CN115948453A - Construction method of viral vector TRVeΔCP expressing three non-fused foreign proteins simultaneously - Google Patents

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

Construction method of viral vector TRVeΔCP expressing three non-fused foreign proteins simultaneously

technical field

The invention relates to the field of molecular biology, and specifically comprises a construction method for simultaneously and rapidly expressing three non-fused foreign proteins in whole plants based on tobacco rattle virus (TRV).

Background technique

With the completion of a large number of plant genome sequencing, a good carrier or technology is urgently needed to quickly analyze the biological functions of new genes or predicted proteins in the genome. At present, the most important way to study genes or proteins with unknown functions is to express the target protein in plants through transgenic technology to confirm its function. Genomic function has increasingly become a trend, and has played an extremely important role in the study of plant functional genomics.

High virus replication/translation efficiency produces a large number of viral proteins in plants, and the genome is small and easy to manipulate. A large number of plant viruses are used as a source for constructing foreign protein expression vectors. Potato virus X (PVX) and tobacco mosaic virus (TMV) are currently the most commonly used viral expression vectors, and their construction strategy is to insert the same virus or different members of the same genus virus into the virus genome. The coat protein (CP) subgenomic promoter (subgenomic promoter, SGP) is used to drive the mRNA transcription of foreign genes, and the viral subgenomic translation strategy is used to express the target protein, but PVX and TMV vectors can only express a single non- Fusion foreign protein. At present, more and more basic and applied plant biology research, such as the functional identification of complexes composed of multiple proteins, the production of drug antibodies or peptides, need to overexpress multiple genes simultaneously in the same single cell, TMV and None of the PVX expression vectors can meet this requirement. A binary expression vector constructed by inserting two CP SGPs into the genomes of PVX and TRV can simultaneously express two non-fused foreign proteins in plants. Due to sequence redundancy caused by multiple additional SGPs, it is easy to cause viral genome The structure is unstable, and the exogenously inserted genes are gradually lost in plants over time, so the two viral vectors cannot stably express the two foreign proteins in plants for a long time. The genome of beet necrotic yellow vein virus (BNYV) is composed of 5 RNA molecules. A vector expressing 4 foreign proteins simultaneously was constructed by using the strategy of peptidase cleavage virus fusion protein, but the virus vector can only be used in plants. The exogenous protein is expressed in the inoculated leaves, which cannot move systematically, and there is an amino acid residue of peptidase at one end of the exogenous target protein. Based on the proteolytic translation strategy of potyvirus multimers, a single polyprotein translated from an open reading frame (ORF) is hydrolyzed into multiple viral functional proteins, and foreign genes are inserted into P1/HC Potato virus A (potato virus A, PVA), soybean mosaic virus (SMV) and turnip mosaic virus (TuMV) among -Pro, HC-Pro/NIb and NIb/CP, etc. Expression vectors constructed from viruses can simultaneously express multiple foreign proteins in the entire host plant, but these recombinant viruses carry foreign genes and have unexpected negative effects on the development of symptoms of the host plant, and the insertion of foreign sequences destroys There are many deficiencies in the integrity of the viral genome structure, fusion co-translational processing, and unintended effects on the intrinsic activity and/or subcellular localization of target proteins. At present, a number of expression vectors for constructing multiple foreign proteins based on negative-strand RNA viruses have been reported, which can simultaneously produce multiple non-fused foreign proteins in the entire host plant, but these expression systems have certain shortcomings: for example, heterologous The expression of protein takes a long time, the large viral genome is not conducive to genetic manipulation, and the virus inoculation process is complicated. At present, there is no better viral vector at home and abroad that can express three non-fused foreign proteins simultaneously and rapidly in the whole plant.

TRV is a typical member of the genus Tobravirus. It has a wide host range and can infect more than 400 kinds of plants, including model plants Arabidopsis, Nicotiana benthamiana, and important crops such as tomato and cotton. TRV is a positive-sense single-stranded RNA (+single strand RNA, +ssRNA) virus, and its genome consists of RNA1 and RNA2 molecules. The four proteins encoded by RNA1 are involved in virus replication, movement and symptom generation; RNA2 encodes CP, 27kDa 2b and 18kDa 2c proteins through a subgenomic strategy. The 2b and 2c genes in TRV are completely deleted, and the virus can still replicate and move systematically in plants. TRV is currently used to construct virus induced gene silencing (VIGS) vectors, express two foreign protein vectors and transcribe guide RNA for gene editing.

