CN115948453A

CN115948453A - Viral vector TRve delta for simultaneously expressing 3 non-fusion foreign proteins CP Method of construction of

Info

Publication number: CN115948453A
Application number: CN202211022069.5A
Authority: CN
Inventors: 廖乾生; 郭歌; 赖家良; 夏涛
Original assignee: Zhejiang Sci Tech University ZSTU
Current assignee: Zhejiang Sci Tech University ZSTU
Priority date: 2022-08-25
Filing date: 2022-08-25
Publication date: 2023-04-11

Abstract

The invention relates to the field of molecular biology, in particular to a Tobacco Rattle Virus (TRV) structureA construction method for simultaneously and rapidly expressing 3 non-fusion foreign proteins in the whole plant. The invention discloses a virus vector TRve delta simultaneously expressed in whole plants by using 3 SGP driving 3 exogenous genes in TRV genome RNA2 ^CP The method of (1). TRve Delta ^CP No obvious symptom reaction is generated in tomato, and TRve delta carrying 3 exogenous genes ^CP Systemically infecting host plants and simultaneously expressing the target protein. The invention firstly utilizes deletion substitution and virus subgenomic translation strategy to construct a plant virus vector TRve delta which can express 3 non-fusion proteins in whole host plants rapidly and in high content ^CP 。

Description

Viral vector TRve delta for simultaneously expressing 3 non-fusion foreign proteins CP Method of construction of

Technical Field

The invention relates to the field of molecular biology, in particular to a construction method for rapidly expressing 3 non-fusion foreign proteins in whole plants based on Tobacco Rattle Virus (TRV) construction.

Background

With the completion of sequencing of a large number of plant genomes, there is an urgent need for a good vector or technique to rapidly analyze new genes in the genome or predict the biological function of proteins. At present, the function of a gene or protein with unknown research function is confirmed by mainly expressing a target protein in a plant through a transgenic technology, but the transgenic operation is complicated, the period is long, the species is limited and other restrictive factors, and the biological function of the protein represented by a plant virus expression vector is increasingly a trend at present, and the gene or protein plays an important role in the research of plant functional genomics.

The virus replication/translation efficiency is high, a large amount of virus proteins are produced in plants, the genome is small and easy to operate, and a large amount of plant viruses are used as a source for constructing a foreign protein expression vector. The Potato Virus X (PVX) and the Tobacco Mosaic Virus (TMV) are currently the most commonly used viral expression vectors, and the construction strategy is to insert a Coat Protein (CP) subgenomic promoter (SGP) of the same virus or different members of the same virus into the genome of the virus to drive mRNA transcription of a foreign gene, and express a target protein by using a viral subgenomic translation strategy, but the PVX and TMV vectors can only express a single non-fusion foreign protein in the whole plant. At present, more and more basic and applied plant biology researches, such as functional identification of a compound consisting of a plurality of proteins, production of drug antibodies or polypeptides, need to simultaneously over-express a plurality of genes in the same single cell, and TMV and PVX expression vectors cannot meet the requirement. The binary expression vector constructed by inserting 2 CP SGPs into PVX and TRV genomes can simultaneously express 2 non-fusion foreign proteins in plants, because a plurality of extra SGPs cause sequence redundancy, the virus genome structure is easy to be unstable, and exogenously inserted genes are gradually lost in the plants along with the time, so that the 2 virus vectors cannot stably express 2 foreign proteins in the plants for a long time. The genome of beet necrotic yellow vein virus (BNYV) is composed of 5 RNA molecules, and a vector for simultaneously expressing 4 foreign proteins is constructed by adopting a peptidase-cleavage virus fusion protein strategy, but the virus vector can only express the foreign proteins in inoculated leaves of plants and can not move systematically, and the amino acid residues of peptidase enzyme are arranged at one end of the foreign target protein. Based on potyvirus (potyvirus) polymer proteolysis reversal strategy, single polyprotein translated by one Open Reading Frame (ORF) is hydrolyzed into a plurality of virus functional proteins, exogenous genes are inserted into potato virus A (potatoto virus A, PVA) Soybean Mosaic Virus (SMV) between P1/HC-Pro, HC-Pro/NIb and NIb/CP, and expression vectors constructed by turnip mosaic virus (TuMV) and other viruses can simultaneously express a plurality of exogenous proteins in the whole host plant, but the recombinant viruses have numerous defects of unexpected negative effects on the symptom development of the host plant after carrying the exogenous genes, the insertion of exogenous sequences breaks the structural integrity of the virus genome, and the fusion co-translation processing has unexpected effects on the activity and/or subcellular localization of target proteins. Many expression vectors for constructing multiple foreign proteins based on negative-strand RNA viruses have been reported to produce multiple non-fused foreign proteins simultaneously in the whole host plant, but these expression systems have certain disadvantages: for example, the expression of heterologous proteins requires a long time, the viral genome is large and unfavorable for genetic manipulation, and the virus inoculation process is complicated. At present, no better viral vector can express 3 non-fusion foreign proteins in whole plants at the same time at home and abroad.

TRV is a typical member of the genus Tobravir (Tobravir), has a broad host range, and can infect more than 400 kinds of plants, including model plants such as Arabidopsis thaliana, nicotiana benthamiana, important crops such as tomato and cotton. TRV is a positive sense single stranded RNA (+ single strand RNA, + ssRNA) virus, whose genome consists of RNA1 and RNA2 molecules. RNA1 encodes 4 proteins involved in viral replication, movement and symptom production; RNA2 encodes CP, 27kDa 2b and 18kDa 2c proteins by a subgenomic strategy. The 2b and 2c genes in TRV are completely deleted, and the virus can also replicate and move systematically in plants. At present, TRV is applied to the construction of virus-induced gene silencing (VIGS) vectors, vectors expressing 2 foreign proteins and guide RNA for transcription gene editing.

