CN100368434C - Tomato RNA virus host factor and its coding gene and application - Google Patents

Abstract

The present invention discloses a host factor of a tomato RNA virus, a coded gene thereof and the application thereof. The present invention provides a host factor of a tomato RNA virus, a coded gene thereof and the application of the host factor in antivirus plant breeding. The host factor of a tomato RNA virus is a protein with one of the following amino acid residue sequences: firstly, SEQ ID No. 1 in a sequence list; secondly, a protein which is used for substituting, deleting or adding one to ten amino acid residues of an amino acid residue sequence of the SEQ ID No. 1 in the sequence list and has the function of supporting virus copy. By transferring an antisense gene fragment or using an RNA interfering method, the coded gene of the protein in a plant body is silenced so as to obtain an antiviral plant. The host factor gene ToTOM3 of a tomato RNA virus and a method for inhibiting the gene expression have great practical meanings and wide application prospects of antivirus plant breeding.

Description

Tomato RNA virus host factor and its coding gene and application

technical field

The invention relates to a protein and its coding gene and application in the field of biotechnology, in particular to a tomato RNA virus host factor and its coding gene and its application in plant anti-virus breeding.

Background technique

Tomato is an important fruit-type vegetable. During its growth and development, it is vulnerable to various diseases, among which viral diseases are the most serious. Most of the viruses infecting tomato reported so far are RNA viruses, of which seven are the most common, namely, cucumber mosaic virus (CMV), tobacco mosaic virus (Tabacco mosaic virus, TMV), tomato Mosaic virus (Tomato mosaic virus, ToMV), potato virus Y (Potato virus Y, PVY), tomato bushy stunt virus (TBSV), tomato infertility virus (Tomato aspermy virus, TAV) and tomato mosaic virus Green spot virus (Tomatochlorotic spot virus, TCSV).

Plant virus disease is one of the important diseases that cause great loss of agricultural production. Since viruses are strictly obligate parasites, there is no strictly selective chemical agent for the prevention and treatment of plant virus diseases. In production practice, preventive measures such as removing the source of the virus and cutting off the transmission route of the virus, such as killing vector insects with chemicals and planting disease-resistant varieties, are mainly used to control the harm of virus diseases. Among them, planting disease-resistant varieties is the most economical and effective. However, the conventional disease-resistant breeding method is cumbersome and requires a lot of manpower and material resources. More importantly, disease-resistant genes are often linked with poor agronomic shape genes, making it difficult to meet the production requirements of disease-resistant and high-quality. On the other hand, the availability of resistant gene resources The lack also limits the application of conventional breeding methods. The application of plant genetic engineering in breeding has opened up a new way for the prevention and treatment of plant virus diseases. At present, there are mainly two methods for obtaining anti-virus transgenic plants: one is to introduce the gene or regulatory sequence of the gene derived from the virus itself (such as the coat protein gene of the virus, the replicase gene and its fragments, the motor protein gene and its 3' or 5' end non-coding region, viral antisense RNA, ribozyme gene and viral satellite RNA, etc.) into recipient plants, thereby obtaining virus-resistant (pathogen-derived resistance) virus-resistant plants of origin (Lomonossoff G.P., 1995. Pathogen -derived Resistance to Plant Viruses, Annu.Rev.Phytopathol., 33:323-343). However, these transgenic plants have certain limitations in antiviral function, such as high specificity, even strain specialization, etc., and there are potential risk factors such as biological safety; another method is to use the plant itself Disease resistance genes (such as N gene) to obtain virus-resistant plants (Goldbach R., Bucher E., and Prins M.2003. Resistance mechani sms to plant viruses: an review. Virus Research., 92: 207-212), but , So far, only a few plant antiviral genes have been cloned and identified, which limits the further development of this strategy.

After the virus invades the cell, it needs to go through the following processes when it replicates and proliferates in the host cell and causes systemic infection: uncapsiding of the virus, genome replication of the virus and protein synthesis, assembly of new virus particles, and virus particle in-cell and inter-cellular transportation etc. In this process, all the convenient conditions provided by the host cell to the virus are called host factors (Host Factors), also known as host proteins or cellular proteins. If these host factors are lacking in the host cell, the virus cannot replicate or the efficiency of replication is greatly reduced (Ahlquist P., Noueiry, A.O., and Lee, W.M., et al., 2003.Host Factor in Positive-Strand RNA Viruses Genome Replication.Journal of Virology, 77(15): 8181-8126.). Therefore, by inhibiting or silencing the genes that support virus replication in host cells, it will be possible to make them unable to support virus replication and maintenance in host cells, thereby obtaining broad-spectrum virus-resistant plants. Therefore, the study of virus host factor genes provides a new way for antiviral genetic engineering.

Contents of the invention

The purpose of the present invention is to provide a tomato RNA virus host factor and its coding gene.

The tomato RNA virus host factor provided by the present invention is called ToTOM3, which is derived from Lycopersicon esculentum Miller and is a protein with one of the following amino acid residue sequences:

1) SEQ ID №: 1 in the sequence listing;

2) Substitution, deletion or addition of one to ten amino acid residues to the amino acid residue sequence of SEQ ID №: 1 in the sequence listing and a protein capable of supporting virus replication.

