 leaves and 10 ul leaves to each mutant seedling, and performing phenotype observation and RT-PCR silencing effect detection on leaves after virus inoculation under the conditions of 18 ℃ and 16h/8h photoperiod long sunlight.

After the study on TCV, the inventors considered that the TCV has the following characteristics: positive strand RNA virus with single-stranded TCV of 4054bp length and no 5^’Headwear and 3^’Poly A tail, convenient for in vitro synthesis; and TCV is one of the most well studied viruses, the functions of 5 genes encoding 5 proteins including P28, P88, P38, P8 and P9 are known. Already, studies show that the replication amount of TCV in model plant Arabidopsis is very high, and a large amount of viral RNA, double-stranded RNA (dsRNA) and vsiRNA can be generated, so that the detection is convenient; also, inhibitors of silencing have been identified in view of TCV encoding. Therefore, the development and design of a VIGS vector for silencing two or even more functional genes simultaneously based on TCV is a relatively easy way to implement.

In general, the idea of constructing the VIGS vector of the double silenced gene in the present application is as follows: firstly, adding specific base to ensure that artificially synthesized 2 PDS fragments are subjected to denaturation and annealing treatment to finally form double-stranded DNA with Kpn I cohesive ends, so that the double-stranded DNA can be directly connected with the TCV mutant CPB subjected to enzyme digestion by Kpn I, and the VIGS vector CPB1B containing the artificially synthesized fragments and only reserving one Kpn I enzyme digestion site is constructed; secondly, another target gene sequence is inserted into the CPB1B vector again to obtain a virus vector CPB 1B-X. Specifically, during detection and verification, after the arabidopsis thaliana leaves are subjected to friction inoculation by virtue of in vitro transcription, not only can target gene silencing be induced, but also a whitening phenomenon generated after a PDS gene is silenced can be used as an indicator to judge whether virus-induced gene silencing occurs or not. When the vector is used for carrying out function identification on an arabidopsis endogenous target gene, the vector has the obvious advantages of intuition, rapidness, high flux, easiness in operation and the like, so that the vector has better practical value and popularization and application significance. Meanwhile, the construction method provides better reference for developing and designing the VIGS vector capable of simultaneously silencing two or even more functional genes by utilizing the artificially synthesized fragments.

Drawings

FIG. 1 is a schematic structural diagram of a virus vector CPB 1B;

FIG. 2 is a 21 day phenotypic observation of the Arabidopsis mutant dcl4 infected with the viral vector CPB 1B;

FIG. 3 shows the silencing effect of a semi-quantitative RT-PCR detection virus vector CPB1B infected Arabidopsis thaliana mutant dcl 4;

FIG. 4 is a 21 day phenotypic observation of the Arabidopsis mutant dcl2DRB4 infected with the viral vector CPB1B-DRB 2;

FIG. 5 shows the silencing effect of a semi-quantitative RT-PCR detection virus vector CPB1B-DRB2 infected Arabidopsis thaliana mutant dcl2DRB 4.

Detailed Description

The present application is further illustrated by the following examples.