The inventor's team's early application of the invention "Construction Method for Simultaneously Expressing Two Foreign Proteins Using Tobacco Fragile Virus" (application number 202110836772.9) informed that the Agrobacterium invasive clone pYL156 based on TRV genomic RNA2, through gene deletion Strategies Construct the vector pTRV2e ³ containing the 2b and 2c SGP of TRV, and obtain the recombinant virus TRVe ³ that simultaneously expresses two non-fused foreign proteins in the whole Nicotiana benthamiana through the two SGPs of TRV itself.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for constructing a viral vector ^TRVeΔCP that rapidly expresses three non-fused foreign proteins simultaneously in the whole plant.

In order to solve the above-mentioned technical problems, the present invention provides a construction method for simultaneously expressing three non-fusion foreign protein viral vectors ^TRVeΔCP (construct method for simultaneously expressing three non-fusion foreign protein vectors ^pTRV2eΔCP ): On the basis of the vector pTRV2e ³ which is not fused with foreign proteins, the gene deletion strategy was used to construct the CP, 2b and 2cSGP vector pTRV2eΔ ^CP containing TRV.

That is, the present invention produces mRNAs of exogenously inserted proteins through the three SGPs of TRV genomic RNA2, and uses the protein translation system of the host plant to express three target proteins rapidly and at high levels. The size of genomic RNA2 in ^pTRV2eΔCP is 1496bp, and the sequence As described in SEQ ID NO:1.

The construction method of simultaneously expressing three non-fused foreign protein vectors ^pTRV2eΔCP of the present invention:

Using the plasmid pTRV2e ³ , the ORF sequence of the CP gene in TRV genomic RNA2 was deleted in the vector pTRV2e ³ and a multiple cloning site was inserted to obtain the vector pTRV2eΔ ^CP containing multiple clone sites (multiple clone site, MCS) 1, 2 and 3.

That is, by using the recombinant plasmid pTRV2e ³ , the pTRV2e ³ vector lacks the ORF sequence of the CP gene of TRV, and introduces restriction sites to obtain the vector pTRV2eΔ ^CP that contains cloning sites downstream of CP, 2b and 2c SGP.

The present invention also provides the recombinant TRV that uses the above-mentioned ^pTRV2eΔCP to prepare green fluorescent protein (GFP) respectively in the whole tomato plant through CP, 2b and 2c SGP, and can simultaneously express TMV coat protein (CP), Arabidopsis three The viral TRVeΔCP- ^CP -CP-GapC-GFP of glyceraldehyde phosphate dehydrogenase (GapC) and GFP, comprising the following steps:

1) GFP was amplified by PCR with 3 pairs of primers containing different restriction sites, the target product was recovered by tapping the gel respectively, and cloned into the plasmid ^pTRV2eΔCP by corresponding double restriction enzyme digestion to obtain 3 vectors ^pTRV2eΔCP -GFP-MCS2-MCS3, ^pTRV2eΔCP -MCS1-GFP-MCS3 and ^pTRV2eΔCP -MCS1-MCS2-GFP;

2) Amplify the ORF of the CP of TMV by PCR, and connect it to the plasmid ^pTRV2eΔCP by double enzyme digestion to obtain pTRV2eΔCP- ^CP -MCS2-MCS3;

3) PCR amplified Arabidopsis GapC, cloned into plasmid ^pTRV2eΔCP by double enzyme digestion, and obtained vector ^pTRV2eΔCP -MCS1-GapC-MCS3;

4) Cloning the PCR product obtained in step 2) into the vector ^pTRV2eΔCP -MCS1-MCS2-GFP obtained in step 1) through double digestion of TMV CP; obtaining the plasmid pTRV2eΔCP ^-CP -CP-MCS2-GFP;

Ligate the PCR product obtained in step 3) through double digestion of GapC to the vector pTRV2eΔCP- ^CP -MCS2-GFP to obtain the vector pTRV2eΔCP- ^CP -CP-GapC-GFP;

5) Transform pTRV2eΔCP- ^CP -GapC-GFP obtained in step 4) into Agrobacterium GV3101, and mix with Agrobacterium pTRV1 to obtain virus TRVeΔCP- ^CP -CP-GapC-GFP.

illustrate:

In the above step 5), the present invention uses ^pTRV2eΔCP , ^pTRV2eΔCP -GFP-MCS2-MCS3, ^pTRV2eΔCP -MCS1-GFP-MCS3, ^pTRV2eΔCP -MCS1-MCS2-GFP, pTRV2eΔCP- ^CP -MCS2-MCS3, pTRV2eΔ ^CP -MCS1-GapC-MCS3 and pTRV2eΔCP- ^CP -GapC-GFP were respectively transformed into Agrobacterium GV3101, and mixed with Agrobacterium pTRV1 according to 1:1, respectively infiltrated and inoculated on two cotyledons of tomato;

The green fluorescent phenotype can be observed in the upper system leaves of tomato infiltrated by viruses TRVeΔ ^CP -GFP-MCS2-MCS3, TRVeΔ ^CP -MCS1-GFP-MCS3 and TRVeΔ ^CP -MCS1-MCS2-GFP, and can be specifically detected by protein hybridization Detect GFP definitively.