The invention of the invention (application No. 202110836772.9) applied by the inventor's team in the early days of the present application, which is a method for constructing a vector for simultaneously expressing two foreign proteins by using tobacco rattle virus, has reported that: agrobacterium infectious clone pYL156 based on TRV genomic RNA2, vector

pTRV2e containing TRV

2b and 2c SGP constructed by gene deletion strategy ³ Obtaining 2 non-fusion foreign protein recombinant viruses TRve which are simultaneously expressed in the whole Nicotiana benthamiana through 2 SGPs of the TRV ³ 。

Disclosure of Invention

The technical problem to be solved by the invention is to provide a virus vector TRve delta for quickly expressing 3 non-fusion foreign proteins in the whole plant ^CP The method of (1).

In order to solve the technical problems, the invention provides a viral vector TRve delta for simultaneously expressing 3 non-fusion foreign proteins ^CP The construction method (simultaneous expression of 3 non-fusion foreign protein vectors pTRV2e delta) ^CP Method for constructing (1): vector pTRV2e for simultaneously expressing 2 non-fusion foreign proteins ³ On the basis, a gene deletion strategy is adopted to construct CP, 2b and 2c SGP vector pTRV2e delta containing TRV ^CP 。

That is, the present invention separately produces mRNA of foreign insert protein by 3 SGPs of TRV genomic RNA2, and rapidly expresses 3 target proteins, pTRV2 e. Delta. In high content by using protein translation system of host plant ^CP The size of the middle genome RNA2 is 1496bp, and the sequence is shown as SEQID NO: 1.

The invention simultaneously expresses 3 non-fusion foreign protein vectors pTRV2e delta ^CP The construction method comprises the following steps:

using plasmid pTRV2e ³ In the vector pTRV2e ³ Deletion of the CP Gene ORF sequence in TRV genomic RNA2 and insertion of a multiple cloning site to obtain vector pTRV2e delta containing Multiple Cloning Sites (MCS) 1, 2 and 3 ^CP 。

Namely, the use of the recombinant plasmid pTRV2e ³ In pTRV2e ³ The ORF sequence of the CP gene lacking TRV is introduced into the vector, and the restriction enzyme cutting site is introduced, so as to obtain the vector pTRV2e delta containing cloning site at the downstream of CP, 2b and 2c SGP ^CP 。

The invention also provides the application of the pTRV2e delta ^CP Preparation of recombinant TRV expressing Green Fluorescent Protein (GFP) in the entire tomato plant by CP, 2b and 2c SGP, respectively, and Virus TRve. DELTA. ^CP -CP-GapC-GFP comprising the steps of:

1) Respectively amplifying GFP by PCR through 3 pairs of primers containing different enzyme cutting sites, respectively tapping and recovering target products, and cloning to plasmid pTRV2e delta through corresponding double enzyme cutting ^CP To obtain 3 vectors pTRV2e delta ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GFP-MCS3 and pTRV2 e. DELTA. ^CP -MCS1-MCS2-GFP；

2) PCR amplification of the ORF of the CP of TMV, double-restriction ligation to the plasmid pTRV2 e.DELTA ^CP To obtain pTRV2 e.DELTA ^CP -CP-MCS2-MCS3；

3) PCR amplification of Arabidopsis GapC, double restriction cloning to plasmid pTRV2e delta ^CP To obtain vector pTRV2 e.DELTA ^CP -MCS1-GapC-MCS3；

4) Cloning the PCR product of the TMV CP subjected to double digestion obtained in the step 2) to the vector pTRV2e delta obtained in the step 1) ^CP -MCS1-MCS2-GFP; obtaining the plasmid pTRV2 e.DELTA ^CP -CP-MCS2-GFP；

The PCR product of the double restriction enzyme GapC obtained in the step 3) is connected to a vector pTRV2e delta ^CP CP-MCS2-GFP vector pTRV2 e. DELTA. ^CP -CP-GapC-GFP；

5) The pTRV2e delta obtained in the step 4) ^CP Transformation of-CP-GapC-GFP into Agrobacterium GV3101 and mixing with Agrobacterium pTRV1 to obtain the virus TRve. DELTA. ^CP -CP-GapC-GFP。

Description of the invention:

in the above step 5), the present invention converts pTRV2e delta ^CP 、pTRV2eΔ ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP -MCS1-GFP-MCS3、pTRV2eΔ ^CP -MCS1-MCS2-GFP、pTRV2eΔ ^CP -CP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GapC-MCS3 and pTRV2 e. DELTA. ^CP -CP-GapC-GFP is transformed into Agrobacterium GV3101 respectively, and is mixed with Agrobacterium pTRV1 according to the ratio of 1: 1, and then is respectively infiltrated and inoculated into 2 cotyledons of tomato;

in the virus TRve. DELTA. ^CP -GFP-MCS2-MCS3、TRVeΔ ^CP MCS1-GFP-MCS3 and TRve Δ ^CP The green fluorescent phenotype was observed in the leaves of the upper system of tomato inoculated with MCS1-MCS2-GFP and GFP could be detected specifically by protein hybridization.

