阅读说明：本技术 CRISPR/RfxCas13d抗植物RNA病毒载体及其构建方法和应用 (CRISPR/RfxCas13d plant RNA virus resistant vector and construction method and application thereof ) 是由 李方方 曹永森 周雪平 于 2020-05-22 设计创作，主要内容包括：本发明公开了一种CRISPR/RfxCas13d抗植物RNA病毒载体及其构建方法和应用,包括pCambia1300-RfxCas13d重组载体,构成如下：CaMV 35S启动子,潮霉素基因,CaMV poly(A)信号终止子,pVS1 RepA,pVS1复制起点,CaMV 35S启动子,RfxCas13d基因,NOS终止子；其中,RfxCas13d基因序列如SEQ ID No.1所示,NOS终止子序列如SEQ ID No.2所示。本发明利用带有GFP荧光标签的芜菁花叶病毒(TuMV-GFP)侵染性克隆来指示RNA病毒自然侵害植物,利用RfxCas13d蛋白和sgRNA结合可以定向特定位置切割RNA。本发明构建的载体能够有效抑制TuMV的侵染,为植物病毒病的防控提供新的策略,具有重要的生产意义。(The invention discloses a CRISPR/RfxCas13d plant RNA virus-resistant vector, a construction method and application thereof, wherein the CRISPR/RfxCas13d plant RNA virus-resistant vector comprises a pCambia1300-RfxCas13d recombinant vector, and comprises the following components: CaMV 35S promoter, hygromycin gene, CaMV poly (a) signal terminator, pVS1 RepA, pVS1 origin of replication, CaMV 35S promoter, RfxCas13d gene, NOS terminator; wherein, the RfxCas13d gene sequence is shown in SEQ ID No.1, and the NOS terminator sequence is shown in SEQ ID No. 2. The invention utilizes turnip mosaic virus (TuMV-GFP) infectious clone with a GFP fluorescent label to indicate that RNA viruses naturally invade plants, and utilizes the combination of RfxCas13d protein and sgRNA to directionally cut RNA at a specific position. The vector constructed by the invention can effectively inhibit TuMV infection, provides a new strategy for preventing and controlling plant virus diseases, and has important production significance.)

1. A CRISPR/RfxCas13d plant RNA virus-resistant vector, comprising: comprises a pCambia1300-RfxCas13d recombinant vector, and consists of the following components: CaMV 35S promoter, hygromycin gene, CaMV poly (a) signal terminator, pVS1 RepA, pVS1 origin of replication, CaMV 35S promoter, RfxCas13d gene, NOS terminator;

wherein, the RfxCas13d gene sequence is shown in SEQ ID No.1, and the NOS terminator sequence is shown in SEQ ID No. 2.

2. The CRISPR/RfxCas13d plant RNA virus-resistant vector according to claim 1, wherein:

the amino acid sequence coded by RfxCas13d is shown as SEQ ID No. 3.

3. The CRISPR/RfxCas13d plant RNA virus-resistant vector according to claim 1, wherein: also comprises a pCambia1300-RfxCas13d-crRNA recombinant vector, which comprises the following components: CaMV 35S promoter, hygromycin gene, cammvpoly (a) signal terminator, pVS1 RepA, pVS1 origin of replication, CaMV 35S promoter, RfxCas13d gene, NOS terminator, AtU6 promoter, crRNA repeat leader sequence, spacer nucleotide sequence containing two BsaI cleavage sites, (T)8 terminator sequence.

4. The method for constructing CRISPR/RfxCas13d plant RNA virus-resistant vector as claimed in any of claims 1 to 3, wherein: the construction method of the pCambia1300-RfxCas13d recombinant vector specifically comprises the steps of artificially synthesizing a 3003bp RfxCas13d gene fragment, cloning the gene fragment to pUC57, and naming the fragment as pUC 57: RfxCas13 d; plasmid pUC57 was purified using BamHI and NcoI: performing double enzyme digestion on RfxCs 13d and pCambia1300-spCas9, recovering a 3003bp RfxCs 13d gene fragment and a pCambia1300 vector skeleton of about 10kb respectively, and connecting the 3003bp RfxCs 13d fragment to the pCambia1300 vector skeleton by using T4 DNA ligase, wherein the plasmid is named as pCambia-Rfxas 13 d; and (4) after sequencing the plasmid, storing the plasmid, and after verification is successful, transferring the plasmid to agrobacterium for storage.