The green fluorescence phenotype can be observed in the upper system leaves of tomato infected by virus TRVeΔ ^CP -CP-CP-GapC-GFP, and CP, GapC and GFP can be detected simultaneously by protein hybridization.

That is, the Agrobacterium transformation of the pTRV2-related vector was set, and each was mixed with Agrobacterium pTRV1 and infiltrated in tomato; the infection activity of the recombinant virus ^TRVeΔCP lacking CP in tomato was recorded, and each SGP in the recombinant virus ^TRVeΔCP expressed GFP respectively situation, and the molecular detection results of simultaneous expression of non-fused CP, GapC and GFP in leaves of the host system.

In the present invention:

The total protein in leaves of TRVeΔ ^CP -CP-CP-GapC-GFP-infected plants was extracted for Western blot analysis, and CP, GapC and GFP could be detected specifically and simultaneously in virus-infected tomato.

The present invention also provides the use of the exogenous protein expression vector ^TRVeΔCP constructed by the above method at the same time: ^TRVeΔCP causes light mosaic symptoms in the host plant, and TRVeΔCP- ^CP -RFP-GFP carrying three exogenous genes in The whole plant expresses CP, GapC and GFP simultaneously;

The TRVeΔ ^CP consists of pTRV1 and pTRV2eΔ ^CP ;

The ^TRVeΔCP -GFP-MCS2-MCS3 is composed of pTRV1 and ^pTRV2eΔCP -GFP-MCS2-MCS3;

The ^TRVeΔCP -MCS1-GFP-MCS3 is composed of pTRV1 and ^pTRV2eΔCP -MCS1-GFP-MCS3;

The ^TRVeΔCP -MCS1-MCS2-GFP consists of pTRV1 and ^pTRV2eΔCP -MCS1-MCS2-GFP;

The TRVeΔCP- ^CP -MCS2-MCS3 is composed of pTRV1 and pTRV2eΔCP- ^CP -MCS2-MCS3;

The ^TRVeΔCP -MCS1-GapC-MCS3 is composed of pTRV1 and ^pTRV2eΔCP -MCS1-GapC-MCS3;

The TRVeΔCP- ^CP -CP-GapC-GFP consists of pTRV1 and pTRV2eΔCP- ^CP -CP-GapC-GFP.

The present invention uses the vector ^pTRV2e3 in the patent application number 202110836772.9 as a material, and constructs the vector ^pTRV2eΔCP containing CP, 2b and 2c SGP by deleting the ORF of the CP gene; after the recombinant virus ^TRVeΔCP is inoculated into the host plant, it passes three subgenomic promoters Different non-fusion foreign proteins were systematically expressed respectively.

The present invention uses the plasmid ^pTRV2e3 as a material, adopts a gene deletion strategy to construct the CP, 2b and 2c subgenomic promoter vector ^pTRV2eΔCP containing TRV; inserts 3 different exogenous genes in the downstream of each SGP of the vector ^pTRV2eΔCP , and constructs them in tomato The viral vector ^TRVeΔCP expresses three non-fusion proteins simultaneously in the whole plant.

The present invention uses the TRV genomic RNA2 of the vectors pYL156, Ppk20, pTRV2e ¹ and TRV2e ² as a control. For detailed information, see https://www.ncbi.nlm.nih.gov/nuccore/AF406991, https://www.ncbi. nlm.nih.gov/nuccore/Z36974 and patent application number 202110836772.9.

CP, 2b, and 2c encoded by TRV genomic RNA2 are translated from three viral subgenomic RNA molecules, and RNA2 lacks 2b and 2c, and the virus still systematically infects many host plants. In the present invention, the ORF of CP in the vector pTRV2e ³ is deleted, and a multiple cloning site is introduced to obtain the plasmid pTRV2eΔ ^CP . In the vector ^pTRV2eΔCP, the ORFs of CP, 2b and CP were completely deleted in TRV genomic RNA2, and only the respective subgenomic promoters were retained. After the recombinant virus ^TRVeΔCP infects plants, four RNA molecules are produced after replication from genomic RNA2, namely genomic RNA2, subgenomic RNA produced by CP SGP, subgenomic RNA produced by 2b SGP and subgenomic RNA produced by 2c SGP. The present invention uses three SGPs in the RNA2 of the virus ^TRVeΔCP to produce the mRNA of the foreign protein, and uses the plant protein translation system to express the three non-fusion proteins of interest.

Technical scheme of the present invention is specifically as follows:

1. Delete the ORF of CP in the pTRV2e ³ vector, and introduce multiple cloning sites to obtain the ^pTRV2eΔCP vector;

Explanation: The preparation methods of pTRV2e ¹ and pTRV2e ³ are clearly informed in the patent 202110836772.9.