Virus TRve Delta ^CP CP-GapC-GFP infected leaves of the upper system of tomato were able to observe the green fluorescent phenotype and CP, gapC and GFP could be detected simultaneously by protein hybridization.

Namely, agrobacterium transformation of pTRV2 related vectors is set, and the agrobacterium pTRV1 and the agrobacterium pTRV are mixed for infiltration and inoculated to tomatoes respectively; recording of CP deleted recombinant Virus TRve Delta ^CP Infection of active, recombinant virus TRve Delta in tomato ^CP Wherein each SGP expresses GFP and expresses non-fused CP, gapC and GFP in the leaf of host system.

In the present invention:

extraction of TRve Delta ^CP Total protein in systemic leaves of-CP-GapC-GFP infected plants was used for Western blot analysis, and CP, gapC and GFP could be specifically and simultaneously detected in virus infected tomato.

The invention also provides the foreign protein expression vector TRve delta constructed by the method ^CP The application of (1): TRve Delta ^CP TRve delta carrying 3 foreign genes causing mild mosaic symptom reaction in host plant ^CP -CP-RFP-GFP expresses CP, gapC and GFP simultaneously throughout the plant;

the TRve Delta ^CP From pTRV1 and pTRV2e delta ^CP Forming;

the TRve Delta ^CP GFP-MCS2-MCS3 from pTRV1 and pTRV2e delta ^CP -GFP-MCS2-MCS3 composition;

the TRve Delta ^CP MCS1-GFP-MCS3 from pTRV1 and pTRV2 e. Delta ^CP -MCS1-GFP-MCS3 composition;

the TRve Δ ^CP MCS1-MCS2-GFP from pTRV1 and pTRV2 e. DELTA. ^CP -MCS1-MCS2-GFP composition;

the TRve Delta ^CP CP-MCS2-MCS3 from pTRV1 and pTRV2e delta ^CP -CP-MCS2-MCS3 composition;

the TRve Δ ^CP MCS1-GapC-MCS3 from pTRV1 and pTRV2 e. Delta ^CP -MCS1-GapC-MCS3 composition;

the TRve Delta ^CP -CP-GapC-GFP consisting of pTRV1 and pTRV2 e. Delta ^CP -CP-GapC-GFP composition.

The invention relates to a vector pTRV2e in the patent application No. 202110836772.9 ³ As a material, a vector pTRV2e delta containing CP, 2b and 2c SGP was constructed by deleting ORF of CP gene ^CP (ii) a Recombinant virus TRve delta ^CP After inoculation of the host plant, different non-fusion foreign proteins were expressed systemically by 3 subgenomic promoters, respectively.

The invention uses the plasmid pTRV2e ³ As a material, a CP, 2b and 2c subgenomic promoter vector pTRV2e delta containing TRV is constructed by adopting a gene deletion strategy ^CP (ii) a In the vector pTRV2 e.DELTA ^CP Respectively inserting 3 different exogenous genes into the downstream of each SGP, and constructing a virus vector TRVe delta for simultaneously expressing 3 non-fusion proteins in the whole plant of tomato ^CP 。

The invention uses vectors pYL156, ppk20 and pTRV2e ¹ And TRV2e ² For the TRV genomic RNA2 as a control, the detailed information is shown in https:// www.ncbi.nlm.nih.gov/nuccore/AF406991, https:// www.ncbi.nlm.nih.gov/nuccore/Z36974, and patent application No. 202110836772.9, respectively.

TRV genomic RNA2 encodes CP, 2b and 2c that are derived from 3 virusesThe subgenomic RNA molecule is translated and RNA2 lacks 2b and 2c, and the virus also systemically infects a number of host plants. The vector pTRV2e of the invention ³ The ORF of CP in (1) was deleted and a multiple cloning site was introduced to obtain plasmid pTRV2 e.DELTA ^CP . In vector pTRV2 e.DELTA ^CP The middle TRV genome RNA2 completely lacks the CP, 2b and CP ORFs, and only retains the respective subgenomic promoters. Recombinant virus TRve delta ^CP After infection of the plant, 4 RNA molecules are produced by replication of genomic RNA2, i.e. genomic RNA2, subgenomic RNA produced by CP SGP, subgenomic RNA produced by 2b SGP and 2c SGP subgenomic RNA. The invention is based on the virus TRve delta ^CP 3 SGPs in RNA2 of (1) to produce mRNA of the foreign protein, and expressing 3 non-fusion proteins of interest using a plant protein translation system.

The technical scheme of the invention is as follows:

1. in pTRV2e ³ The vector was deleted for CP ORF and introduced into the multiple cloning site to obtain pTRV2 e.DELTA ^CP A carrier;

description of the invention: pTRV2e ¹ And pTRV2e ³ The preparation method is specifically disclosed in patent 202110836772.9.

2. Plasmid pTRV2e delta ^CP After being transferred into agrobacterium GV3101, the bacillus is mixed with agrobacterium pTRV1 according to the proportion of 1: 1 to be infiltrated and inoculated into 2-leaf-stage tomatoes with TRve delta ^CP Does not cause significant symptoms in the host plant; with TRV, TRve ¹ And TRve ³ For control, host plants were observed for phenotypic changes after inoculation.

