阅读说明：本技术 适用于表达异源蛋白的病毒载体的构建及其使用方法 (Construction of viral vectors suitable for expression of heterologous proteins and methods of use thereof ) 是由 谷红仓 许佩松 王云飞 车仙荣 于 2020-04-23 设计创作，主要内容包括：本发明提供了一种适用于表达异源蛋白的病毒载体的构建及其使用方法,其特征在于：菜豆荚斑驳病毒(BPMV)载体,其包含编码RNA2多蛋白开放阅读框(ORF)的核酸序列。本发明通过对质粒载体和病毒序列进行一系列修改,发明了一种BPMV全长的cDNA重组质粒载体pGG7R2。同时,在该质粒载体上引入荧光报告集团GFP蛋白,用于追踪和分析外源蛋白的表达水平。BPMV载体也可用于病毒诱导的基因沉默和异源多肽的表达方法。本发明还提供了病毒诱导的基因沉默方法,适用于确定目的基因的功能。(The invention provides a construction method of a virus vector suitable for expressing heterologous protein and a using method thereof, which are characterized in that: a phaseolopsis variegated virus (BPMV) vector comprising a nucleic acid sequence encoding an RNA2 polyprotein Open Reading Frame (ORF). The invention discloses a BPMV full-length cDNA recombinant plasmid vector pGG7R2 through carrying out a series of modifications on a plasmid vector and a virus sequence. Meanwhile, a fluorescent reporter group GFP protein is introduced into the plasmid vector and is used for tracking and analyzing the expression level of the foreign protein. BPMV vectors are also useful in methods of virus-induced gene silencing and expression of heterologous polypeptides. The invention also provides a virus-induced gene silencing method which is suitable for determining the function of a target gene.)

1. The virus vector construction method suitable for the expression of the exogenous genes in the soybeans is characterized by comprising the following steps: a phaseolopsis variegated virus (BPMV) vector comprising a nucleic acid sequence encoding an RNA2 polyprotein Open Reading Frame (ORF).

2. The method of claim 1, wherein the RNA2 polyprotein ORF sequence comprises a first and a second QM cleavage site, the difference between the nucleic acid sequences being sufficient to reduce homologous recombination between the nucleic acid sequences of the QM cleavage sites.

3. The method of claim 2, wherein the QM cleavage site is a GlinMet-containing protease cleavage site located between the Motor Protein (MP) and the coat protein (L-CP) encoded by the RNA2 polyprotein.

4. The method of claim 3, wherein each codon encoding the QM cleavage site differs between the nucleic acid sequences encoding the first QM cleavage site and the second QM cleavage site, but the amino acid sequences are the same.

5. The method of claim 2, wherein the QM cleavage site comprises 8 MP-derived C-terminal amino acids and 19L-CP-derived amino acids, and the amino acid sequence is QYNEVAQMELTNLFKLSLDDVETPKGS (SEQ ID NO: 1).

6. The vector of claim 2, wherein the vector comprises restriction enzyme sites for insertion of foreign sequences between the QM cleavage sites, and a BPMV full-length cDNA recombinant plasmid pGG7R2 is constructed.

7. The method as claimed in claim 7, wherein the plasmid pGG7R2 is modified and the fluorescent reporter protein GFP is introduced to construct a green fluorescent protein GFP infectious recombinant plasmid pCG7R2-GFP and pGHoR 2-GFP.

8. The method of claim 13, wherein the pGG7R2-GFP of GFP structure is modified to remove most of GFP sequence and insert two new endonuclease restriction sites to generate a new vector named pGG7R2-V in order to generate a suitable BPMV RNA2 vector for cloning and expressing foreign genes.

9. The method as claimed in claim 14, wherein the pGG7R2-V vector is modified to generate pGG7R2-Bar for evaluation of biological activity in plants in order to evaluate the biological activity of the inserted foreign gene.

10. The method of claim 14, wherein a method for virus-induced gene silencing in soybean plants and a vector pGG7R2-PDS useful for virus-induced gene silencing are constructed in order to study the role of BPMV vectors as virus-induced gene silencing (VIGS) in soybean gene function studies.