2. After the plasmid ^pTRV2eΔCP was transferred into Agrobacterium GV3101, it was mixed with Agrobacterium pTRV1 at a ratio of 1:1 and co-infiltrated and inoculated on two cotyledon stage tomatoes. ^TRVeΔCP did not cause obvious symptoms in the host plant; with TRV, TRVe ¹ and TRVe ³ were used as controls to observe the phenotypic changes of host plants after inoculation.

3. Insert GFP into the MCS1, MCS2 and MCS3 sites of the vector ^pTRV2eΔCP respectively to obtain the vectors ^pTRV2eΔCP -GFP-MCS2-MCS3, ^pTRV2eΔCP -MCS1-GFP-MCS3 and ^pTRV2eΔCP -MCS1-MCS2-GFP.

4. Construct vectors pTRV2eΔ ^CP -CP-MCS2-MCS3, pTRV2eΔ ^CP -MCS1-GapC-MCS3 and pTRV2eΔ ^CP -CP-GapC-GFP, wherein CP is the TMV coat protein gene, and GapC is the glyceraldehyde triphosphate desorbent gene of Arabidopsis thaliana hydrogenase gene.

5. Agrobacterium transformation of pTRV2-related plasmids, and respectively mixed with TRV1 to infiltrate tomatoes; 10 days after inoculation, observe and record tomato symptom response, GFP fluorescence phenotype and virus genome RNA hybridization detection, the results show that: the recombinant virus ^TRVeΔCP system When sexually infecting tomato, CP, 2b and 2c SGP can all drive the expression of GFP in the whole tomato plant.

6. Tomato total protein extraction, protein electrophoresis, membrane transfer and hybridization detection, recombinant virus ^TRVeΔCP expressing exogenous protein in plants, protein hybridization showed that: ^TRVeΔCP vector can simultaneously express CP, GapC and GFP in the whole tomato plant.

The present invention constructs a virus vector TRVeΔ ^CP that simultaneously and rapidly expresses three non-fused foreign proteins in whole tomato plants based on TRV. In the recombinant virus ^TRVeΔCP genomic RNA2, the ORFs of CP, 2b and 2c were completely deleted, and three SGP cis-acting element sequences were retained, and the deleted regions were replaced by different foreign genes without structural integrity. The technical advantages are: based on the characteristics of high virus reproduction efficiency and the ability to move systematically, TRVeΔ ^CP expresses three non-fused ectoproteins rapidly and at high levels in the whole host plant.

To sum up, the present invention adopts the strategy of deletion replacement and viral subgenome translation in plant virus expression vectors, and utilizes TRV to construct a viral vector that expresses three non-fused foreign proteins at the same time, rapidly and at high levels in the whole plant.

The invention discloses a construction method for simultaneously expressing viral vector ^TRVeΔCP in whole plants by using three SGPs in TRV genome RNA2 to drive three foreign genes. TRVeΔ ^CP did not produce obvious symptoms in tomato, and TRVeΔ ^CP carrying 3 exogenous genes systematically infected host plants and expressed the target protein at the same time. For the first time, the present invention uses deletion replacement and viral subgenome translation strategies to construct a plant virus vector TRVeΔ ^CP that expresses three non-fusion proteins rapidly and at high levels simultaneously in the whole host plant.

The present invention has the following beneficial effects: 1) The present invention adopts the deletion and replacement construction strategy of the plant virus expression vector; completely deletes the ORFs of the CP, 2b and 2c genes in the TRV genomic RNA2, and replaces them with 3 foreign genes, thereby not affecting the virus Genome structural integrity; 2) ^TRVeΔCP does not cause obvious symptoms in tomato, and can be systematically extended to the whole plant after carrying 3 foreign genes, and the expression of foreign proteins in the host plant has the characteristics of high expression amount and fast expression time.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 is a schematic diagram of the structure of TRV genome RNA2 in pTRV2-related vectors;

Figure 2 is a comparison chart of symptoms and responses caused by TRV, TRVe ¹ , TRVe ³ and ^TRVeΔCP infiltration inoculated with tomato for 10 days;

Fig. 3 is the Northern blot photo graph of TRV, TRVe ¹ , TRVe ³ and ^TRVeΔCP genome RNA2;

In Fig. 3, rRNA is 28s RNA, is used for determining the sample amount of total RNA;

Figure 4 is a schematic diagram of the expression of GFP driven by each SGP ^{of TRVeΔCP} ;

Fig. 5 is the fluorescence photograph of tomato plants after 10 days of infiltration and inoculation, using CP, 2b and 2c SGP in ^TRVeΔCP to express GFP respectively;

Fig. 6 is a photograph of GFP expression driven by each SGP of ^TRVeΔCP detected by protein hybridization at 10 days after infiltration and inoculation;

In Fig. 6, Rubisco is the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Ribulose bisphosphatecarboxylase oxygenase, Rubisco), which is used to determine the loading amount of total plant protein;

Fig. 7 is a vector schematic diagram of the recombinant virus ^TRVeΔCP expressing CP, GapC and GFP simultaneously in the host plant;

Fig. 8 is the photograph diagram that Western blot detects that ^TRVeΔCP expresses CP, GapC and GFP simultaneously in whole tomato;

In Fig. 8, Rubisco is the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is used to determine the loading amount of total plant protein.