3. In the vector pTRV2 e.DELTA ^CP GFP is respectively inserted into the middle MCS1, MCS2 and MCS3 sites to obtain a vector pTRV2e delta ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GFP-MCS3 and pTRV2 e. DELTA. ^CP -MCS1-MCS2-GFP。

4. Construction of vector pTRV2 e. DELTA. ^CP -CP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GapC-MCS3 and pTRV2 e. DELTA. ^CP -CP-GapC-GFP, wherein CP is TMV coat protein gene and GapC is arabidopsis thaliana glyceraldehyde triphosphate dehydrogenase gene.

5. Agrobacterium transformation of pTRV 2-related plasmids and co-infiltration with TRV1, respectivelyEggplant (E); inoculation 10d, observation and recording of symptom response of tomato, GFP fluorescence phenotype and RNA hybridization detection of viral genome, results indicated: recombinant virus TRve delta ^CP Systemically infecting tomatoes, CP, 2b and 2c SGP all drive GFP expression throughout tomato plants.

6. Extracting total tomato protein, protein electrophoresis, membrane transfer and hybridization detection, and recombining virus TRve delta ^CP In the case of expressing foreign proteins in plants, protein hybridization shows: TRve Delta ^CP The vector can simultaneously express CP, gapC and GFP in the whole tomato plant.

The invention constructs a virus vector TRve delta which can express 3 non-fusion foreign proteins in tomato whole plants simultaneously and rapidly based on TRV ^CP . In the recombinant virus TRve delta ^CP The ORFs of CP, 2b and 2c in genomic RNA2 were completely deleted, all retaining 3 SGP cis-acting element sequences, and the deleted region was replaced with a different foreign gene without its structural integrity. The technical advantages are that: based on the characteristics of high virus propagation efficiency and capability of systematically moving, TRve delta ^CP The 3 non-fused exoproteins were expressed rapidly in high amounts throughout the host plant.

In conclusion, the invention adopts deletion substitution in plant virus expression vectors and a virus subgenomic translation strategy, and utilizes TRV to construct a virus vector which can express 3 non-fusion foreign proteins in whole plants rapidly and in high content.

The invention discloses a virus vector TRve delta simultaneously expressed in whole plants by using 3 SGP driving 3 exogenous genes in TRV genome RNA2 ^CP The method of (1). TRve Δ ^CP No obvious symptom reaction is generated in tomato, and TRve delta carrying 3 exogenous genes ^CP Systemically infecting host plants and simultaneously expressing the target protein. The invention constructs a plant virus vector TRve delta which can express 3 non-fusion proteins in whole host plants at high speed and high content for the first time by utilizing deletion substitution and a virus subgenomic translation strategy ^CP 。

The invention has the following beneficial effects: 1) The invention adopts the deletion replacement construction strategy of the plant virus expression vector; complete deletion of CP, 2b in TRV genomic RNA2And 2c gene, and 3 foreign genes, so as not to affect the structural integrity of the viral genome; 2) TRve Delta ^CP Tomato does not cause obvious symptoms, can be systematically expanded to the whole plant after carrying 3 exogenous genes, and has the characteristics of high expression quantity and quick expression time when expressing exogenous proteins in host plants.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a schematic diagram showing the structure of TRV genomic RNA2 in a pTRV 2-related vector;

FIG. 2 shows TRV and TRve ¹ 、TRVe ³ And TRve Δ ^CP A contrast graph of symptom response caused by infiltrating inoculated tomatoes for 10 d;

FIG. 3 shows TRV and TRve ¹ 、TRVe ³ And TRve Δ ^CP Northern blot photograph of genomic RNA 2;

in FIG. 3, rRNA is 28s RNA and was used to determine the loading of total RNA;

FIG. 4 shows TRve Δ ^CP Schematic representation of the driving of GFP expression by each SGP;

FIG. 5 shows infiltration inoculation 10d with TRve Δ ^CP Fluorescent photograph pictures of tomato plants after medium CP, 2b and 2c SGP respectively express GFP;

FIG. 6 shows the immersion inoculation 10d, protein hybridization assay TRve Delta ^CP Photographic images of the respective SGPs driving GFP expression;

in FIG. 6, rubisco is the large subunit of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) used to determine the loading amount of total plant protein;

FIG. 7 shows the recombinant virus TRve. DELTA. ^CP Schematic vector for simultaneous expression of CP, gapC and GFP in host plants;

FIG. 8 shows the detection of TRve. Delta. By Western blot ^CP Photographic images of simultaneous expression of CP, gapC and GFP throughout tomato;

in FIG. 8, rubisco is the large subunit of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) used to determine the loading of total plant protein.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1, pTRV2 e. DELTA. ^CP Vector construction

With plasmid pTRV2e ³ Materials, methods for their preparation are explicitly described in patent 202110836772.9. Deletion of the plasmid pTRV2e by PCR ³ CP of middle RNA2 to obtain pTRV2e delta ^CP And (3) a carrier.

With plasmid pTRV2e ³ Using a primer pair P1/P2 to carry out PCR amplification to obtain f1 as a template, using a primer pair P3/P4 to carry out PCR amplification to obtain f2, carrying out tapping recovery on 2 PCR amplification fragments, and using Hind III/Xba I double enzyme digestion vector pTRV2e ³ And tapping the rubber. PCR products 1, 2 and HindIII/XbaI were digested with pTRV2e according to the instructions for the use of the products ³ Passing of the vector

The Ultra One Step Cloning Kit (Vazyme) was ligated together and transformed into E.coli to obtain the vector pTRV2 e. Delta ^CP 。

The reaction system for PCR amplification of fragment 1 is: 5 XQ 5 reaction buffer 8. Mu.L, dNTP (2.5 mmol/L) 3.2. Mu.L, P1/P2 (10. Mu. Mol/L) each 2. Mu.L, pTRV2e ³ Template 10ng, Q5 polymerase (1U/. Mu.L) 0.4. Mu.L, ddH ₂ O is complemented to 40 mu L; the PCR reaction program is: at 98 ℃ 3min,98 ℃ 10s,58 ℃ 45s,72 ℃ 45s,35 cycles, at 72 ℃ 5min.