Technical Field

The invention relates to the field of Plant molecular biology, in particular to a construction method for expressing heterologous genes by Plant virus-based vectors.

Background

Plant viral vectors express foreign proteins in plants, providing a very promising biotechnological tool for supplementing conventional breeding and transgenic technologies. The use of viral vectors provides an attractive and economically efficient means for overproduction of valuable proteins and rapid evaluation of new traits in plants, given the rate of formation of viral infections throughout plants and the high yield of virus-encoded proteins accumulated in plants.

Several different types of positive-stranded RNA plant viruses have been developed as vectors for the production of recombinant proteins and polypeptides ((Pogue et al, Annu. Rev. Phytopathol.40: 45-74 (2002)). depending on the viral structure, genomic replication and methods of expression involved, a number of viral vector methods have been used, including gene replacement, gene insertion, epitope presentation and complementation Means for expressing novel properties of proteins that confer disease/pest resistance and/or enhance the commercial value of soybean.

Plant viral vectors provide valuable tools for expression of foreign proteins in plants and gene function studies. None of the previous viral vectors are suitable for use on soybean and, therefore, there is a need for a method that can be effectively used in plants such as soybean to express heterologous proteins and determine plant gene function. Although the CPMV-RNA2 vector is likely to be an expression vector for soybean, it is unstable and CPMV infection of soybean causes severe symptoms. Furthermore, soybean is not a natural host for CPMV, and thus, CPMV-based vectors cannot be released in the field in countries where CPMV is not prevalent in practical applications.

The present invention satisfies these needs and provides related advantages as well, including the construction of a legume mottle virus (BPMV) vector for the expression of heterologous proteins in plants such as soybean; BPMV vectors can also be used for virus-induced gene silencing; methods of expressing heterologous polypeptides in plants such as soybean.

Disclosure of Invention

The present invention addresses the deficiencies of the prior art by providing a pod mottle virus (BPMV) vector useful in plants such as soybean. The BPMV vector can be used for efficiently expressing heterologous proteins in plants such as soybeans. BPMV vectors are also useful for virus-induced gene silencing. The invention also provides methods for expressing heterologous polypeptides in plants such as soybean. The present invention provides the first plant viral vector suitable for expressing foreign proteins in soybean. The invention also provides methods of virus-induced gene silencing, particularly in soybean plants, which can be used to determine the function of a gene of interest. The BPMV vectors of the invention advantageously allow for efficient systemic expression of exogenous polypeptides and nucleic acids in soybean.

In order to achieve the above objects and other related objects, the technical solution of the present invention is as follows:

1. the invention provides a method for vector construction and in vitro transcription, in particular to construction of a BPMV full-length cDNA recombinant plasmid, and the specific scheme is as follows:

1) RNA extraction of BPMV strains KY-Hopkins 1(K-Hol), KY-Hancock 1(K-Hal) and KY-Graves 7 (K-G7);

2) first strand cDNA synthesis of viral RNA was performed using Superscript II reverse transcriptase kit (Invitrogen);

3) full-length cDNA clones of genomic RNA from strains K-G7 (group I) and K-Hal (group II) were constructed using RT-PCR method; constructing near full-length cDNA of RNA1 and RNA2 of which the 5' -end is 21 and 14 nucleotides from a strain K-Hol deletion;

4) designing a PCR primer to amplify cDNA, wherein the specific sequences of the 5' ends of the three strains are the same, and designing a primer addition promoter sequence for transcription expression;

further, since the 5' -end 46 nucleotides of both genomic RNAs were identical for all 3 strains, the cDNA amplification was performed using the same forward (sense) primer (F1) in each case.

Further, the F1 primer contained a modified T7 promoter sequence, an additional G base and 39 nucleotides from the 5' end of the genomic RNA. The reverse (antisense) primer (R1) contains 18 (dT) residues (complementary to the poly (a) tail of the viral RNA), a restriction enzyme SaII site and 21 additional nucleotides for efficient restriction enzyme digestion.

5) The purified PCR products (RNA 1cDNA of strain K-Hol and RNA2cDNA of all three strains) were cloned into pGEM-T easy vector to generate plasmids pGHoR1, pGHoR2, pGG7R2 and pGHaR2, respectively;

further, PCR products of RNA1cDNA of strains K-G7 and K-Hal were cloned into pCR2.1-TOPO vector (Invitro gen) to generate plasmids pCRG7R1 and PCRHAR1, respectively.

