CN110295192B - Bivalent RNAi expression vector for constructing TYLCV and ToCV by Gateway technology and application thereof - Google Patents

Abstract

The invention discloses a bivalent RNAi expression vector for constructing TYLCV and ToCV by using Gateway technology and application thereof. The bivalent RNAi expression vector is named pRNAi-TOCV-TY. The nucleotide sequence of the RNAi expression vector is shown as SEQ ID NO. 5. According to the invention, a great deal of research work is carried out by utilizing RNAi technology, a double RNAi expression vector of the coat protein genes of TYLCV and ToCV is constructed by using Gateway technology, and transgenic tomato plants resistant to two viruses are obtained by experimental methods such as genetic transformation, resistance identification and the like. The invention further discloses application of the pRNAi-TOCV-TY expression vector in effectively preventing and treating tomato infected by ToCV and TYLCV. In particular to the application of pRNAi-TOCV-TY expression vector in effectively preventing the replication of tomato viruses in plants.

Description

Bivalent RNAi expression vector for constructing TYLCV and ToCV by Gateway technology and application thereof

Technical Field

The invention provides a novel plant material for quickly and simply constructing an anti-TYLCV and ToCV virus bivalent RNAi expression vector by using Gateway technology and inhibiting infection of the TYLCV and ToCV viruses. Belongs to the technical field of genetic engineering.

Background

Tomato (tomato)Lycopersicon esculentum Mill.) is one of the main vegetable crops cultivated in facilities in China, and plays an important role in annual supply of vegetables. However, with the growing expansion of tomato planting area and the diversification of introduction channels, tomato virus diseases are increasingly serious, and become the bottleneck for restricting tomato development.

At present, tomato chlorosis virus @Tomato chlorosis virus ToCV) and tomato yellow leaf curl virusTomato yellow leaf curl virus,TYLCV) The damage to tomatoes is most serious, and complex infection often occurs, aggravates symptoms and causes serious reduction of yield and quality. At present, the tomato yellow leaf curl virus is mainly cultivated by using wild resistance resources. This approach is not capable of tomato chlorosis, as so far no resistance gene for this virus has been found worldwide. Therefore, the cultivation of tomato chlorosis resistant viruses has become a difficult problem to be solved urgently.

In recent years, RNAi silencing technology is used as a defense mode for resisting foreign gene invasion, and becomes the most effective method in genetic engineering disease-resistant breeding. The method for constructing the plant RNAi expression vector basically comprises two methods, namely, constructing a traditional plant RNAi expression vector based on enzyme digestion connection; another plant RNAi expression vector based on homologous recombination is constructed. The construction method of the homologous recombination RNAi expression vector represented by Gateway is widely applied by the characteristics of high efficiency and rapidness. Research on cultivating antiviral varieties by the method also reports that stable disease-resistant strains are obtained by using coat protein genes of cucumber green mottle mosaic virus (CGMCCV) such as Kamachi and the like; yan Peijiang et al constructed RNAi vectors using the coat protein gene of TMV and transformed into tobacco, and transgenic plants exhibited good resistance. However, in tomato, TYLCV and ToCV frequently undergo complex infection, and it is necessary to construct vectors against both viruses to develop tomato material with multiple resistances.

The invention utilizes Gateway technology to construct bivalent RNAi plant expression vector of the coat protein gene conservation section of TYLCV and ToCV, further converts tomato cultivation antiviral material, and provides new resources for disease resistance breeding of tomato.

Disclosure of Invention

The tomato yellow leaf curl virus and tomato chlorosis virus are frequently infected by a compound. Because the tomato chlorosis has no resistance source, the current control of the disease can only prevent the propagation of bemisia tabaci, but the bemisia tabaci has small volume and strong drug resistance, and the method is difficult to be effective in tomato production. Aiming at the problems, the inventor performs a great deal of research work by utilizing RNAi technology, constructs a double RNAi expression vector of the coat protein genes of TYLCV and ToCV by using Gateway technology, and obtains transgenic tomato plants resisting two viruses by using experimental methods such as genetic transformation, resistance identification and the like.