The reaction system for PCR amplification of fragment 2 is: 5 XQ 5 reaction buffer 8. Mu.L, dNTP (2.5 mmol/L) 3.2. Mu.L, P3/P4 (10. Mu. Mol/L) each 2. Mu.L, pTRV2e ³ Template 10ng, Q5 polymerase (1U/. Mu.L) 0.4. Mu.L, ddH ₂ O is complemented to 40 mu L; the PCR reaction program is: at 98 ℃ for 3min,98 ℃ for 10s,60 ℃ for 45s,72 ℃ for 10s,35 cycles, at 72 ℃ for 5min.

Primer P1: TTGGGCCCGGCGCGCCAAGCTTG

And (3) primer P2: TGATTGATTGATCGACAAATTCCCTTGTTGATTAGGACGCAAGCATGCAGTGATTCAGTAAC

And (3) primer P:

GATAGGTACGATGAATCAactagtCTCGAGgagctcGGTCCGATACGTCCTAATCCCTAG

the underlined in the primer P3 are Spe I, xho I and Sac I enzyme digestion sites in sequence;

primer P4: taacgcgtgaatttctctagaagc;

pTRV2eΔ ^CP vectors pYL156, pTRV2e involved in the preparation ¹ 、pTRV2e ³ Patent 202110836772.9 specifically reports that pTRV2e delta prepared by the invention ^CP And related vectors TRV genomic RNA2 are schematically shown in FIG. 1.

Example 2 expression of foreign proteins pTRV2 e. DELTA ^CP Construction of related vectors

2.1 construction of vectors for expressing GFP separately for CP, 2b and 2c SGP

GFP is amplified by 3 pairs of primers containing different enzyme cutting sites through PCR, target fragments are respectively recovered by tapping, and are respectively cloned to a vector pTRV2e delta through Spe I/Sac I, xba I/Mlu I and BamH I/Sma I ^CP To obtain vector pTRV2 e.DELTA ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GFP-MCS3 and pTRV2 e. DELTA. ^CP MCS1-MCS2-GFP, (see vector scheme 4). The size of GFP is 717bp, and the sequence is shown as SEQ ID NO. 2.

The GFP reaction system for PCR amplification is as follows: 5 XQ 5 reaction buffer 8. Mu.L, dNTP (2.5 mmol/L) 3.2. Mu.L, each of primer pairs P5/P6, P7/P8 or P9/P10 (10. Mu. Mol/L), GFP template 10ng, Q5 polymerase (1U/. Mu.L) 0.4. Mu.L, ddH ₂ O is complemented to 40 mu L; the PCR reaction program is: 98 ℃ 3min,98 ℃ 10s,58 ℃ 25s,72 ℃ 30s,35 cycles, 72 ℃ 5min.

Vector pTRV2 e.DELTA ^CP Primers corresponding to GFP-MCS2-MCS 3:

primer P5: gactagatATGAGTAAAGGAAGAACTTTTCACTG

Primer P6: gcgagcctatttgtatagttcatgcatgccat;

pTRV2eΔ ^CP -primers corresponding to MCS1-GFP-MCS 3:

and (3) primer P7: GCtctagaaTGAGTAAGAACTTTTCACTG

And (3) primer P8: CGacgcgtCTATTTGTATAGTTCATCCATGCCATGCCAT

pTRV2eΔ ^CP -primers corresponding to MCS1-MCS 2-GFP:

and (3) primer P9: CGggatccataTGAGTAAGGAGAACTTTCACTG

Primer P10: TCCccccgggcTATTTTGTATAGTTCATCCATGCCAT

The lowercase letters in the above primers represent enzyme cleavage sites, the same applies below.

2.2 construction of vectors related to expression of 3 foreign proteins simultaneously

Construction of the CP SGP vector pTRV2e delta for driving the expression of TMV CP cloning ^CP CP-MCS2-MCS3, 2b SGP drive glyceraldehyde triphosphate dehydrogenase gene (GapC) expression vector pTRV2e delta ^CP MCS1-GapC-MCS3 and vector pTRV2e delta for simultaneous expression of 3 foreign proteins ^CP CP-GapC-GFP, vector schematic as shown in FIG. 7.

2.2.1 vector pTRV2 e.DELTA ^CP Construction of-CP-MCS 2-MCS3 vector

The CP size of TMV is 480bp, and the sequence is shown as SEQ ID NO. 3.

The CP reaction system for the PCR amplification of TMV is as follows: 5 XQ 5 reaction buffer 8. Mu.L, dNTP (2.5 mmol/L) 3.2. Mu.L, each primer pair P11/12 (10. Mu. Mol/L) 2. Mu.L, the CP template of TMV 10ng, Q5 polymerase (1U/. Mu.L), ddH ₂ O is complemented to 40 mu L; the PCR reaction program is: at 98 ℃ for 3min, at 98 ℃ for 10s, at 60 ℃ for 15s, at 72 ℃ for 30s, for 35 cycles, at 72 ℃ for 5min. The PCR product is recovered by tapping and cloned to the vector pTRV2e delta through SpeI/SacI double enzyme digestion ^CP Obtaining the vector pTRV2e delta ^CP -CP-MCS2-MCS3。

Primer P11: gactagtATGTCCTTAACTACTCTCCGAG;

primer P12: GCgagcctaAGTAGCCGGAGTTGTGTGCC.