6) After in vitro transcription, the results of the transcription reaction mixtures were analyzed by electrophoresis on a 1% agarose gel to assess yield and quality;

further, unless otherwise indicated, pGHoR1 transcripts from plasmids of full-length infectious RNA1cDNA (type I, RNA 1) and transcripts from recombinant plasmids of full-length pGG7R2 or pGHoR2 were used in all inoculations.

2. The invention provides a construction method of a recombinant plasmid, in particular to the construction of a green fluorescent protein GFP recombinant plasmid, and the scheme is as follows:

1) amplifying a 5' -half fragment of BPMV RNA2cDNA from plasmids pGG7R2 or pGHoR2 by using a primer pair F1 and SWAL-REV R2, and cloning the PCR product into a pGEM-T Easy vector;

2) carrying out Swal and NcoI digestion on the clone obtained in the step 1), and selecting two clones pGG7R2-1 and pGHoR2-1 after restriction enzyme digestion and nucleotide sequencing verification;

3) clones pGG7R2-1 and pGHoR2-1 were digested with Aat II, blunt-ended and self-ligated to remove the Aat restriction site in the vector and generate new recombinant pGG7R2-2 and pGHoR 2-2;

4) plasmid pZGFP (Soldevila and Ghabrial, j.virol.74: 997-1003(2000)) as a template, and primers GFP-For and GFP-Rev amplify a GFP5 gene;

5) cloning the PCR product in the step 4) into a pGEM-T Easy vector, and sequencing to confirm that the PCR product is cloned pGGFP-1;

6) double digestion of recombinant pGG7R2-2 and pGHoR2-2 with Swal and SalI and ligation to the same digested pCGFP-1 to generate constructs pCG7R2-3 and pGHoR2-3, respectively;

7) amplifying a 3' -half fragment of BPMV RNA2cDNA from plasmids pGG7R2 or pGHoR2 by using a primer pair Aati I-for-R2 and R1, and cloning a PCR product onto a pGEM-T Easy vector to obtain clones pGG7R2-4 and pGHoR 2-4;

8) cloning pGG7R2-4 and pGHoR2-4 by digestion with SacI and PstI, blunt end and self-ligation, respectively, removing the SaII site of the vector to obtain the clones pGG7R2-5 and pGHoR2-5

9) Finally, clones pGG7R2-5 and pGHoR2-5 were digested with AatII and SalI, respectively, and smaller fragments were isolated and ligated to AatII/SalI digested pCG7R2-3 and pGHoR2-3, respectively, to construct infectious recombinant plasmids pCG7R2-GFP and pGHoR 2-GFP.

3. The invention provides a construction method of recombinant plasmid, in particular to the construction of DsRed recombinant plasmid, and the scheme is as follows:

1) using plasmid pDsRed2-C1(Clontech) as a template, and using primers RFP-For and RFP-Rev to amplify the DsRed target gene by PCR;

2) cloning the PCR product into a pGEM-T Easy vector to obtain a clone pGdsRed-1, and sequencing to confirm the clone pGdsRed-1;

3) the DSRed gene is released from pGdsRed-1 by double digestion and is connected to plasmids pGG7R2-GFP and pGHoR2-GFP which are digested by SwaI/AatII to replace the GFP gene, so that infectious recombinant plasmids pGG7R2-DsRed and pGHoR2-DsRed are constructed.

4. The invention provides a method for modifying a vector, in particular to a method for modifying a recombinant plasmid pCG7R2-GFP, which has the following scheme:

1) to generate a suitable BPMV RNA2 vector for cloning and expressing foreign genes, pGG7R2-GFP (fig. 1) of the GFP structure was modified to remove most of the GFP sequence and insert two new endonuclease restriction sites;

2) PCR amplification was performed using primers VecModi-for1 and VecModi-Rev1 partially annealed to each other, the product was cloned into a pGEM-T Easy vector, a BamHI cleavage site was introduced, and sequencing was confirmed to be pVecModi-1;