Detailed ways

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1, ^pTRV2eΔCP vector construction

Using the plasmid pTRV2e ³ material, its preparation method is clearly informed in the patent 202110836772.9. The CP of RNA2 in the plasmid ^pTRV2e3 was deleted by PCR to obtain the ^pTRV2eΔCP vector.

Ultra One Step Cloning Kit(Vazyme)连接在一起，并转化至大肠杆菌中，获得载体pTRV2eΔ^CP。Using the plasmid pTRV2e ³ as a template, the primer pair P1/P2 was used for PCR amplification to obtain f1, and the primer pair P3/P4 was used for PCR amplification to obtain f2, and the 2 PCR amplified fragments were recovered by tapping gel, and HindⅢ/XbaⅠ double enzyme digestion vector pTRV2e ³ and tap rubber. According to the product instructions, PCR products 1 and 2 and HindⅢ/XbaⅠ digested pTRV2e ³ vector were passed

The Ultra One Step Cloning Kit (Vazyme) was ligated together and transformed into Escherichia coli to obtain the vector pTRV2eΔ ^CP .

The reaction system of PCR amplified fragment 1 is: 5×Q5 reaction buffer 8μL, dNTP (2.5mmol/L) 3.2μL, P1/P2 (10μmol/L) each 2μL, pTRV2e ³ template 10ng, Q5 polymerase (1U/μL) 0.4 μL, supplemented with ddH ₂ O to 40 μL; PCR reaction program: 98°C for 3 min, 98°C for 10 s, 58°C for 45 s, 72°C for 45 s, 35 cycles, 72°C for 5 min.

The reaction system for PCR amplified fragment 2 is: 5×Q5 reaction buffer 8μL, dNTP (2.5mmol/L) 3.2μL, P3/P4 (10μmol/L) 2μL each, pTRV2e ³ template 10ng, Q5 polymerase (1U/μL) 0.4 μL, supplemented with ddH ₂ O to 40 μL; PCR reaction program: 98°C for 3 min, 98°C for 10 s, 60°C for 45 s, 72°C for 10 s, 35 cycles, 72°C for 5 min.

Primer P1: TTGGGCCCGGCGCGCCAAGCTTG

Primer P2: TGATTGATCGTACAAATCTCCCTTGTTGATTAGCGCAAGTGATTCAGTAAC

Primer P3:

GATAGGTACGATGAATCA actagtCTCGAGgagctc GGTCCGATACGTCCTAATCCCTAG

The underlined sequences in primer P3 are SpeI, XhoI and SacI restriction sites;

Primer P4: TAACGCGTGAATTCTCTAGAAAGC;

The vectors pYL156, pTRV2e ¹ , and pTRV2e ³ involved in the preparation of pTRV2eΔ ^CP are clearly informed in the patent 202110836772.9. The schematic diagram of the structure of TRV genomic RNA2 in pTRV2eΔ ^CP and related vectors prepared by the present invention is shown in Figure 1.

Example 2. Construction of ^pTRV2eΔCP -related vectors expressing foreign proteins

2.1 Construction of CP, 2b and 2c SGP vectors expressing GFP respectively

GFP was amplified by PCR with 3 pairs of primers containing different restriction sites, and the target fragments were recovered by tapping the gel, and cloned into the vector ^pTRV2eΔCP by SpeI/SacI, XbaI/MluI and BamHI/SmaI, respectively, to obtain the vector ^pTRV2eΔCP -GFP-MCS2 -MCS3, ^pTRV2eΔCP -MCS1-GFP-MCS3 and ^pTRV2eΔCP -MCS1-MCS2-GFP, (see vector schematic 4). The size of GFP is 717bp, and the sequence is as SEQ ID NO:2.

The PCR amplification GFP reaction system is: 5×Q5 reaction buffer 8μL, dNTP (2.5mmol/L) 3.2μL, primer pair P5/P6, P7/P8 or P9/P10 (10μmol/L) each 2μL, GFP template 10ng, Q5 polymerase (1U/μL) 0.4 μL, ddH ₂ O to make up to 40 μL; PCR reaction program: 98°C for 3min, 98°C for 10s, 58°C for 25s, 72°C for 30s, 35 cycles, 72°C for 5min.