SEQ ID No. 1 in the sequence listing consists of 295 amino acid residues. Containing multiple highly hydrophobic regions, it is speculated that the protein is a transmembrane protein with 7 transmembrane domains, and the homology with Arabidopsis AToTOM3 at the amino acid level is 76.9%.

The polynucleotide encoding the protein sequence of SEQ ID №: 1 in the sequence listing also belongs to the protection scope of the present invention.

The gene encoding the host factor of tomato RNA virus (ToTOM3) includes the cDNA gene of host factor of tomato RNA virus and the genome gene of host factor of tomato RNA virus. Its genome gene is one of the following nucleotide sequences:

1) The DNA sequence of SEQ ID №: 2 in the sequence listing;

2) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID No. 2 in the sequence listing under high stringency conditions.

SEQ ID № in the sequence listing: 2 consists of 8129 bases, has 11 exons and 10 introns, and the 1245th-1487th bases from the 5′ end are the first exon of the genome gene The 3519-3609th base from the 5' end is the second exon of the genome gene, and the 3765-3817th base from the 5' end is the third exon of the genome gene, since 5' The 3895-3987th base at the 'end is the fourth exon of the genome gene, and the 4065-4136th base from the 5' end is the fifth exon of the genome gene, and the 4791st base from the 5' end Base -4863 is the sixth exon of the genome gene, bases 4939-4984 from the 5' end are the seventh exon of the genome gene, bases 6259-6322 from the 5' end The base is the eighth exon of the genome gene, the 6403-6456 base from the 5' end is the ninth exon of the genome gene, and the 6790-6867 base from the 5' end is the genome The tenth exon of the gene, the 7730-8129th base from the 5' end is the eleventh exon of the genome gene, and the 1317-1319th base from the 5' end is the beginning of the genome gene The start codon ATG, bases 7821-7822 from the 5' end are the stop codon TGA of the genome gene; bases 1488-3518 from the 5' end are the first intron of the genome gene, since Bases 3610-3764 at the 5' end are the second intron of the genome gene, bases 3818-3894 at the 5' end are the third intron of the genome gene, bases at the 5' end are the third intron Bases 3988-4064 are the fourth intron of the genome gene, bases 4137-4790 from the 5' end are the fifth intron of the genome gene, bases 4864-4938 from the 5' end The base is the sixth intron of the genome gene, the 4985-6258th base from the 5' end is the seventh intron of the genome gene, and the 6323-6402th base from the 5' end is the The eighth intron of the genome gene, the 6457-6789th base from the 5' end is the ninth intron of the genome gene, and the 6868-7729th base from the 5' end is the first intron of the genome gene Ten introns.

Its cDNA sequence is one of the following nucleotide sequences:

1) The DNA sequence of SEQ ID №: 3 in the sequence listing;

2) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID No. 3 in the sequence listing under high stringency conditions.

SEQ ID №: 3 in the sequence listing consists of 1282 bases, and its open reading frame (ORF) is the 73-957th base from the 5′ end, encoding the amino acid residues with SEQ ID №: 1 in the sequence listing sequence of proteins.

The above-mentioned high stringency conditions can be 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution, hybridization at 65° C. and washing the membrane.

The expression vector containing the gene encoding the host factor of tomato RNA virus, the transgenic cell line and the host bacterium, and the primers for amplifying any fragment of the gene encoding the host factor of tomato RNA virus also belong to the protection scope of the present invention.

Another object of the present invention is to provide a method for breeding virus-resistant plants.

The method for cultivating anti-virus plants provided by the present invention is to introduce the antisense RNA of ToTOM3 or the RNA interference expression vector of ToTOM3 into plants to obtain transgenic plants.

The antisense RNA of ToTOM3 or the RNA interference expression vector of ToTOM3 can be introduced into transformed plant cells or tissues according to conventional methods in the field of plant genetic engineering, such as Agrobacterium-mediated transformation, gene gun-mediated transformation or pollen tube passage method wait. The transformed plant host can be a plant of Solanaceae, Brassicaceae or Cucurbitaceae.

The tomato RNA virus host factor ToTOM3 provided by the present invention is a related protein for the survival of RNA viruses in plants, and the antiviral effect can be obtained by silencing the RNA virus host factor gene in plants by transfecting antisense RNA or RNA interference vectors. plant. Experiments have shown that compared with non-transgenic plants, the accumulation of CMV and TMV in transgenic tomato plants transfected with ToTOM3 antisense RNA and RNA interference vectors is significantly reduced, the onset time is significantly delayed, and even symptoms are asymptomatic, indicating that tomato ToTOM3 is the CMV and TMV A host factor gene, CMV- and TMV-resistant plant varieties can be obtained by inhibiting or silencing ToTOM3. The tomato RNA virus host factor gene ToTOM3 and the method for inhibiting the gene expression of the present invention have great practical significance and broad application prospects in the cultivation and breeding of anti-virus plants.