3) PCR amplification was performed using primers VecModi-For2 and VecModi-Rev2 that partially anneal to each other, the product was cloned into a pGEM-T Easy vector, MscI cleavage sites were introduced, and sequencing confirmed to be pVecModi-2;

4) carrying out double enzyme digestion on plasmid pGG7R2 GFP by using SwaI and MscI, separating out a larger fragment, and connecting the larger fragment to pVecModi-1 subjected to the same enzyme digestion to obtain plasmid pGG7R 2-6;

5) plasmid pGG7R2-6 was digested with ClaI and AatII and the large fragment isolated and ligated into pVecModi-2, which was likewise digested, to generate the BPMV-RNA2 vector, designated pGG7R 2-V;

5. the invention provides a construction method of a recombinant plasmid, in particular to the construction of the recombinant plasmid of a bar gene, and the scheme is as follows:

1) plasmid pBG-GD (Straubinger et al, future Genet. Newsl.39: 82-83(1992)) after BglII, Klenow large fragment DNA polymerase fill-in, BamHI cleavage, the bar gene encoding phosphinothricin acetyltransferase is released;

2) the DNA fragment containing the Bar gene was gel purified and ligated to MscI and BamHI-digested pGG7R2-V to obtain pGG7R 2-Bar.

6. The invention provides a construction method of recombinant plasmid, in particular to the construction of recombinant plasmid of RNA silencing inhibitor, the scheme is as follows:

1) a.the primer pair TBSV-P19-For and TBSV-P19-Rev was used to generate a primer pair from plasmid PZPTBSVvp 19 (Quet al, J.Virol.77: 511-522(2003)) to amplify the TBSV P19 gene; b. cloning the PCR product into a pGEM-T Easy vector, selecting a clone with a correct insertion direction, and carrying out BamHI and MscI double enzyme digestion; c. the enzyme-cleaved P19 gene was cloned into BamHI/MscI-cleaved pGG7R2-V to obtain pGG7R2-P19 recombinant plasmid.

2) amplifying a turnip shrunken virus Coat Protein (CP) gene from a plasmid PZP-TCVCP (Quet al, supra, 2003) by using a primer pair TCV-CP-For and TCV-CP Rev; b. cloning the PCR product into pGEM-T Easy vector, selecting clone with correct insertion direction, and carrying out BamHI and EcoRV double enzyme digestion. c. The CP gene released by enzyme digestion was cloned into BamHI/MscI digested pGG7R2-V to obtain pGG7R2-TCP recombinant plasmid.

3) a.from plasmid pTEV7D containing full-length cDNA of tobacco etch virus (TEV-RNA) with primer pair TEV-P2-For and TEV-P2-Rev (Dolja et al, Proc. Natl. Acad. Sci. U.S.A.89: 10208-10212(1992)) to amplify the coding region of Tobacco Etch Virus (TEV) HC-Pro; b. cloning the PCR product into a pGEM-T Easy vector, and carrying out enzyme digestion on the clone with the correct direction by using BamHI and EcoRV; c. the HC-Pro gene released by digestion was cloned into BamHI/MscI digested pGG7R2-V to obtain pGG7R2-HCPro (T) recombinant plasmid.

4) Cloning of the SMV HC-Pro coding region by RT-PCR: a. first strand cDNA synthesis was performed in total RNA of SMV strain G6 or G7 infected soybean leaf using a reverse primer (SMVBr); b. performing PCR amplification by using the first strand cDNA as a template and two pairs of amplification primers (SMV-Ar and SMV-Af, SMV-Br and SMV-Bf) respectively to amplify two overlapped cDNA fragments (the fragments A and B cover 5 'and 3' half parts respectively) containing the whole HC-Pro sequence; c. performing a second PCR amplification with equimolar amounts of fragments A and B, primers SMV-Br and SMV-Af.; d. cloning the PCR product into a pGEM-T Easy vector, and verifying the correctness of the cloning direction by sequencing; e. the genes derived from the G6 and G7 HCPro were digested with BamHI and EcoRV, respectively, and ligated to BamHI/MscI digested pCG7R2-V to obtain recombinant plasmids pGG7R2-HCPro (S6) and pGG7R2-HCPro (S7), respectively.