2.2.2 vector pTRV2 e.DELTA ^CP Construction of-MCS 2-GapC-MCS3

The sequence size of the arabidopsis thaliana glyceraldehyde triphosphate dehydrogenase gene (GapC) is 1017bp, and the sequence is shown as SEQ ID NO:4.

The GapC reaction system for PCR amplification of Arabidopsis thaliana is: 5 XQ 5 reaction buffer 8. Mu.L, dNTP (2.5 mmol/L) 3.2. Mu.L, each 2. Mu.L of primer pair P13/14 (10. Mu. Mol/L), arabidopsis GapC template 10ng, Q5 polymerase (1U/. Mu.L), ddH ₂ O is complemented to 40 mu L; the PCR reaction program is: at 98 ℃ 3min,910s at 8 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles at 72 ℃ for 5min. The PCR product is recovered by tapping and cloned to the vector pTRV2e delta through Xba I/Mlu I double enzyme digestion ^CP Obtaining the vector pTRV2e delta ^CP -MCS1-GapC-MCS3。

Primer P13: GCtctagaatggctgacagaaagattcagaatc

Primer P14: cgACGCGTctaagcgtaatctggaacatcgtatgggtaGGCCTTTGACATGTGAACG”，

The HA tag sequence is underlined in primer P14.

2.2.3 pTRV2eΔ ^CP Construction of-CP-GapC-GFP vector

The PCR product of the SpeI/SacI-digested TMV CP obtained in 2.2.1 was ligated to the plasmid pTRV2 e. Delta. Obtained in 2.1 ^CP MCS1-MCS2-GFP vector pTRV2 e. DELTA. ^CP -CP-MCS2-GFP；

The PCR product of the Arabidopsis thaliana GapC digested with XbaI/Mlu I in 2.2.2 was cloned into pTRV2 e. Delta ^CP -CP-MCS2-GFP to obtain pTRV2 e. Delta ^CP -CP-GapC-GFP vector.

EXAMPLE 3 Agrobacterium transformation and culture of pTRV 2-related vectors

Plasmid pTRV2e delta ^CP 、pTRV2eΔ ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP -MCS1-GFP-MCS3、pTRV2eΔ ^CP -MCS1-MCS2-GFP、pTRV2eΔ ^CP -CP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GapC-MCS3 and pTRV2 e. DELTA. ^CP Transforming Agrobacterium GV3101 with-CP-GapC-GFP to obtain Agrobacterium pTRV2e delta ^CP 、pTRV2eΔ ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP -MCS1-GFP-MCS3、pTRV2eΔ ^CP -MCS1-MCS2-GFP、pTRV2eΔ ^CP -CP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GapC-MCS3 and pTRV2 e. DELTA. ^CP -CP-GapC-GFP. Wherein the agrobacterium pTRV1, pYL156, pTRV2e ¹ And pTRV2e ³ The specific method for activating, expanding culture and collecting thallus refers to patent 201810261990.2. Centrifuging all overnight enlarged Agrobacterium culture solution at 5000r/min for 5min to collect thallus; 5mL of infiltration inoculation buffer (10 mmol/L MgCl) ₂ 10mmol/L MES and 200mmol/L acetosyringone), centrifuging at 5000r/min for 5min, and discarding the supernatant; finally, using infiltration inoculation buffer solutionThe cell concentration was adjusted to OD ₆₀₀ Is 0.2. Therefore, the final products obtained in this example 3 were Agrobacterium pTRV2 e. Delta. Respectively ^CP 、pTRV2eΔ ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP -MCS1-GFP-MCS3、pTRV2eΔ ^CP -MCS1-MCS2-GFP、pTRV2eΔ ^CP -CP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GapC-MCS3 and pTRV2 e. DELTA. ^CP -CP-GapC-GFP。

Example 4 Agrobacterium infiltration inoculation

The OD obtained in example 3 was taken ₆₀₀ pYL156, pTRV2e at 0.2 ¹ 、pTRV2e ³ 、pTRV2eΔ ^CP 、pTRV2eΔ ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP -MCS1-GFP-MCS3、pTRV2eΔ ^CP -MCS1-MCS2-GFP、pTRV2eΔ ^CP -CP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GapC-MCS3 and pTRV2 e. DELTA. ^CP 5mL of-CP-GapC-GFP agrobacterium liquid and 5mL of-CP-GapC-GFP agrobacterium liquid with concentration of 0.2OD ₆₀₀ Mixing the pTRV1 agrobacterium; pretreating the Agrobacterium mixture in the dark at room temperature for 4h, infiltrating the back of leaves of 2 pieces of leaf tomato with a syringe without needle and 1mL, and culturing all inoculated plants in a plant growth room at 25 deg.C in the dark for 16h and 8h in the light period.