Description of drawings

Figure 1 shows the results of agarose gel electrophoresis detection of the 3'RACE product of ToTOM3

Figure 2 is the result of enzyme digestion and identification of the cloning vector containing the 3'RACE PCR fragment of ToTOM3

Figure 3 shows the results of agarose gel electrophoresis detection of the 5'RACE product of ToTOM3

Figure 4 is the result of enzyme digestion identification of the cloning vector containing the 5'RACE PCR fragment of ToTOM3

Figure 5 is the agarose gel electrophoresis detection result of the PCR amplified fragment of ToTOM3

Figure 6 is the Southern hybridization result of the total tomato DNA and the PCR amplified fragment (probe) of ToTOM3

Figure 7 is the positive clone obtained by colony in situ hybridization

Fig. 8 is the agarose gel electrophoresis detection result of ToTOM3 genomic gene 3' end fragment PCR product

Figure 9 is the agarose gel electrophoresis detection result of the recombinant plasmid connected with the 3' end fragment of the ToTOM3 genome gene

Figure 10 is the agarose gel electrophoresis detection result of the ToTOM3 full-length genome gene fragment amplified by PCR

Figure 11 shows the enzyme digestion identification of the recombinant plasmid of ToTOM3 full-length genome fragment

Fig. 12 is the agarose gel electrophoresis detection result of ToTOM3 antisense RNA expression plasmid

Figure 13 is the PCR identification result of antisense RNA expression plasmid

Fig. 14 obtains RNAi (A) fragment for PCR amplification

Figure 15 is the identification result of BglII and SpeI double enzyme digestion of RNA interference plasmid pUToT3R(A-)

Figure 16 is the result of double enzyme digestion identification of RNA interference plasmid pUToT3R(A)SalI and XhoI

Figure 17 is the BglII digestion identification result of the RNA interference expression plasmid pBIToT3R (A)

Figure 18 is the PCR identification result of RNA interference expression plasmid pBIToT3R (A)

Figure 19 is the agarose gel electrophoresis detection result of the RNAi (B) fragment obtained by double digestion of pTTOM3-3 with BglII and SpeI

Figure 20 is the identification result of RNA interference plasmid pUToT3R(B-)BglII and SPeI double enzyme digestion

Fig. 21 is the identification result of SalI and XhoI double enzyme digestion of RNA interference plasmid pUToT3R(B)

Figure 22 is the BglII digestion identification result of the recombinant RNA interference expression plasmid pBIToT3R (B)

Figure 23 is the PCR identification result of recombinant RNA interference expression plasmid pBIToT3R (B)

Figure 24 is the PCR detection result of the antisense RNA expression plasmid pBIToT3 (A) transgenic tomato

Figure 25 is the result of Southern hybridization analysis of antisense RNA expression plasmid pBIToT3 (A) transgenic tomato (total DNA BamHI single enzyme cut)

Fig. 26 is the PCR detection result of RNA interference plasmid pBIToT3R (A) transgenic tomato

Figure 27 is the Southern hybridization analysis result of transgenic tomato (total DNAEcoRI single enzyme digestion) of 8B. transfer RNA interference plasmid pBIToT3R (A)

Figure 28 is the PGR detection result of 9A. transfer RNA interference plasmid pBIToT3R(B) transgenic tomato

Figure 29 is the Southern hybridization analysis result of transgenic tomato (total DNA EcoRII single enzyme digestion) of 9B. transfer RNA interference plasmid pBIToT3R (B)

Figure 30A is the symptom of transgenic tomato seedlings inoculated with TMV for 10 days

Figure 30B is the symptoms of non-transgenic tomato seedlings inoculated with TMV for 10 days

Figure 30C is the symptom of transgenic tomato seedlings inoculated with CMV for 10 days

Figure 30D is the symptoms of non-transgenic tomato seedlings inoculated with CMV for 10 days

Figure 31A is the symptom of 90-day transgenic tomato seedlings inoculated with TMV

Figure 31B shows the symptoms of non-transgenic tomato seedlings inoculated with TMV for 90 days

Figure 31C is the symptom of transgenic tomato seedlings inoculated with CMV for 90 days

Figure 31D shows the symptoms of 90-day non-transgenic tomato seedlings inoculated with CMV

Figure 32 is the physical map of the plant expression vector pBI121

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified, and the tomato variety used is "Super Star" purchased from Guangxi Academy of Agricultural Sciences.

Embodiment 1, the cloning of the full-length cDNA sequence of tomato host factor gene ToTOM3

1. Cloning of ToTOM3 3' end cDNA

According to the tomato EST sequence BI923910 (250nt) (gi: 16225384) found in GenBank that has a certain homology with Arabidopsis ATOM3 (gi: 15425640), two forward primers were designed to amplify the cDNA sequence at the 3' end of ToTOM3. The primer sequences are as follows :

ttom3-1: 5'-CGGAGATGGTTGTAGGCCCG-3';

ttom3-A: 5'-TGAATGATGCAATCAATTGG-3'