5. The method for constructing a CRISPR/RfxCas13d plant RNA virus-resistant vector of claim 3, wherein the CRISPR/RfxCas13 comprises: the construction method of pCambia1300-RfxCas13d-crRNA recombinant vector specifically comprises the steps of artificially synthesizing a AtU6-crRNA gene fragment of 377bp, as shown in SEQ ID No.4, cloning the gene fragment to pUC57, and naming the pUC 57: AtU 6-crRNA; pUC57 was first treated with HindIII and SbfI: AtU6-crRNA and pCambia1300-RfxCas13d are subjected to double digestion, a 377bp fragment and a 12.5kb linearized vector skeleton are respectively recovered, and a 3003bp crRNA fragment is connected to pCambia1300-RfxCas13d by using T4 DNA ligase, so that the named pCambia1300-RfxCas13d-crRNA is obtained.

6. Use of a CRISPR/RfxCas13d plant RNA virus-resistant vector according to any of claims 1 to 3, characterized in that: the pCambia1300-RfxCas13d-crRNA was targeted according to the target.

7. Use of a CRISPR/RfxCas13d plant RNA virus-resistant vector according to claim 6, characterized in that: use of a CRISPR/RfxCas13d anti-plant RNA viral vector for silencing at least one of an endogenous gene in the genome of nicotiana benthamiana, and/or an exogenous RNA silencing of an RNA virus in the genome of a plant RNA virus.

8. Use of a CRISPR/RfxCas13d plant RNA virus-resistant vector according to claim 8, characterized in that: application in resisting against turnip mosaic virus.

9. Use of a CRISPR/RfxCas13d plant RNA virus-resistant vector according to claim 9, characterized in that:

1) transforming agrobacterium with the constructed vector, infiltrating leaves of the Nicotiana benthamiana plant after the positive identification of the agrobacterium, simultaneously inoculating infectious clone of the turnip mosaic virus, observing the phenotype and disease symptoms of the system leaves after 7 days, and sampling;

2) and (3) carrying out molecular detection after sampling, and detecting the change of the virus accumulation amount by using a Western blot method in a detection test on the protein level.

Technical Field

The invention relates to the technical field of biology, in particular to a high-efficiency site-specific editing CRISPR/RfxCas13d plant virus-resistant vector and a construction method and application thereof.

Background

The genome site-directed editing technology is an effective high-quality technical means, and can be used for plant functional genome research and crop molecular genetic breeding. The genome editing technology is mainly realized by the following three artificial endonucleases: zinc finger nucleases, ZFNs, transcription activator-like effector nucleases, TALENs, and RNA-guided endonucleases based on CRISPR/Cas systems. The gene editing process for DNA is the generation of double stranded DNA breaks (DSBs) at the genomic target site that can be repaired by non-homologous end joining (NHEJ) and homologous recombination (HDR). Endonucleases in the RNA-guided endonuclease system of CRISPR-Cas systems, such as SpCas9 and LbCpf1, have been demonstrated to be multifunctional tool enzymes for plant gene editing and regulation. RNA gene editing was developed in recent years to discover new Cas proteins (mainly Cas13 protein). Once cleaved, the RNA is directly degraded.

In nature, the phenomenon of plant virus infection of plants is quite common. Plant viruses can infect many important crops and are one of the important factors affecting agricultural production. Plant viruses are widely distributed around the world, with the exception of a small proportion of DNA viruses, the majority of plant viral genomes being single-stranded rna (ssrna) viruses. Among them, turnip mosaic virus (TuMV) is a single-stranded, positive-sense plant RNA virus belonging to potyviridae. TuMV can infect various vegetable crops and oil crops of the ten-seed flower family and is an important pathogen in agricultural production in China and the world. Currently, there is no effective means to control TuMV infestation.

Disclosure of Invention

The invention aims to provide a CRISPR/RfxCas13d plant RNA virus resistant vector, a construction method and application thereof.

The invention directly targets the RNA sequence of TuMV by utilizing a gene editing technology, so that the target RNA is cut and degraded, and the antiviral application is realized. The invention uses bacterial protein RfxCas13d protein from Ruminococcus flavefaciens strain XPD3002 to develop CRISPR plant genome editing tool based on RfxCas13d system. The tool can cut RNA more efficiently and recognize specific genome sequences more widely, and is a tool for resisting plant RNA viruses efficiently.

A CRISPR/RfxCas13d plant RNA virus resistant vector comprises a pCambia1300-RfxCas13d recombinant vector, and comprises the following components: CaMV 35S promoter, hygromycin gene, CaMV poly (a) signal terminator, pVS1 RepA, pVS1 origin of replication, CaMV 35S promoter, RfxCas13d gene, NOS terminator;

wherein, the RfxCas13d gene sequence is shown in SEQ ID No.1, and the NOS terminator sequence is shown in SEQ ID No. 2; the amino acid sequence coded by RfxCas13d is shown as SEQ ID No. 3.