Wherein,

the mixture of pTRV1 and pYL156 Agrobacterium is virus TRV,

pTRV1 and pTRV2e ¹ The Agrobacterium mixture is the virus TRve ¹ ，

pTRV1 and pTRV2e ³ The Agrobacterium mixture was TRve ³ ，

pTRV1 and pTRV2e delta ^CP The Agrobacterium mixture is the virus TRve delta ^CP ，

Other viruses are named and so on; namely:

pTRV1 and pTRV2e delta ^CP The mixture of GFP-MCS2-MCS3 Agrobacterium is TRve. DELTA. ^CP -GFP-MCS2-MCS3；

pTRV1 and pTRV2e delta ^CP The Agrobacterium mixture MCS1-GFP-MCS3 is TRve. DELTA. ^CP -MCS1-GFP-MCS3；

pTRV1 and pTRV2e delta ^CP Mixture of MCS1-MCS2-GFP Agrobacterium into TRve. DELTA. ^CP -MCS1-MCS2-GFP；

pTRV1 and pTRV2e delta ^CP -CP-MCS2-MCS3 Agrobacterium mixture TRve Delta ^CP -CP-MCS2-MCS3；

pTRV1 and pTRV2e delta ^CP The Agrobacterium mixture MCS1-GapC-MCS3 is TRve. DELTA. ^CP -MCS1-GapC-MCS3; pTRV1 and pTRV2e delta ^CP -CP-GapC-GFP Agrobacterium mixture TRve Delta ^CP -CP-GapC-GFP。

Example 5 recombinant TRV in tomato symptom response

Viruses TRV, TRve ¹ 、TRVe ³ And TRve Δ ^CP Respectively infiltrating and inoculating cotyledons of 2 cotyledons of tomatoes, and inoculating an infiltration buffer solution as a control Mock; the symptomatic response of the host plant after 10d was: at TRve ¹ 、TRVe ³ And TRve Δ ^CP Causing a light flower leaf phenotype, and causing necrosis of the apical systemic leaves of tomato by TRV (see FIG. 2), the recombinant virus TRve delta constructed by the present invention ^CP The tomato extract causes no obvious symptoms in tomato, and is beneficial to high-efficiency expression of exogenous target protein in plants.

Example 6 Northern blot assay of TRV genomic RNA in host

Infiltrating and inoculating Agrobacterium for 10d, and respectively taking viruses TRV and TRve ¹ 、TRVe ³ And TRve Δ ^CP 0.1g of upper systemic leaves of tomatoes are infected, and the total RNA of the plants is extracted by Trizol according to the specification of Trizol products. TRV genome Membrane transfer and hybridization methods referring to the product instructions of digoxin marker detection kit II (Roche, switzerland), hybridization probes were a stretch of nucleotides complementary to the 3' end of RNA2 (5 ' -CTTCAGACACGATCTACTTAAAGAACCGTTTAATGTCTTCGGGAC-DIG-3 '), synthesized by Shanghai bioengineering, inc.

The result of RNA hybridization detection shows that:

no viral genomic RNA, TRV, TRve could be detected in control Mock inoculated host plants ¹ 、TRVe ³ And TRve Δ ^CP Virus genome RNA is detected from infected plant system leaves, and the content of 4 virus genomes is not obviously different; at TRV and TRve ¹ Infected plants produce 3 RNA molecules (genomic RNA2, CP subgenomic RNA and 2b subgenomic RNA), TRVe ³ And TRve Δ ^CP Infection of the host plant results in genomic RNA2, CP subgenomic RNA, 2b subgenomic RNA, and 2c subgenomic RNA (see FIG. 3). The above results confirm TRve. DELTA. ^CP Tomato can be systemically infected and 3 subgenomic RNA molecules are produced.

Example 7 TRve. DELTA. In tomato ^CP Fluorescent phenotypic observations exhibited by individual SGPs driving GFP expression

TRVeΔ ^CP -GFP-MCS2-MCS3、TRVeΔ ^CP -MCS1-GFP-MCS3 and TRve Delta ^CP MCS1-MCS2-GFP inoculated tomato 10d, and the fluorescent phenotype in the systemic leaves of each virus-inoculated plant was observed in the dark using a long-wave portable ultraviolet lamp (Black Ray model B100 AP/R, uplank, USA), the results being shown in FIG. 5. FIG. 5 shows: 3 GFP carrying proteins at TRve. DELTA. ^CP The green fluorescent phenotype was produced in tomato, while the control TRve. DELTA. ^CP No GFP phenotype was observed in the inoculated and Mock plants. The above results indicate that the virus TRve Delta ^CP Middle CP, 2b and 2c SGP all drive GFP expression.

Example 8 Western blot analysis TRve. DELTA. ^CP Expression of foreign proteins in hosts

Infiltrating and inoculating Agrobacterium for 10d, and respectively taking virus TRve delta ^CP 、TRVeΔ ^CP -GFP-MCS2-MCS3、TRVeΔ ^CP -MCS1-GFP-MCS3、TRVeΔ ^CP -MCS1-MCS2-GFP、TRVeΔ ^CP -CP-MCS2-MCS3、TRVeΔ ^CP -MCS1-GapC-MCS3 and TRve Δ ^CP Inoculating 0.1g of tomato system leaves with-CP-GapC-GFP, grinding the tomato system leaves into powder by liquid nitrogen, adding PBS buffer solution containing 2% of beta-mercaptoethanol, grinding the tomato system leaves into uniform liquid, centrifuging, taking the supernatant, adding 2 × loading buffer, boiling in water bath at 95 ℃ for 10min, and taking the supernatant for later use at 10000r/min for 5min. Methods for protein transmembrane, antibody hybridization and substrate visualization reference is made to the compiled molecular biology laboratory guidelines (third edition), using polyclonal antibodies specific for GFP, TMV CP and HA tags.