The cDNA at the 3' end of ToTOM was synthesized using the 3'RACE System for Rapid Aplication of cDNA Ends kit (Invitrogen, catalog number 18373-027). Total RNA was extracted from tomato, and its first-strand cDNA was synthesized by reverse transcription. The reaction system and reaction conditions were: 8 μL ddH ₂ O (RNase-Free), 1 μL AP (10 μM) (AP: 5'-GCCACGCGTCGACTAGTACT(T) ₁₆ - 3'), 3 μL tomato total RNA (about 5 μg) and 1 μL dNTP (10 mM), after mixing, bathe in water at 72°C for 10 minutes, quickly put it on ice for 5 minutes, then add 4 μL 5× first-strand buffer and 2 μL 0. 1M DTT, mixed evenly, after 3 min in water bath at 42°C, 1 μL of SuperScript II RT (Invitrogen Company) was added, and reacted at 42°C for 1 hour. After the reaction, heat at 70°C for 15 minutes to inactivate the transcriptase. Using the first-strand cDNA synthesized by reverse transcription as a template, under the guidance of primer ttom3-1 and the primer AUAP: 5'-GGCCACGCGTCGACTAGTAC-3' provided by the above kit, the first PCR was carried out, and then the first PCR The product is a template, and under the guidance of primer ttom3-A and primer AUAP, the second PCR is carried out. After the reaction, the PCR product is detected by 0.8% agarose gel electrophoresis, and the detection result is as shown in Figure 1 (swimming lane M: 100bp DNA Ladder lane 1: 3'RACE PCR product of ToTOM3), indicating that a specific band with a length of about 1200bp was amplified, which was consistent with the expected result. The specific PCR product was recovered, and the 3'RACE PCR product was cloned using TOPO TA Cloning Kits (Invitrogen Company). The reaction system was: 2 μL PCR product, 0.5 μL Salt Solution and 0.5 μL pCR 2.1-TOPO vector. Put the reaction solution at room temperature for 30 minutes, connect the target fragment with the carrier pCR 2.1-TOPO, and then transform the ligated product into E.coli DH5α competent cells. Dicer enzyme EcoR I was used for enzyme digestion identification, and the results of enzyme digestion identification are shown in Figure 2 (lane M: GeneRuler 1 kb DNA ladder, lane CK1: EcoRI digestion product of vector pCR2.1-TOPO, lane CK2: 3' of ToTOM3 RACE PCR fragment, swimming lane 1-3: the EcoR I digestion product of the recombinant plasmid), indicating that the 3' RACE PCR fragment of ToTOM3 with a length of about 1200bp cloned has been correctly inserted into the vector pCR 2.1-TOPO, and the recombinant vector was named is pCToT3-3. Carry out DNA sequence determination to pCToT3-3, the sequencing result shows that the length of the inserted DNA fragment is 1149bp, has the DNA sequence of the 134th-1282th position from the 5' end of SEQ ID №: 3 in the sequence table, and the 3' of the DNA fragment The end has a poly(A) tail of 15 bases in length.

2. Cloning of ToTOM35' end cDNA

According to the tomato EST sequence BI923910 (250nt) (gi: 16225384) found in GenBank that has a certain homology with Arabidopsis ATOM3 (gi: 15425640), two reverse primers were designed to amplify the cDNA sequence at the 5' end of ToTOM. The primer sequences are as follows :

ttom3-2: CCATTCACAAAGAAATTGAG;

ttom3-B: GAGCAACGACGGCGACAACGCCGTAGAG

The cDNA at the 5' end of ToTOM was synthesized using the SMART ^TM RACE cDNA Ampiification kit (Clontech, catalog number K1881-1). The total RNA of tomato was extracted, and its first-strand cDNA was synthesized by reverse transcription. The reaction system and reaction conditions were: 3 μL tomato total RNA (1 μg/μL), 1 μL 5'-CDS primer (5'-(T) ₂₅ N _-1 N -3') (10 μM) and 1 μL SMART II A Oligo (5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3') (10 μM), after mixing, bathe in water at 72°C for 2 minutes, quickly put it on ice for 2 minutes, and then add 2 μL of 5×first Strain buffer, 1 μL 20mM DTT, 1 μL 10mM dNTP Mix and 1 μL Power Script reverse transcriptase, mix well, react in 42°C water bath for 90 minutes, add 100 μL Tricine-EDTA buffer, 72°C water bath for 7 minutes, then place on ice to stop the reaction. Using the first-strand cDNA synthesized by reverse transcription as a template, under the guidance of primer ttom3-2 and the primer UPM provided by the above kit: 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3', the first PCR was carried out, and then the first PCR The product is used as a template. Under the guidance of primer ttom3-B and primer NUP: 5'-AAGCAGTGGTATCAACGCAGAGT-3', the second PCR is carried out. After the reaction, the PCR product is detected by 1.2% agarose gel electrophoresis. The detection result is shown in the figure As shown in 3 (lane M: 1kb DNA Ladder, lane 1: 5'RACE PCR fragment of ToTOM3), it shows that a specific band with a length of about 250bp was amplified, which is consistent with the expected result. Recover the specific PCR product, use TOPO TA Cloning Kits to clone the 5'RACE PCR product, and first connect the target fragment to the vector pCR2.1-TOPO (the reaction system and reaction conditions are the same as the cloning of the ToTOM3' end cDNA in step 1) , and then the ligation product was transformed into E.coli DH5α competent cells, and the white single colony was selected to extract the plasmid after the blue-white screening, and the restriction endonuclease EcoR I was used for enzyme digestion identification, and the enzyme digestion identification results are shown in Figure 4 (swimming lane M: GeneRuler 1kb DNA ladder, CK1: 5'RACE PCR fragment, CK2: pCR2.1-TOPO (3913bp), lanes 1-5: EcoR I digestion product of recombinant plasmid connected with 5'RACE PCR fragment), indicating The cloned 5'RACE PCR fragment of ToTOM3 with a length of about 250bp has been correctly inserted into the vector TOPO, and the recombinant vector was named pCToT3-5. DNA sequence determination was carried out on pCToT3-5, and the sequencing results showed that the length of the 5'RACE PCR fragment was 241bp, and it had the DNA sequence of nucleotides 1-241 from the 5' end of SEQ ID No. 3 in the sequence listing.