The construction method of the pCambia1300-RfxCas13d recombinant vector specifically comprises the steps of artificially synthesizing a 3003bp RfxCas13d gene fragment, cloning the gene fragment to pUC57, and naming the fragment as pUC 57: RfxCas13 d; plasmid pUC57 was purified using BamHI and NcoI: performing double enzyme digestion on RfxCs 13d and pCambia1300-spCas9, recovering a 3003bp RfxCs 13d gene fragment and a pCambia1300 vector skeleton of about 10kb respectively, and connecting the 3003bp RfxCs 13d fragment to the pCambia1300 vector skeleton by using T4 DNA ligase, wherein the plasmid is named as pCambia-Rfxas 13 d; and (4) after sequencing the plasmid, storing the plasmid, and after verification is successful, transferring the plasmid to agrobacterium for storage.

The plasmid pCambia1300-RfxCas13d-crRNA was constructed as follows: CaMV 35S promoter, hygromycin gene, CaMV poly (a) signal terminator, pVS1 RepA, pVS1 origin of replication, CaMV 35S promoter, RfxCas13d gene, NOS terminator, AtU6 promoter, crRNA repeat leader sequence, spacer nucleotide sequence containing two BsaI cleavage sites, (T)8 terminator sequence.

The construction method of pCambia1300-RfxCas13d-crRNA recombinant vector specifically comprises the steps of artificially synthesizing a AtU6-crRNA gene fragment of 377bp, as shown in SEQ ID No.4, cloning the gene fragment to pUC57, and naming the pUC 57: AtU 6-crRNA; pUC57 was first treated with HindIII and SbfI: AtU6-crRNA and pCambia1300-RfxCas13d are subjected to double digestion, a 377bp fragment and a 12.5kb linearized vector skeleton are respectively recovered, and a 3003bp crRNA fragment is connected to pCambia1300-RfxCas13d by using T4 DNA ligase, so that the named pCambia1300-RfxCas13d-crRNA is obtained.

The application of CRISPR/RfxCas13d anti-plant RNA virus vector, pCambia1300-RfxCas13d-crRNA according to the target design. Taking crGFP as an example, a primer crGFP-F1 (shown as SEQ ID No. 5) is phosphorylated and then annealed with crGFP-R1 (shown as SEQ ID No. 6) to pCambia1300-RfxCas13d-crRNA, which is named pCambia1300-RfxCas13 d-crGFP.

Use of a CRISPR/RfxCas13d anti-plant RNA viral vector for silencing at least one of an endogenous gene in the genome of nicotiana benthamiana, and/or an exogenous RNA silencing of an RNA virus in the genome of a plant RNA virus.

Further, the application of the CRISPR/RfxCas13d plant RNA resistant virus vector in resisting turnip mosaic virus.

Compared with the prior art, the invention has the outstanding effects that:

TuMV is a pathogen seriously harming agricultural production, and no effective prevention and treatment means exists at present. The invention utilizes infectious clone TuMV-GFP to indicate that RNA virus naturally invades plants, and utilizes RfxCas13d protein and sgRNA expression vector to bind and cut virus RNA at specific positions. The vector constructed by the invention can effectively inhibit TuMV infection, provides a new strategy for preventing and controlling plant virus diseases, and has important production significance.

The recombinant vector pCambia1300-RfxCas13d constructed by the invention has the following characteristics and advantages:

(1) the bacterial protein RfxCas13d does not affect the normal physiological activities of plants;

(2) the protein can express specific Cas13d protein to be combined with sgRNA, and can be used for cutting certain specific sequences on RNA to realize accurate targeting;

(3) cas13d can cleave RNA, thus achieving site-directed cleavage of not only plant endogenously produced mRNA but also viral genomic RNA.

The CRISPR/RfxCas13d anti-plant RNA viral vector, the construction method and the application thereof are further described in the following description and specific examples in combination with the attached drawings.

Drawings

FIG. 1 is a schematic diagram of a vector for pCambia1300-RfxCas13d (Cas13 d).

FIG. 2 is a vector diagram of pCambia1300-RfxCas13d-crRNA (Cas13 d-crRNA).

FIG. 3 is the viral genome of a TuMV-GFP invasive clone with green fluorescent protein GFP in dark grey box.

FIG. 4 is a graph showing the inhibition of TuMV-GFP invasion and accumulation of TuMV Capsid Protein (CP) protein by pCambia1300-RfxCas13 d-crGFP.

(A) The co-inoculated TuMV-GFP invasive clone was applied to Nicotiana benthamiana plants with pCambia1300-RfxCas13d-crGFP (Cas13 d-crGFP, the same applies below) or with Cas13d, and photographs of the Nicotiana benthamiana plants were taken 10 days after inoculation using a portable UV lamp.

(B) Analyzing the accumulation amount of the CP protein in the TuMV by using the Western blot through Cas13d-crGFP compared with Cas13d, and using ponceau red stained Rubisco large subunit as a loading control, which shows that the Cas13d-crGFP can effectively reduce the accumulation amount of the CP in the TuMV.

Detailed Description