In order to achieve the above object, the present invention discloses a TYLCV and ToCV bivalent RNAi expression vector constructed by Gateway technology, which is named pRNAi-TOCV-TY. The nucleotide sequence of the RNAi expression vector is shown in SEQ ID NO. 5:

RNAi sequence:

atggagaaca gtgctgttgc aaacactggt gataacggtg gtggccgcaa tcctctggtt

agaccgttag atgatggcgt agatgacgag gtgcagaact tgggcaggag ggacgattcg

acatctctta ttccggctaa tcctaatcga tcttccagtt gggctttgtt gaacccggat

actattaatt ataacgagtt aaggaaattg aaggtacact ccactagggg tgatactctt

accttgactc aggaagagga gttcgagaag atactcgaat ccttttgcag gcgaataatc

ggtgagaccc cgatgacgga taagattttc gctggtttct acatgtctat gtgtcaggcc

attgtaaacc aagggacctc agttaaagca cccggtaata acagtcttga aaactacttt

gaggtagatg gtgatg tcgaagcg ac caggcgatat aatcatttcc acgcccgcct cgaaggttcg

ccgaaggctg aacttcgaca gcccatacag cagccgtgct gctgtcccca ttgtccaagg

cacaaacaag cgacgatcat ggacgtacag gcccatgtac cggaagccca gaatatacag

aatgtatcga agccctgatg ttcctcgtgg atgtgaaggc ccatgtaaag tccagtctta

tgagcaacgg gatgatatta agcatactgg tattgttcgt tgtgttagtg atgttactcg

tggatctgga attactcaca gagtgggtaa gaggttctgt gttaaatcga tatatttttt

aggtaaagtc tggatggatg aaaatatcaa gaagcagaat cacactaatc agg

the invention further discloses a construction method of pRNAi-TOCV-TY expression vector, which is characterized by comprising the following steps:

(1) Extracting nucleic acid from tomato leaf, namely collecting tomato leaf with TYLCV disease, and extracting total DNA; tomato leaves with ToCV pathogenesis are collected, RNA is extracted by using a DALIANBAO biological company (TaKaRa) RNA extraction kit, and cDNA is synthesized by reverse transcription by using a random primer Oligo dT.

(2) Acquisition of ToCV and TYLCV CP Gene fragments

Respectively selecting CP gene conservation sequence regions of ToCV and TYLCV, designing synthetic primers, such as SEQ ID NO. 1-4, and amplifying the CP fragments of ToCV by the primers ToCV-F/R; primer TY-F/R amplifies CP fragment of TYLCV;

(3) Obtaining of tandem ToCV and TYLCV CP

Mixing the PCR products of the TYLCCP and the ToCVCP, performing PCR amplification by using primers ToCV-F and TY-R to obtain a tandem gene ToCV-TY fragment of the TYLCCP and the ToCVCP, performing electrophoresis on the amplified product by using 1% agarose gel, recovering the obtained tandem gene fragment to be named as TOCV-TY, and transferring the TOCV-TY connection into a pMD18-T vector to obtain a recombinant plasmid P-TOCV-TY;

(4) Acquisition of tandem Gene with attB linker

PCR was performed using the recombinant plasmid P-ToCV-TY as a template and attB-TOCV-F and attB-TY-R primers, and B sequences were added upstream and downstream of the tandem gene, respectively;

(5) Obtaining entry vectors using Gateway technology

The PCR product with attB linker was purified, recovered and sequenced, and the PCR product was used with pDONR ^TM 221 Carrying out BP reaction by Vector to obtain an entry Vector pDONR221-TOCV-TY, converting DH5 alpha by the reacted solution, culturing 16 h at 37 ℃, carrying out PCR detection by using M13F/R primer, screening positive clone, and carrying out sequencing identification on the obtained positive clone;