3. Obtaining the full-length cDNA of ToTOM3

Sequence analysis was performed on the 5'RACE PCR fragment and 3'RACE PCR fragment of ToTOM3 obtained in steps 1 and 2, and the analysis results showed that there was a restriction endonuclease at the 148th base from the 5' end of the 5'RACE PCR fragment The cleavage site of the enzyme Mun I overlaps with the 3'RACE PCR fragment. There is an Xba I restriction site on the vector pCToT3-5, and the Mun I and Xba I restriction sites are used to connect the cDNA of 3'RACE and 5'RACE to obtain the full-length cDNA of ToTOM3. The full-length cDNA is sequenced, and the sequencing results show that the sequence has the nucleotide sequence of SEQ ID №: 3 in the sequence listing. SEQ ID №: 3 in the sequence listing consists of 1282 nucleotides, and the 3' end has a length of 15 base poly(A) tail. Sequence analysis was performed on the full-length cDNA sequence of ToTOM3 with Vector NTI software, and the analysis results showed that its open reading frame (ORF) was 73-957 bases from the 5' end, and the length of the 5' non-coding region was 72 bases base, the length of the 3' non-coding region is 307 bases, the amino acid residue sequence of SEQ ID №: 1 in the coding sequence listing, and the SEQ ID №: 1 in the sequence listing is composed of 295 amino acid residues. ToTOM3 and The homology comparison of the amino acid residue sequence encoded by Arabidopsis AtTOM3 showed that the homology between ToTOM3 and Arabidopsis AtTOM3 at the amino acid level was 76.9%. The method of Kyte & Doolittle (Kyte & Doolittle, A simple method for displaying the hydropathic character of a protein, Journal Molecular Biology, 157(1): 105-132) was used to analyze the hydrophobicity of the protein encoded by ToTOM3, and the results showed that ToTOM3 encoded The protein has a highly hydrophobic region, and the online software TMPred (http://www.ch.embnet.org/cgi-bin/TMPREN-FORM-PARSER) is used to predict the transmembrane structure region, and it is inferred that the protein is a protein with Transmembrane protein with 7 transmembrane domains.

Cloning of embodiment 2, ToTOM3 genome gene

According to the obtained full-length cDNA sequence of ToTOM3, primers were designed to amplify the genome sequence of ToTOM3. The primer sequences are as follows:

ttom3-1: 5'-CGGAGATGGTTGTAGGCCCG-3';

ttom3-2: 5'-CCATTCACAAAGAAATTGAG-3'