At TRve Delta ^CP 、TRVeΔ ^CP -GFP-MCS2-MCS3、TRVeΔ ^CP -MCS1-GFP-MCS3、TRVeΔ ^CP (iii) MCS1-MCS2-GFP infecting systemic leaf samples of host plants, GFP antibody being specifically detectable for the target stripBelt, control inoculation TRve Δ ^CP No GFP signal was detected in tomato (see fig. 6), further demonstrating that CP, 2b and 2c SGP all drive GFP expression throughout the host.

To determine the recombinant virus TRve. DELTA. ^CP Whether 3 foreign proteins, TRve delta, can be expressed simultaneously in tomato ^CP -CP-MCS2-MCS3、TRVeΔ ^CP -MCS1-GapC-MCS3、TRVeΔ ^CP -MCS1-MCS2-GFP and TRve Delta ^CP And (3) infiltrating and inoculating the tomatoes with the-CP-GapC-GFP for 10d, and respectively extracting total proteins of systemic leaves infected by each virus for Western blot detection, wherein the result is shown in a figure 8. FIG. 8 shows: TRve Δ ^CP CP-GapC-GFP infected leaves of the tomato system can simultaneously detect CP, gapC and GFP, and TRve delta carrying a single exogenous gene ^CP Only one target protein can be detected by infection, which indicates that the recombinant virus TRve delta ^CP 3 non-fused foreign proteins can be simultaneously and rapidly expressed in a host plant.

It is to be noted that the above-listed are only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by the person skilled in the art from the present disclosure are to be considered within the scope of the present invention.

Claims

1. Viral vector TRve delta for simultaneously expressing 3 non-fusion foreign proteins ^CP The method for constructing (1) is characterized in that: vector pTRV2e for simultaneously expressing 2 non-fusion foreign proteins ³ As a material, a CP, 2b and 2c subgenomic promoter vector pTRV2e delta containing TRV is constructed by adopting a gene deletion strategy ^CP 。

2. The method of claim 1, wherein the plasmid pTRV2e is used ³ The method is characterized in that:

in the vector pTRV2e ³ Deletion of the ORF sequence of the CP gene in the TRV genomic RNA and insertion of a multiple cloning site to obtain a vector pTRV2e delta containing multiple cloning sites MCS1, MCS2 and MCS3 ^CP 。

3. By using as followsThe vector pTRV2 e.DELTA as claimed in claim 1 or 2 ^CP Preparation of a Virus TRve Delta capable of simultaneously expressing Tobacco Mosaic Virus (TMV) CP protein, arabidopsis thaliana glyceraldehyde triphosphate dehydrogenase GapC and GFP ^CP -CP-GapC-GFP method, characterized in that it comprises the following steps:

1) PCR amplification is carried out by 3 pairs of primers containing different enzyme cutting sites to PCR amplify GFP, and the PCR products are respectively cloned into the vector pTRV2e delta after different double enzyme cutting ^CP To obtain pTRV2 e.DELTA ^CP -GFP-MCS2-MCS3、pTRV2eΔ ^CP MCS1-GFP-MCS3 and pTRV2 e. DELTA. ^CP -MCS1-MCS2-GFP；

2) PCR amplification of the ORF of the CP of TMV, double ligation to the plasmid pTRV2 e.DELTA ^CP To obtain pTRV2 e.DELTA ^CP -CP-MCS2-MCS3；

3) PCR amplification of Arabidopsis thaliana glyceraldehyde triphosphate dehydrogenase gene (GapC), double restriction cloning to plasmid pTRV2e delta ^CP To obtain vector pTRV2 e.DELTA ^CP -MCS1-GapC-MCS3；

4) Cloning the PCR product of the CP subjected to double digestion obtained in the step 2) to the vector pTRV2e delta obtained in the step 1) ^CP Obtaining pTRV2 e. DELTA. In-MCS 1-MCS2-GFP ^CP -CP-MCS2-GFP；

Cloning the PCR product of GapC obtained by double enzyme digestion in step 3) to a vector pTRV2e delta ^CP -CP-MCS2-GFP to obtain vector pTRV2 e. Delta ^CP -CP-GapC-GFP；

4. The method of claim 3, further comprising the steps of:

5. The foreign protein viral vector TRVe delta constructed according to the method of claim 1 ^CP The use of (a), characterized by: TRve Delta ^CP TRve delta carrying 3 foreign genes causing mild mosaic symptom reaction in host plant ^CP CP-RFP-GFP expresses CP, gapC and GFP simultaneously in the whole plant.

6. Use according to claim 5, characterized in that:

the TRve Delta ^CP From pTRV1 and pTRV2e delta ^CP Forming;

the TRve Δ ^CP GFP-MCS2-MCS3 from pTRV1 and pTRV2e delta ^CP -GFP-MCS2-MCS3 composition;

the TRve Delta ^CP MCS1-MCS2-GFP from pTRV1 and pTRV2 e. DELTA. ^CP -MCS1-MCS2-GFP composition;

the TRve Delta ^CP CP-MCS2-MCS3 from pTRV1 and pTRV2e delta ^CP -CP-MCS2-MCS3 composition; the TRve Δ ^CP MCS1-GapC-MCS3 from pTRV1 and pTRV2 e. Delta ^CP -MCS1-GapC-MCS3 composition;

the TRve Delta ^CP CP-GapC-GFP consisting of pTRV1 and pTRV2e delta ^CP -CP-GapC-GFP composition.