(6) Expression vector acquisition

Performing LR reaction on the obtained entry clone and an expression vector pK7 GWIGG 2 (I) 0 to obtain a pK7 GWIGG 2 (I) 0-TOCV-TY expression vector, namely the RNAi expression vector is named pRNAi-TOCV-TY, transforming a reaction product into escherichia coli DH5 alpha, and performing colony PCR by using primers p35S and TY-R. Positive colonies were sequenced forward and reverse with primers P35SP and T35SP to verify the correctness of the ligation orientation. Extracting pRNAi-TOCV-TY plasmid from positive cloned and correctly oriented colony, and transforming agrobacterium GV3101;

the invention further discloses application of TYLCV and ToCV bivalent RNAi expression vector pRNAi-TOCV-TY in effectively preventing and treating composite infection tomato of ToCV and TYLCV. The effective prevention and treatment of composite infection of ToCV and TYLCV on tomato refers to effective inhibition of tomato virus replication in plants. The experimental results show that: after the transgenic tomato plants are inoculated with the bemisia tabaci population carrying ToCV and TYLCV, the transgenic plants show the target CP genes for efficiently silencing the ToCV and TYLCV viruses, and meanwhile, part of the transgenic plants show delayed disease resistance. The double RNAi tomato plant transformed with the ToCV CP gene and the TYLCV CP gene effectively blocks the replication of viruses in the plant body, and provides a new thought and practice method for preventing and treating the composite infection tomato of the ToCV and the TYLCV.

The invention mainly solves the practical problem that the development of the tomato industry is hindered by the serious diseases of ToCV and TYLCV compound infection on tomato production, and mainly considers the effect of the lengths of different coding genes and different interference fragments of ToCV and TYLCV on silencing exogenous virus infection, and has the main difficulties that: construction of a tandem RNAi expression vector of two gene fragments and obtaining of transgenic plants.

Compared with the prior art, the method for constructing the bivalent RNAi expression vector of TYLCV and ToCV by using Gateway technology has the following positive effects:

(1) Solves the long-standing threat of environmental pollution and food safety caused by killing insect media bemisia tabaci by using chemical agents for preventing TYLCV and ToCV.

(2) Since resistance genes to ToCV have not been found from tomato resources worldwide, the problem is effectively solved by using innovative materials against RNAi obtained by RNAi technology.

Drawings

FIG. 1ToCV and TYLCV CP gene fragments were amplified; m DL2000; toCV CP fragment; a tylcv CP fragment;

FIG. 2 PCR identification of ToCV-TY tandem genes; m.dl2000;1. PCR products with ToCV-F and TY-R as primers;

FIG. 3 PCR identification of entry vector; m.dl2000; M13F/M13R amplification products

FIG. 4 PCR identification of pRNAi-TOCV-TY transferred Agrobacterium GV3101; M.DL2000, 1-2. P35S and TY-R primers;

FIG. 5 Agrobacterium tumefaciens transformed tomato; A. cotyledon transformation; B. hypocotyl transformation; C. inducing adventitious buds; D. inducing adventitious roots; E. regenerating plants;

PCR detection of transgenic plants of fig. 6; M.DL2000, 1-2. P35S and TY-R primer amplification

FIG. 7 shows the performance of vaccination with TYLCV; 1. 2-5, disease-resistant transgenic plants;

FIG. 8 blade performance; 1.2, controlling new leaves of plants; 3. control plant old leaves; 4. disease-resistant transgenic plant old leaves;

FIG. 9 virus molecule detection; A. PCR amplification of TY-F/R primer; PCR amplification of ToCV CPF/R primer; m, DL 2000. Control plants, 2-6.4, 9, 10, 11, 13; 7-13.1, 2, 3, 5, 8, 12, 14.

Detailed Description

The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are all commercially available.