With tomato total DNA as template, under the guidance of primer ttom3-1 and primer ttom3-2, carry out PCR amplification, carry out 0.8% agarose gel electrophoresis detection to PCR after reaction finishes, and detection result is as shown in Figure 5 (swimming lane M: 1KbDNAMarker, lane 1: PCR-amplified gene fragment of ToTOM3), indicating that a gene fragment of ToTOM3 with a size of about 2.3kb was amplified, and the DNA fragment was sequenced, and the sequencing results showed that the fragment contained a partial sequence of ToTOM3. The tomato total DNA was digested with the restriction endonuclease EcoR V, and the digested product was subjected to 0.7% agarose gel electrophoresis and then transferred to a nylon membrane, and the 2.3kb DNA fragment amplified by the above-mentioned PCR labeled with ³² P Southern hybridization detection was carried out, and the results are shown in Figure 6 (swimming lane M: 1Kb DNA Marker, swimming lane 1: hybridization band), indicating that a hybridization band with a size of about 6.5 kb was obtained. Digest total tomato DNA with restriction endonuclease EcoR V, separate by 0.7% agarose gel electrophoresis, and recover fragments with a size of 6.0-6.5kb. The phosphorylated vector pSL118 (Promega Company) was transformed into Escherichia coli DH5a to obtain a partial gene library of tomato genomic DNA EcoR V digestion products. About 10,000 transformants were screened by colony in situ hybridization, and a positive clone was obtained (Fig. 7), which was named pSLToT3-6K. The DNA sequence of the positive clone was determined by duplex duplex, and the results showed that the size of the EcoR V restriction fragment It is 6533bp, and has the nucleotide sequence from the 1st to the 6533rd position of the 5' end of SEQ ID No.: 2 in the sequence listing. Using tomato total DNA as a template, under the guidance of primer ttom3-RNAi-IF: CCTCAGATCTGGGCTGAGATATACTAC and primer ttom3-G2: GAGAACAACGTGAAGTTTCAGGAG, PCR amplified the 3' end fragment of ToTOM3 genomic gene, and after the reaction, PCR was performed on 0.8% agarose gel Gel electrophoresis detection, the detection results are shown in Figure 8 (swimming lane M: 1Kb DNA Marker, swimming lane 1: PCR product), indicating that a specific band with a size of about 4.0kb was obtained. The specific amplification product was recovered, connected to the T carrier pCR2.1-TOPO, and the connected product was detected by 0.8% agarose gel electrophoresis, and the detection results were shown in Figure 9 (swimming lane CK: pCR2.1-TOPO, Swimming lanes 1-3: the recombinant plasmids connected with the 3' end fragment of the ToTOM3 genome gene) to obtain 3 recombinant vectors larger than the control pCR2. The result shows that the size of the PCR product is 4165bp, and has the nucleotide sequence of 3965-8129 from the 5' end of SEQ ID No. 2 in the sequence table, wherein the 5595th base from the 5' end is a restriction The restriction site of endonuclease Sac I has a BamHI restriction site on the sequence of the vector pCR2.1-TOPO (Invitrogen Company), utilizes these two restriction sites to convert the above-mentioned two sizes of 6533bp and 4165bp The DNA fragments were connected to obtain the full-length genome gene fragment of tomato ToTOM3, and the sequencing results showed that its genome gene had the nucleotide sequence of SEQ ID №: 2 in the sequence listing, and SEQ ID №: 2 in the sequence listing consisted of 8129 bases Base composition, with 11 exons and 10 introns, bases 1245-1487 from the 5' end are the first exon of the genome gene, bases 3519-3609 from the 5' end It is the second exon of the genome gene, the 3765-3817th base from the 5' end is the third exon of the genome gene, and the 3895-3987th base from the 5' end is the genome gene The fourth exon, bases 4065-4136 from the 5' end are the fifth exon of the genome gene, and bases 4791-4863 from the 5' end are the sixth exon of the genome gene Exons, bases 4939-4984 from the 5' end are the seventh exon of the genome gene, bases 6259-6322 from the 5' end are the eighth exon of the genome gene, Bases 6403-6456 from the 5' end are the ninth exon of the genome gene, bases 6790-6867 from the 5' end are the tenth exon of the genome gene, and bases from the 5' end are the tenth exon of the genome gene. The 7730-8129th base is the eleventh exon of the genomic gene, the 1317-1319th base from the 5' end is the start codon ATG of the genomic gene, and the 7821-7822nd from the 5' end The first base is the stop codon TGA of the genome gene; the 1488-3518th base from the 5' end is the first intron of the genome gene, and the 3610-3764th base from the 5' end is the genome The second intron of the gene, the 3818-3894th base from the 5' end is the third intron of the genome gene, and the 3988-4064th base from the 5' end is the fourth intron of the genome gene The 4137-4790th base from the 5' end is the fifth intron of the genome gene, and the 4864-4938th base from the 5' end is the sixth intron of the genome gene , bases 4985-6258 from the 5' end are the seventh intron of the genome gene, bases 6323-6402 from the 5' end are the eighth intron of the genome gene, and bases from the 5' end are the eighth intron of the genome gene The 6457-6789 bases at the end are the ninth intron of the genome gene, and the 6868-7729 bases from the 5' end are the tenth intron of the genome gene.

Embodiment 3, the acquisition of transgenic tomato plants

1. Construction of plant expression vectors

1. Construction of plant expression vectors containing ToTOM3 antisense RNA

Primers were designed according to the 5' and 3' UTR sequences of the full-length cDNA of tomato ToTOM3, and the primer sequences were as follows:

ttom3-F: GTGAGTTTGATTTTGGAATCTCCG;