Primers corresponding to the vector ^pTRV2eΔCP -GFP-MCS2-MCS3:

Primer P5: GactagtATGAGTAAAGGAGAAGAACTTTTCACTG

Primer P6: GCgagctcCTATTTGTATAGTTCATCCATGCCAT;

Primers corresponding to pTRV2eΔCP- ^MCS1 -GFP-MCS3:

Primer P7: GCtctagaATGAGTAAAGGAGAAGAACTTTTCACTG

Primer P8: CGacgcgtCTATTTGTATAGTTCATCCATGCCAT

Primers corresponding to ^pTRV2eΔCP -MCS1-MCS2-GFP:

Primer P9: CGggatccATGAGTAAAGGAGAAGAACTTTTCACTG

Primer P10: TCCcccgggCTATTTGTATAGTTCATCCATGCCAT

The lowercase letters in the above primers represent restriction sites, and the following are similar.

2.2 Construction of related vectors for simultaneous expression of three foreign proteins

Construct the expression vector pTRV2eΔ ^CP -CP-MCS2-MCS3 of CP SGP-driven TMV clone expression, 2b SGP-driven glyceraldehyde triphosphate dehydrogenase gene (GapC) expression vector pTRV2eΔ ^CP -MCS1-GapC-MCS3 and simultaneously express three foreign genes The source protein vector pTRV2eΔ ^CP -CP-GapC-GFP, the schematic diagram of the vector is shown in FIG. 7 .

2.2.1 Construction of vector pTRV2eΔCP- ^CP -MCS2-MCS3 vector

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

The CP reaction system for PCR amplification of TMV is: 5×Q5 reaction buffer 8μL, dNTP (2.5mmol/L) 3.2μL, primer pair P11/12 (10μmol/L) 2μL each, TMV CP template 10ng, Q5 polymerase (1U /μL) 0.4 μL, made up to 40 μL with ddH ₂ O; PCR reaction program: 98°C for 3 min, 98°C for 10 s, 60°C for 15 s, 72°C for 30 s, 35 cycles, 72°C for 5 min. The PCR product was recovered by tapping rubber, and cloned into the vector ^pTRV2eΔCP by SpeI/SacI double enzyme digestion to obtain the vector pTRV2eΔCP- ^CP -MCS2-MCS3.

Primer P11: GactagtATGCCTTATACAATCAACTCTCCGAG;

Primer P12: GCgagctcCTAAGTAGCCGGAGTTGTGGTCC.

2.2.2 Construction of vector ^pTRV2eΔCP -MCS2-GapC-MCS3

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

The GapC reaction system for PCR amplification of Arabidopsis thaliana is: 5×Q5 reaction buffer 8μL, dNTP (2.5mmol/L) 3.2μL, primer pair P13/14 (10μmol/L) 2μL each, Arabidopsis GapC template 10ng, Q5 Polymerase (1U/μL) 0.4 μL, ddH ₂ O to make up to 40 μL; PCR reaction program: 98°C for 3 min, 98°C for 10 s, 56°C for 30 s, 72°C for 30 s, 35 cycles, 72°C for 5 min. The PCR product was recovered by tapping rubber, and cloned into the vector pTRV2eΔ ^CP by Xba Ⅰ/Mlu Ⅰ double enzyme digestion to obtain the vector pTRV2e Δ ^CP -MCS1-GapC-MCS3.

Primer P13: GCtctagaATGGCTGACAAGAAGATCAGAATC

Primer P14: cgACGCGT ctaagcgtaatctggaacatcgtatgggtaGGCCTTTGACATGTGAACG ",

The underlined sequence in primer P14 is the HA tag sequence.

2.2.3 Construction of pTRV2eΔCP- ^CP -GapC-GFP vector

Ligate the PCR product of TMV CP obtained by SpeI/SacI digestion in 2.2.1 to the plasmid ^pTRV2eΔCP -MCS1-MCS2-GFP obtained in 2.1 to obtain the vector pTRV2eΔCP- ^CP -MCS2-GFP;

The PCR product of Arabidopsis GapC digested with XbaI/MluI in 2.2.2 was cloned into pTRV2eΔCP- ^CP -CP-MCS2-GFP to obtain the pTRV2eΔCP- ^CP -CP-GapC-GFP vector.