Example 1

1. Materials and methods

1.1 materials

Tomato leaves of tomato yellow leaf curl virus and tomato chlorosis virus with typical characteristics are collected in a solar greenhouse tomato cultivation field of the sixth Fu in the western green area of Tianjin, for subsequent experiments of nucleic acid extraction. Tomato susceptible inbred line J501 was maintained for this study.

pDONR ^TM 221 Vector, BP Clonase II enzyme Mix, LR Clonase II enzyme Mix, reverse transcriptase were purchased from Invitrogen, USA. rTaq DNA polymerase, dNTPs, DNA recovery and purification kit, RNA extraction kit were purchased from Dalianbao bioengineering Co., ltd, and plasmid miniprep kit and Agrobacterium GV3101 were purchased from Tiangen Biochemical Co., ltd. PCR primers were synthesized by Dalianbao bioengineering Co., ltd. pK7 GWIGG 2 (I) and 2X SG PCR Master Mix are products of Beijing Xinnogen Biotechnology Co., ltd. Carrying TYLCV and ToThe bemisia tabaci population of CV was raised on tomato seedlings from the agricultural biotechnology research center in the Tianjin city.

1.2 method

1.2.1 nucleic acid extraction

Taking leaves of TYLCV disease plants, and extracting total DNA by a CTAB method; leaves of ToCV disease plants are extracted by using a DALIANBAO biological company (TaKaRa) RNA extraction kit, and cDNA is synthesized by reverse transcription with random primer Oligo dT.

1.2.2 Acquisition of ToCV and TYLCV CP Gene fragments

Synthetic primers were designed by selecting the CP gene conserved sequence regions of ToCV and TYLCV, respectively (Table 1). Amplifying ToCV CP fragments by using a primer ToCV-F/R; primer TY-F/R amplified the CP fragment of TYLCV. The total volume of PCR was 25. Mu.L, which included 10 Xreaction buffer (Mg-containing ^2＋ ) 2.5. Mu.L, dNTP mixture (2.5 mmol/L) 0.5. Mu.L, upstream primer (10. Mu. Mol/L) 2.5. Mu.L, downstream primer (10. Mu. Mol/L) 2.5. Mu.L, template 0.5-2. Mu.L, DNA polymerase (5U/. Mu.L) 0.5. Mu.L, the remainder being made up with water. The reaction procedure is 94 ℃ denaturation for 5min, 94 ℃ 30 s, 54 ℃ 30 s, 72 ℃ 1.5 min, 35 cycles; 72. extending at 10min. The target bands were recovered, sequenced and analyzed using the DNA recovery purification kit, respectively.

1.2.3 obtaining of two Gene concatemers of ToCV and TYLCV CP

Mixing the PCR products of the TYLCCP and the ToCVCP, and carrying out PCR amplification by using the primers ToCV-F and TY-R to obtain the tandem gene ToCV-TY fragment of the TYLCCP and the ToCVCP. The total volume of PCR was 50. Mu.L, 2X SG PCR Master Mix. Mu.L, 1. Mu.L each for the template, 1. Mu.L each for the upstream and downstream primers, and the remainder was made up with water. The reaction procedure is: 94. denaturation at 2 min; 94. 30℃ s, 54 DEG C30 s, 72 DEG C1.5 min, 35 cycles; 72. extending at 10min. The amplified product is subjected to agarose gel electrophoresis with concentration of 1%, the obtained tandem gene fragment is recovered and named as TOCV-TY, and the TOCV-TY is connected and transferred into a pMD18-T vector to obtain the recombinant plasmid P-TOCV-TY.

1.2.4 acquisition of the tandem Gene with attB linker

PCR was performed using the recombinant plasmid P-ToCV-TY as a template and attB-TOCV-F and attB-TY-R primers, and the B sequence was added upstream and downstream of the tandem gene, respectively. The reaction system: the total volume of PCR was 50. Mu.L, of which 2 XSG PCR Master mix was 25. Mu.L, template was 1. Mu.L, each of the upstream and downstream primers was 1. Mu.L, and the remainder was made up with water. The reaction procedure is: 94. denaturation at 2 min; 94. 30℃ s, 55 DEG C30 s, 72 DEG C1.5 min, 35 cycles; 72. extending at 10min.