7. The invention provides a construction method of a recombinant plasmid, in particular to the construction of the recombinant plasmid of phytoene desaturase, and the scheme is as follows:

1) extracting genomic DNA from leaves of the soybean variety 'Essex' (Srinivasa et al, Phytopathology 91: 831-838 (2001));

2) performing PCR amplification on PDS-sen5-For and PDS-sen5-Rev by using primers to generate a PDS fragment of 318 bp;

3) the PCR product was digested with BamHI and EcoRV and ligated to BamHI/MscI digested pGG7R2-V to construct pGG7R2 PDS recombinant plasmid.

Drawings

FIG. 1 shows a schematic diagram of the construction of BPMV RNA2 vector.

FIG. 2 shows an immunoblot analysis of total protein from soybean plants infected with GFP vector. FIG. 2a shows Western blot analysis using antiserum against GFP. Total protein samples (15ug) were extracted from soybean plants as mock inoculations (lane 1), wild type BPMV K-G7 infection (lane 2), pGG7R2 GFP infection (3 and 4 lanes), and pGHoR2-GFP infection (5 and 6 lanes), respectively. Purified His 6-tagged GFP protein (50ng) was contained in lane 7. Lane M contains a low molecular weight protein Marker. In fig. 2 b. The protein loading tested in FIG. 2a was analyzed by SDS-PAGE and Coomassie blue staining.

FIG. 3 shows the stability of GFP and Dsked genes expressed from BPMV vectors. FIGS. 3a and 3b show Northern blot hybridization analysis for assessing insertion stability of foreign genes. RNA extracted from purified virions of soybean plants previously inoculated with the following viral isolates or transcripts was used: 1. wild type strain K-HO 1; 2, wild-type strain K-G7: 3, pGHoR1+ pGG7R2-GFP transcript; 4, pGHoR + pGG7R2 DsRed transcript; ghorr 1+ pGHoR2-GFP transcript: and 6, p.GHoR1+ pGHoR2-DsRed transcript. In FIG. 3a, a probe specific for K-HO 1RNA2 (type II) was used. In FIG. 3b, a K-G7RNA2 (type I) specific probe was used. Note that the recombinant RNA2 vector containing GFP or DsRed (channels 3-6) was larger in size than the wild-type RNA2 (channels 1 and 2). In fig. 4, RNA loading levels were assessed by ethidium bromide staining of viral RNA.

Figure 4 shows the severity of symptoms in soybean plants infected with BPMV vectors carrying known RNA silencing viral inhibitors. These photographs were taken 2 weeks after inoculation. Leaf extract used for infection in the figure was from pGHoR1 transcript plus from: mock inoculation (FIG. 4-1); pCG7R2 (FIG. 4-2); pGG7R2-P19 (FIGS. 4-3); pGG7R2-TCVCP (FIGS. 4-4): pGG7R2-HCPro (S7) (FIGS. 4-5); transcripts of pGG7R2-HCPro (T) (FIGS. 4-6).

FIG. 5 shows green fluorescence on leaves of inoculated soybean plants. After 4 generations of continuous passage, the extract of the leaves of the plant infected by BPMV-GFP is inoculated to the primary leaves of the soybean seedlings. Another approach is to inoculate primary leaves with wild-type K-HO-1 isolate or to simulate inoculation with buffer only. The first (fig. 5a) and second (fig. 5b) leaves of soybean plants previously inoculated with BPMV GFP vector showed strong green fluorescence under uv light. No fluorescence was detected on mock-inoculated primary leaves (FIG. 5c) or on the second third leaflet of K-HO-1 infected plants (FIG. 5 d). The leaves in FIGS. 5a, 5b and 5d show typical symptoms of infection with extract K-Hol, mosaic and necrosis on inoculated leaves, and mottle on systemic leaves. After 11 days of inoculation, all leaves were photographed under uv light.

Detailed Description

The invention proves that the vector based on the BPMV is suitable for the high-efficiency expression of the soybean foreign protein for the first time. In order to enable scientific researchers in the related field to better understand the scheme of the invention, the following detailed description is combined with the embodiment mode and the attached drawings. However, the present invention is not limited to the following embodiments.