Agrobacterium transformation and cultivation of embodiment 3, pTRV2 related vector

Plasmids ^pTRV2eΔCP , ^pTRV2eΔCP -GFP-MCS2-MCS3, ^pTRV2eΔCP -MCS1-GFP-MCS3, ^pTRV2eΔCP -MCS1-MCS2-GFP, pTRV2eΔCP- ^CP -MCS2-MCS3, ^pTRV2eΔCP -MCS1-GapC-MCS3, and pTRV2eΔ ^CP -CP-GapC-GFP transformed Agrobacterium GV3101 to obtain Agrobacterium pTRV2eΔ ^CP , pTRV2eΔ ^CP -GFP-MCS2-MCS3, pTRV2eΔ ^CP -MCS1-GFP-MCS3, pTRV2eΔ ^CP -MCS1-MCS2-GFP, pTRV2eΔ ^CP -CP- MCS2-MCS3, ^pTRV2eΔCP -MCS1-GapC-MCS3 and pTRV2eΔCP- ^CP -GapC-GFP. Among them, the specific methods of Agrobacterium pTRV1, pYL156, pTRV2e ¹ and pTRV2e ³ and their activation, expansion culture and cell collection refer to the invention patent 201810261990.2. All overnight expanded Agrobacterium cultures were collected by centrifugation at 5000r/min for 5min; 5mL infiltration inoculation buffer (10mmol/L MgCl ₂ , 10mmol/L MES and 200mmol/L acetosyringone) was used to suspend the bacteria, centrifuged at 5000r/min for 5min, The supernatant was discarded; finally, the concentration of each bacterial cell was adjusted to OD ₆₀₀ of 0.2 with infiltration inoculation buffer. Therefore, the final products obtained in Example 3 are respectively Agrobacterium ^pTRV2eΔCP , ^pTRV2eΔCP -GFP-MCS2-MCS3, ^pTRV2eΔCP -MCS1-GFP-MCS3, ^pTRV2eΔCP -MCS1-MCS2-GFP, pTRV2eΔCP- ^CP -MCS2- MCS3, ^pTRV2eΔCP -MCS1-GapC-MCS3 and pTRV2eΔCP- ^CP -GapC-GFP.

Embodiment 4, Agrobacterium infiltration inoculation

_pYL156 , pTRV2e ¹ , pTRV2e ³ , pTRV2eΔ ^CP , pTRV2eΔ ^CP -GFP-MCS2-MCS3, pTRV2eΔ ^CP -MCS1-GFP-MCS3, pTRV2eΔ CP -MCS1-MCS2-GFP, pTRV2eΔ ^CP -MCS1-MCS2-GFP, 5 mL of pTRV2eΔ ^CP -CP-MCS2-MCS3, pTRV2eΔ ^CP -MCS1-GapC-MCS3 and pTRV2eΔ ^CP -CP-GapC-GFP Agrobacterium liquid were mixed with 5 mL of pTRV1 Agrobacterium with a concentration of 0.2OD ₆₀₀ ; After pretreatment at room temperature and darkness for 4 hours, infiltrate the back of 2 cotyledon tomato leaves with a needle-free 1 mL sterilized syringe, and place all inoculated plants in a plant growth room at 25°C with a light period of 16 hours and a dark period of 8 hours.

in,

The mixture of pTRV1 and pYL156 Agrobacterium is the virus TRV,

The mixture of pTRV1 and pTRV2e ¹ Agrobacterium is virus TRVe ¹ ,

pTRV1 and pTRV2e ³ Agrobacterium mixture is TRVe ³ ,

The mixture of pTRV1 and ^pTRV2eΔCP Agrobacterium is the virus ^TRVeΔCP ,

Other viruses are named in the same way; namely:

The mixture of pTRV1 and ^pTRV2eΔCP -GFP-MCS2-MCS3 Agrobacterium is ^TRVeΔCP -GFP-MCS2-MCS3;

The mixture of pTRV1 and ^pTRV2eΔCP -MCS1-GFP-MCS3 Agrobacterium is ^TRVeΔCP -MCS1-GFP-MCS3;

The mixture of pTRV1 and ^pTRV2eΔCP -MCS1-MCS2-GFP Agrobacterium is ^TRVeΔCP -MCS1-MCS2-GFP;

The mixture of pTRV1 and pTRV2eΔCP- ^CP -MCS2-MCS3 Agrobacterium is ^TRVeΔCP -CP-MCS2-MCS3;

The mixture of pTRV1 and pTRV2eΔCP ^- MCS1-GapC-MCS3 is ^TRVeΔCP- MCS1-GapC-MCS3; the mixture of pTRV1 and pTRV2eΔCP- ^CP -CP-GapC-GFP is TRVeΔCP- ^CP -CP-GapC-GFP.

Embodiment 5, recombinant TRV in tomato symptom response

Viruses TRV, TRVe ¹ , TRVe ³ and ^TRVeΔCP were respectively infiltrated and inoculated on two cotyledon stage tomato cotyledons, and the infiltration buffer was inoculated as a control Mock; the symptom response of the host plant after 10 days was: in TRVe ¹ , TRVe ³ and ^TRVeΔCP Cause light mosaic phenotype, and TRV then causes the systematic leaf of tomato top to produce necrosis symptom (see Fig. 2), the recombinant virus TRVeΔ ^CP that the present invention constructs does not cause obvious symptom in tomato, helps its efficient in plant Express foreign protein of interest.