TABLE 1 PCR primers used in this study

1.2.5 Obtaining entry vectors using Gateway technology

The PCR product with attB linker was purified, recovered and sequenced. By using the PCR product and pDONR ^TM 221 The Vector was subjected to BP reaction, 10. Mu.L of the system was constructed according to the instructions of BP Clonase II enzyme Mix kit, and the system was left at 25℃for 1 hour, 1. Mu.L of protease K was added thereto, and the mixture was homogenized at 37℃for 10 minutes. The reaction solution was transformed into DH 5. Alpha. Competent cells. The transformed cells were uniformly spread on LB medium containing 50ug/ml kanamycin, cultured at 37℃for 16 h, then selected and monoclonal in LB liquid medium containing kanamycin at the same concentration, shake cultured at 37℃for 16 h, then PCR detection was performed by using M13F/R primers, positive clones were screened, the obtained positive clones were subjected to sequencing and identification, and the correct clone was identified as pENTR-TOCV-TY.

1.2.6 obtaining of expression vectors

The obtained entry clone vector pENTR-TOCV-TY was subjected to LR reaction with the expression vector pK7 GWIGG 2 (I), a 10. Mu.L reaction system was constructed according to the instructions of LR Clonase II enzyme Mix kit, and left at 25℃for 1 hour, 1. Mu.L of protease K was added, and mixed well, and at 37℃for 10 minutes, to obtain pK7 GWIGG 2 (I) -TOCV-TY expression vector, the reaction product was transformed into E.coli DH 5. Alpha. Competent cells, the transformed cells were uniformly spread on LB solid medium containing 50ug/ml of spectinomycin, after 16 h culture at 37℃were performed, and after 16 h shake culture at 37℃were performed, colonies were subjected to PCR using primers p35S and TY-R. Forward and reverse sequencing was performed with P35SP and T35SP, and positive clones were selected in the correct direction, namely the RNAi expression vector designated pRNAi-TOCV-TY.

1.2.7 acquisition of Agrobacterium

Plasmids were extracted from DH 5. Alpha. Containing pRNAi-TOCV-T expression vector, agrobacterium GV3101 was transformed by freeze thawing, transformed cells were spread evenly on YEB solid medium containing spectinomycin (Spe, 50 ug/ml) and rifampicin (Rif, 50 ug/ml), after 2 days of culture at 28℃were picked up and were subjected to bacterial liquid PCR with specific primers p35S and TY-R, respectively, after 2 days of culture at 28℃to identify positive clones. After tomato is transformed by the expression vector, a hairpin RNA structure is formed, so that the effect of interfering viral RNA replication is achieved.

1.2.8 Identification of Agrobacterium-transformed tomato and transgenic plants

Tomato was transformed using agrobacterium-mediated leaf disc transformation. Soaking seeds of the tomato infectious disease inbred line J501 in 70% ethanol for 30 min, sterilizing with 10% sodium hypochlorite for 6min, washing with sterile water for 3 times, and inoculating on an MS culture medium for two weeks for culture; taking cotyledons or hypocotyls of the aseptic tomato seedlings as explants, and soaking the explants with an agrobacterium tumefaciens bacterial solution with OD600 = 0.5 or so for 5min. The explants were removed, placed on sterile filter paper to blot residual bacterial solution, transferred to MS medium containing IAA (O.2 mg/L) and 6-BA (2.0 mg/L) for co-cultivation for 3 days, and then transferred to screening medium containing cephalosporin (500 mg/L) and kanamycin (50 mg/L) for cultivation every 2 weeks. Transferring the regenerated buds to 1/2MS rooting culture medium containing (IBA 1.0 mg/L) and cephalosporin (400 mg/L) when the regenerated buds grow to about 3 cm; transplanting the plant to soil after the root system is developed.

The CTAB method is used for extracting total DNA of tomato regenerated plant leaves, and primers p35S and TY-R are used for PCR detection, so that non-transformed tomatoes are used as a control.