ttom3-G2: GAGAACAACGTGAAGTTTCAGGAG

Using the total tomato DNA as a template, under the guidance of primers ttom3-F and ttom3-G2, PCR amplifies the full-length genome gene fragment of ToTOM3. After the reaction, the PCR was tested by 0.8% agarose gel electrophoresis. The test results are shown in the figure As shown in 10 (lane M: 1Kb DNA Marker, lane 1: PCR product), it shows that a specific band of about 6.5kb was amplified. Reclaim this specific PCR product, connect it with carrier pCR2.1-TOPO (Invitrogen Company), obtain 3 kinds of recombinant plasmids bigger than control pCR2.1-TOPO as a result, further use restriction endonuclease BamHI and XhoI It was digested, and the digested products were detected by 0.8% agarose gel electrophoresis, and the detection results were shown in Figure 11 (lane M: 1Kb DNA Marker, lane 1-3: recombinant plasmid, lane CK1: pCR2.1- BamH I and XhoI digestion products of TOPO, lane CK2: PCR product of ToTOM3 full-length genomic gene), indicating that all three groups of recombinant plasmids contain exogenous DNA fragments with a size of 6.5 kb. The clones shown in swimming lane 2 and swimming lane 3 were sequenced with forward and reverse universal primers M13F and M13R, and the sequencing results showed that the 6.5kb PCR product amplified was the genomic DNA of ToTOM3, and they were all inserted forward (universal primer The M13F sequencing sequence corresponds to the ToTOM3 5' end cDNA sequence), and the recombinant plasmid was named pCToT3-6K. Digest pCToT3-6K with restriction endonuclease Xho I, fill in with _T4 polymerase, and then digest with BamHI to recover a 6.5kb exogenous DNA fragment, which is directional cloned into the restricted On the carrier pBI121 (Clontech) digested with Dicerase Sma I and BamH I, transform Escherichia coli, select a single colony that grows out, shake and culture overnight in LB liquid medium containing kanamycin (50mg/L), and extract the plasmid 0.8% agarose gel electrophoresis detection was carried out on the proposed plasmid, and the detection results are shown in Figure 12 (swimming lane CK: pBI121, swimming lane 1-2: recombinant expression plasmid), and 2 bands larger than the control pBI121 were obtained. Using the plasmid as a template, carry out PCR identification under the guidance of primer ttom3-F and primer ttom3-G2. After the reaction, the PCR product is detected by 0.8% agarose gel electrophoresis, and the detection results are as shown in Figure 13 (swimming lane M : 1Kb DNA Marker, lane CK: pBI121, lane 1-2: recombinant expression plasmid), a 6.5kb specific band was amplified, indicating that the full-length ToTOM3 had been inserted into the vector pBI121, and the recombinant plasmid was named pBIToT3- a.

2. Construction of ToTOM3 RNA interference plasmid pBIToT3R-A

According to the full-length cDNA sequence of tomato ToTOM3, a pair of primers were designed to amplify the coding gene of the small interfering RNA of ToTOM3. The primer sequences are as follows:

ttom3-RNAi-1F: 5'- CCTCAGA TCTGGGCTGAGATATACTAC-3' (underlined part of the base is the restriction endonuclease Bgl II recognition site);

ttom3-RNAi-1R: 5'- TCTCACTA GTAGAGGAGAAATCCCAATG-3' (the bases underlined are the recognition site of the restriction endonuclease Spe I).

Using the 3'RACE cloning plasmid pTTOM3-3 of ToTOM3 as a template, PCR amplification was carried out under the guidance of primers ttom3-RNAi-1F and primers ttom3-RNAi-1R, and the two ends of the amplified products were respectively added with restriction endogenous Dicer Bgl II and Spe I recognition sites. The PCR product was detected by 1.2% agarose gel electrophoresis, and the detection result is shown in Figure 14 (swimming lane M: 100bp DNA ladder, swimming lane 1: PCR product), and a specific fragment (RNAi (A) fragment) with a size of about 200bp was obtained. The obtained PCR product was double-digested with restriction endonucleases BglII and SpeI, and the RNA interference plasmid pUCGA was double-digested with the same enzymes (construction method: using total potato DNA as a template, primer GA-F: 5'-AGG GAGCTC CTCGAG ACTAGT AGATCTG GTACGGACCGTACTACTCTATTCG-3' (the bases underlined are the recognition sites of restriction enzymes Sac I, Xho I, Spe I and Bgl II) and primer GA-R: 5'-AGG GGATCC CTATATAATTTAAGTGGAAAAAAAAGGTTAAC- Carry out PCR amplification under the guidance of 3' (underlining part base is the recognition site of restriction endonuclease BamHI), obtain the first intron fragment (199bp) of potato GA20 oxidase gene, this fragment is used Sac I and BamH I digestion, and the carrier pUC18 (TaKaRa company) that is double digested with the same enzyme is connected, and the recombinant vector obtained is called pUCGA) connection, the connection product is transformed into Escherichia coli, coated with ampicillin (100 mg /L) on LBA medium for 12-24 hours. Select the 3 single colonies that grew out and shake and culture them in LB liquid medium containing ampicillin (50mg/L) for 12-24 hours, extract the recombinant plasmid and carry out enzyme digestion and identification with restriction endonucleases BglII and SpeI, the results are shown in the figure As shown in 15 (lane M: 100bp DNA ladder, lane CK1: RNAi (A) fragment, lane CK2: pUCGA, lanes 1-3: recombinant plasmid), enzyme digestion releases a fragment with a length of about 200bp, indicating that PCR amplified RNAi (A) The fragment has been inserted into the pUCGA vector, and the recombinant plasmid is named pUToT3R(A-). pUToT3R (A-) was double-digested with restriction enzymes Xba I and BamH I, and then ligated with the RNAi (A) fragment double-digested with restriction enzymes BglII and SpeI. The ligation product was transformed into Escherichia coli, spread on LBA medium containing ampicillin (100 mg/L) and cultured for 12-24 hours. Pick 3 single bacterium colonies and shake culture overnight in the LB liquid medium containing ampicillin (50mg/L), extract plasmid and carry out enzyme digestion identification with restriction endonuclease SalI and XhoI, the result is as shown in Figure 16 (swimming lane M: 1kb DNA ladder, lane CK: pUToT3R(A-), lane 1-3: recombinant plasmid), the three recombinant plasmids can all release fragments with a length of about 600bp, and the plasmid pUToT3R(A-) obtained by the first ligation released The fragment is about 400bp, indicating that the RNAi (A) fragment of ToTOM3 is connected to pUCGA in both forward and reverse directions, and the recombinant RNA interference intermediate plasmid is named pUToT3R(A). Digest pUToT3R(A) with restriction endonuclease Sal I, blunt the ends with T ₄ DNA polymerase, then digest with restriction endonuclease SpeI, and recover a small 600bp fragment after agarose gel electrophoresis Fragments, the recovered fragments were ligated with the carrier pBI121 double-digested by SmaI and XbaI, the ligated products were transformed into Escherichia coli, spread on LBA medium containing kanamycin (100 mg/L) and cultivated for 12-24 hours. Pick 5 single colonies and shake them overnight in LB liquid medium containing kanamycin (50mg/L), and extract plasmids for identification by enzyme digestion. Since there is a BglII restriction site at the 2349th and 9149th bases of pBI121 (the physical map is shown in Figure 32), the RNA interference fragment of ToTOM3 has a BglII site. If the fragment is inserted into the polyclonal Site, cut the recombinant RNA interference expression plasmid with the restriction endonuclease BglII to obtain three fragments, one of which is 7948bp, and the length of the other two fragments is about 3700bp. The results of enzyme digestion identification are shown in Figure 17 (lane M: 1Kb DNA Marker, lane 1-2: recombinant RNA interference expression plasmid, lane 3: pBI121), after digestion with restriction endonuclease BglII, the recombinant plasmid was cut into Among the three fragments, one fragment has the same size as the 7948bp fragment of the control pBI121, and the other two fragments are about 3700bp in length (electrophoresis results show that these two fragments overlap). Using the recombinant plasmid as a template, under the guidance of the specific primer ttom3-RNAi-1F and the primer 35S-F: 5'-AATCTTCGTCAACATGGTGGAGCAC-3', PCR detection was carried out, and the PCR product was detected by 1.2% agarose gel electrophoresis to detect The results are shown in Figure 18 (lane M: pUC Mix8 DNAMarker, lanes 1-2: recombinant RNA interference expression plasmid), a specific band with a length of about 640bp was amplified, indicating that the RNA interference fragment had been connected to the expression vector pBI121 , and named the RNA interference vector of ToTOM3 as pBIToT3R-A.