Example 6, Northern blot detection of TRV genomic RNA in the host

10 days after Agrobacterium infiltration and inoculation, virus TRV, TRVe ¹ , TRVe ³ and ^TRVeΔCP were respectively used to infect 0.1 g of tomato upper system leaves, and the total plant RNA was extracted with Trizol. The method was carried out by referring to the Trizol product manual. For the TRV genome transfer and hybridization methods, refer to the product instruction manual of the Digoxigenin Labeling Detection Kit II (Roche, Switzerland). '), synthesized by Shanghai Bioengineering Co., Ltd.

The results of RNA hybridization detection showed:

No virus genome RNA could be detected in the host plants inoculated with Mock, but virus genome RNA was detected in the leaves of plants infected with TRV, TRVe ¹ , TRVe ³ and ^TRVeΔCP , and there was no significant difference in the genome content of the four viruses; ¹ Infected plants produce 3 RNA molecules (genomic RNA2, CP subgenomic RNA and 2b subgenomic RNA), and TRVe ³ and ^TRVeΔCP infect host plants to produce genomic RNA2, CP subgenomic RNA, 2b subgenomic RNA and 2c Subgenomic RNA (see Figure 3). The above results confirmed that ^TRVeΔCP can systemically infect tomato and produce three subgenomic RNA molecules.

Example 7. Observation of fluorescence phenotypes presented by each SGP of ^TRVeΔCP driving GFP expression in tomato

TRVeΔ ^CP -GFP-MCS2-MCS3, TRVeΔ ^CP -MCS1-GFP-MCS3 and TRVeΔ ^CP -MCS1-MCS2-GFP inoculated tomatoes for 10 days, and used a long-wave portable ultraviolet lamp (Black Ray model B 100AP/R, Upland , USA) to observe the fluorescence phenotypes in the systemic leaves of each virus-inoculated plant, the results are shown in Figure 5. Figure 5 shows that three GFP-carrying plants produced a green fluorescent phenotype in ^TRVeΔCP tomato plants, while no GFP phenotype was observed in plants inoculated with control ^TRVeΔCP and Mock. The above results indicated that CP, 2b and 2c SGP in virus ^TRVeΔCP could all drive the expression of GFP.

Example 8, Western blot analysis ^TRVeΔCP expresses foreign protein in the host

After 10 days of Agrobacterium infiltration and inoculation, the viruses TRVeΔ ^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 TRVeΔCP- ^CP -GapC-GFP inoculated tomato system leaves 0.1g, ground into powder with liquid nitrogen, added PBS buffer containing 2% β-mercaptoethanol and ground to a homogeneous liquid, centrifuged Take the supernatant and add 2×loading buffer, cook in a water bath at 95°C for 10 minutes, 10000r/min for 5 minutes, take the supernatant for later use. The methods of protein transfer, antibody hybridization and substrate color development refer to the refined Molecular Biology Experiment Guide (Third Edition), and the antibodies used are polyclonal antibodies specific for GFP, TMV CP and HA tags.

In the systematic leaf samples of TRVeΔ ^CP , TRVeΔ ^CP -GFP-MCS2-MCS3 , TRVeΔ ^CP -MCS1-GFP-MCS3 , TRVeΔ ^CP -MCS1-MCS2-GFP infected host plants, the GFP antibody can specifically detect the target band , while no GFP signal could be detected in ^TRVeΔCP tomato (see Figure 6), which further indicated that CP, 2b and 2c SGP could all drive the expression of GFP in the whole host.

In order to determine whether the recombinant virus TRVeΔ ^CP could simultaneously express three foreign proteins in tomato, TRVeΔ ^CP -CP-MCS2-MCS3, TRVeΔ ^CP -MCS1-GapC-MCS3, TRVeΔ ^CP -MCS1-MCS2-GFP and TRVeΔ ^CP -CP -GapC-GFP was infiltrated and inoculated with tomato for 10 days, and the total leaf protein of each virus-infected system was extracted for Western blot detection. The results are shown in Figure 8. Figure 8 shows: TRVeΔ ^CP -CP-CP-GapC-GFP infected tomato system leaves can detect CP, GapC and GFP at the same time, TRVeΔ ^CP infection carrying a single exogenous gene can only detect one target protein, indicating that the recombinant virus TRVeΔ ^CP can simultaneously and rapidly express three non-fused foreign proteins in host plants.

It should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (6)

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；

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。

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

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.

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-GFP-MCS3 from pTRV1 and pTRV2 e. Delta ^CP -MCS1-GFP-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.

Images