1.2.9 identification of disease resistance of transgenic tomato by Bemisia tabaci virus-transferring inoculation

Control and positive transgenic plants were planted in the insect control greenhouse. And (3) placing the leaves with the period of 1 heart of the 4 leaves and the control in an insect-raising net, and carrying the bemisia tabaci population carrying T YLCV and ToCV in the net for insect-transmission inoculation. After one week of inoculation, the sprayed insecticide is removed, then tomatoes are planted in the flowerpot, and the disease condition is observed periodically. PCR and RT-PCR assays were performed 15 and 30 days after inoculation, and TYLCV and ToCV were detected using primers ShangghaiF/R and ToCVCPF/R, respectively.

Results and analysis

2.1 cloning of ToCV and TYLCV CP Gene fragments

Specific RT-PCR and PCR reactions were performed using total RNA and total DNA of plants infected with ToCV and TYLCV viruses, respectively, as templates, to obtain two conserved fragments of 433bp and 406bp, respectively (FIG. 1). Sequencing results showed 100% identity with the Tianjin isolate.

Obtaining a tandem Gene fragment with TYLCV

Since there is a sequence on the TY-F primer that is complementary to the reverse primer ToCV-R of ToCV, the ends of the amplified 2 fragments have complementary sequences that can be complementary during annealing in the amplification process after mixing. Then, PCR was performed using ToCV-F and TY-R as primers. Two viral gene tandem sequences TOCV-TY of 839bp in size were obtained in one step (FIG. 2).

Expression vector construction

And performing BP reaction on the pDONR221 vector and a TOCV-TY tandem gene PCR product with attB sites to obtain the pENTR-TY PCR vector. After transformation of the bacteria, colony PCR was performed using the primer M13F/R, the product size was 1100bp, significantly larger than the insert, indicating that the tandem gene fragment ToCV-TY had been ligated into the vector (FIG. 3). The obtained entry vector was subjected to LR reaction with expression vector pK7 GWIGG 2 (I) to obtain pRNAi-TOCV-TY expression vector. The obtained expression vector was transformed into DH 5. Alpha. Colony PCR verification was performed using primers p35S and TY-R, resulting in an amplified band of approximately 1000bp, significantly larger than the insert, consistent with the expected size. Forward and reverse sequencing was performed using P35SP and T35SP, and the results indicated that the direction and sequencing were correct, indicating that RNAi vector construction was successful. The pRNAi-TOCV-TY expression vector was transformed into Agrobacterium strain GV3101, and the expected band was amplified by identification using primers p35SP and TY-R, demonstrating successful transfer of the expression vector into Agrobacterium (FIG. 4)

2.4 plant expression vector transformation of tomato

A total of 30 regenerated plants were obtained by Agrobacterium-mediated transformation of tomato J501 cotyledon and hypocotyl explants. Control and regenerated plant genomic DNA was extracted and PCR was performed using p35SP and TY-R primers. The results show that 15 plants can amplify about 1000bp bands, while the control plants have no amplified bands, which indicates that the exogenous target gene is integrated into the tomato genome.

5 identification of disease resistance of transgenic tomato

The 15 positive T0 transgenic tomato plants were inoculated with the toxic bemisia tabaci, and the results indicate that ToCV and TYLCV resistances were significantly improved in the T0 material (Table 1).

Neither the 10 controls nor the 15 transgenic plants showed symptoms at 15 days of inoculation and no bands were amplified by molecular detection with PCR. After 4 weeks of inoculation, 9 control plants grew slowly, old leaves showed yellowing, new leaves showed wrinkling and the same symptoms (4, 9, 10, 11, 13) were seen with 5 transgenic plants, 3 symptoms (6, 7, 15) were seen with 1 control at 5-7 weeks of inoculation, and none of the remaining 7 transgenic plants had symptoms (1, 2, 3, 5, 8, 12, 14) after 10 weeks of inoculation. Therefore, 7 plants of the high-resistance TYLCV plant are selected from the T0 transgenic plants, and the high-resistance TYLCV plant accounts for 46.7% of the total investigation plants; 3 plants of the delayed infection plants accounting for 20 percent; 5 plants of the infected plant account for 30 percent. No plants exhibiting disease resistance after inoculation of the non-transgenic plants (fig. 7, 8). Preliminary shows that the bivalent RNAi vector constructed by the invention can effectively silence the target gene CP genes of ToCV and TYLCV viruses.

TABLE 2 disease resistance of transgenic plants

Extracting total DNA and total RNA of leaf, and respectively performing PCR and RT-PCR detection by using specific primers shanghai F/R of TYLCV virus and specific primers ToCV CPF/R of ToCV. The results showed that control plants and diseased plants after virus inoculation amplified the band of interest of approximately 575bp and 840bp, whereas transgenic plants exhibiting disease resistance failed to amplify the band (FIG. 9).

The invention provides application of transgenic tomato plants capable of expressing ToCV CP genes and TYLCV CP genes dsRNA in control of ToCV and TYLCV. After the transgenic tomato plants are inoculated with bemisia tabaci carrying ToCV and TYLCV, 46.7% of the transgenic plants show high-efficiency silencing of the target CP genes of ToCV and TYLCV viruses and 30% of the transgenic plants show delayed disease resistance compared to the control treatment. The double RNAi tomato plant transformed with the ToCV CP gene and the TYLCV CP gene effectively blocks the replication of viruses in the plant body, and provides a new thought and practice method for preventing and treating the composite infection tomato of the ToCV and the TYLCV.

SEQUENCE LISTING

<110> Tianjin agricultural biotechnology research center

<120> construction of TYLCV and ToCV bivalent RNAi expression vectors using Gateway technology and use thereof

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> artificial sequence

<400> 1

atggagaaca gtgctgttgc a 21

<210> 2

<211> 20

<212> DNA

<213> artificial sequence

<400> 2

caccatctac ctcaaagtag 20

<210> 3

<211> 39

<212> DNA

<213> artificial sequence

<400> 3

ctactttgag gtagatggtg atgtcgaagc gaccaggcg 39

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<400> 4

cctgattagt gtgattctgc 20

<210> 5

<211> 839

<212> DNA

<213> artificial sequence

<400> 5

atggagaaca gtgctgttgc aaacactggt gataacggtg gtggccgcaa tcctctggtt 60

agaccgttag atgatggcgt agatgacgag gtgcagaact tgggcaggag ggacgattcg 120

acatctctta ttccggctaa tcctaatcga tcttccagtt gggctttgtt gaacccggat 180

actattaatt ataacgagtt aaggaaattg aaggtacact ccactagggg tgatactctt 240

accttgactc aggaagagga gttcgagaag atactcgaat ccttttgcag gcgaataatc 300

ggtgagaccc cgatgacgga taagattttc gctggtttct acatgtctat gtgtcaggcc 360

attgtaaacc aagggacctc agttaaagca cccggtaata acagtcttga aaactacttt 420

gaggtagatg gtgatgtcga agcgaccagg cgatataatc atttccacgc ccgcctcgaa 480

ggttcgccga aggctgaact tcgacagccc atacagcagc cgtgctgctg tccccattgt 540

ccaaggcaca aacaagcgac gatcatggac gtacaggccc atgtaccgga agcccagaat 600

atacagaatg tatcgaagcc ctgatgttcc tcgtggatgt gaaggcccat gtaaagtcca 660

gtcttatgag caacgggatg atattaagca tactggtatt gttcgttgtg ttagtgatgt 720

tactcgtgga tctggaatta ctcacagagt gggtaagagg ttctgtgtta aatcgatata 780

ttttttaggt aaagtctgga tggatgaaaa tatcaagaag cagaatcaca ctaatcagg 839

Claims (3)

1. TYLCV and ToCV bivalent RNAi expression vector constructed by Gateway technology is named pRNAi-TOCV-TY; the construction method of the expression vector comprises the following steps:

(1) Extracting nucleic acid from tomato leaf, namely collecting tomato leaf with TYLCV disease, and extracting total DNA; collecting tomato leaves with ToCV pathogenesis, extracting RNA by using a TaKaRa RNA extraction kit of Dalianbao biological company, and performing reverse transcription by using a random primer Oligo dT to synthesize cDNA;

(2) Acquisition of ToCV and TYLCV CP Gene fragments

Respectively selecting CP gene conservation sequence regions of ToCV and TYLCV, designing synthetic primers, and amplifying CP fragments of the ToCV by the primers ToCV-F/R; primer TY-F/R amplifies CP fragment of TYLCV; the sequence of the primer ToCV-F is shown as SEQ ID NO. 1; the sequence of the primer ToCV-R is shown as SEQ ID NO. 2; the sequence of the primer TY-F is shown as SEQ ID NO. 3; the sequence of the primer TY-R is shown as SEQ ID NO. 4;

(3) Obtaining of tandem ToCV and TYLCV CP

Mixing the PCR products of the TYLCV CP and the ToCV CP, performing PCR amplification by using primers ToCV-F and TY-R to obtain a tandem gene ToCV-TY fragment of the TYLCV and the ToCVCP, performing 1% agarose gel electrophoresis on the amplified product, recovering the obtained tandem gene fragment to be named as TOCV-TY, and transferring the TOCV-TY connection into a pMD18-T vector to obtain a recombinant plasmid P-ToCV-TY;

(4) Acquisition of tandem Gene with attB linker

PCR was performed using the recombinant plasmid P-ToCV-TY as a template and attB-ToCV-F and attB-TY-R primers, and B sequences were added upstream and downstream of the tandem gene, respectively;

the sequence of attB-ToCV-F is shown in SEQ ID NO. 5;

the sequence of attB-TY-R is shown as SEQ ID NO. 6;

(5) Obtaining entry vectors using Gateway technology

The PCR product with attB linker was purified, recovered and sequenced, and the PCR product was used with pDONR ^TM 221 The Vector carries out BP reaction to obtain the entry Vector pDONR221-TOCV-TY, transforming DH5 alpha from the reaction solution, culturing at 37 ℃ for 16 h, performing PCR detection by using M13F/R primers, screening positive clones, and sequencing and identifying the obtained positive clones;

the sequence of M13F is shown as SEQ ID NO. 9;

the sequence of M13R is shown as SEQ ID NO. 10;

identifying the correct clone designated pENTR-TOCV-TY;

(6) Expression vector acquisition

Carrying out LR reaction on the obtained entry cloning vector pENTR-TOCV-TY and an expression vector pK7 GWIGG 2 (I), constructing a 10 mu L reaction system according to the instruction of a LR Clonase II enzyme Mix kit, standing at 25 ℃ for 1 hour, adding 1 mu L of protease K, uniformly mixing, carrying out 10min at 37 ℃ to obtain a pK7 GWIGG 2 (I) -TOCV-TY expression vector, transforming the reaction product into escherichia coli DH5 alpha competent cells, uniformly coating the transformed cells on LB solid medium containing 50ug/ml of spectinomycin, culturing at 37 ℃ for 16 h, picking up monoclonals into LB liquid medium containing the same concentration of spectinomycin, carrying out colony PCR by using primers p35S and TY-R after shake culturing at 37 ℃ for 16 h;

forward and reverse sequencing is carried out by using P35SP and T35SP, and positive clones in the correct direction are selected, namely the RNAi expression vector is named pRNAi-TOCV-TY;

the sequence of P35S is shown as SEQ ID NO. 7;

the sequence of T35SP is shown in SEQ ID NO. 8.

2. Use of TYLCV and tolcv bivalent RNAi expression vectors pRNAi-ToCV-TY according to claim 1, for effectively controlling a composite infection of tolcv and TYLCV with tomato.

3. The use of claim 2, wherein said effective control of a tomato infection by a combination of ToCV and TYLCV is effective to prevent tomato virus replication in plants.

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