3. Construction of ToTOM3 RNA interference plasmid pBIToT3R-B

The 3'RACE clone plasmid pTTOM3-3 of ToTOM3 was digested with restriction endonucleases BglII and SpeI, and the digested product was subjected to 1.2% agarose gel electrophoresis detection, and the detection results were shown in Figure 19 (lane M: 100bp DNAladder , lane 1: RNAi (B) fragment), recovering and purifying the RNAi (B) fragment with a size of 334bp, connecting the 334bp fragment with the RNA interference vector pUCGA double-digested with restriction endonucleases BglII and SpeI , the ligation product was transformed into Escherichia coli, spread on the LBA medium containing ampicillin (100 mg/L) and cultivated for 12-24 hours. Pick 3 single colonies for further culture, extract the plasmid and carry out enzyme digestion and identification with restriction endonucleases BglII and SpeI, the results are shown in Figure 20 (swimming lane M: 1kb DNA ladder, swimming lane CK1: RNAi (A) fragment, swimming lane CK2 : pUCGA, lane 1-2: recombinant plasmid), a fragment of about 300bp in length can be released after double digestion with BglII and SpeI, indicating that the RNAi (B) fragment has been inserted between the BglII and SpeI restriction sites of pUCGA, and The recombinant plasmid was named pUToT3(B-). pUToT3(B-) was digested with restriction endonucleases XbaI and BamHI and ligated with the RNAi(B) fragment, the ligated product was transformed into Escherichia coli, and spread on the LBA medium containing ampicillin (100mg/L) Incubate for 12-24 hours. Pick 2 single colonies for further culture, extract the plasmid and use restriction endonucleases SalI and XhoI for further digestion and identification, the results are shown in Figure 21 (lane M: 100bp DNAladder plus, lane CK: pUToT3R(B-), Swimming lane 1-2: recombinant plasmid), the two obtained recombinant plasmids can release fragments with a length of about 900bp, and the fragment released by the plasmid pUToT3R (B-) obtained for the first ligation is about 600bp, indicating that the RNAi of ToTOM3 ( B) The fragments were connected to the vector pUCGA in both positive and negative directions, and the recombinant RNA interference intermediate plasmid of ToTOM3 was named pUToT3R(B).

Digest the above-mentioned RNA interference intermediate plasmid pUToT3R(B) with restriction endonuclease SalI, fill in with _T4 DNA polymerase, digest with restriction endonuclease SpeI, and recover by 0.8% agarose gel electrophoresis A small fragment of 900bp was ligated with the carrier pBI121 that had been cut with SmaI and XbaI, and the ligated product was transformed into Escherichia coli, and 5 single colonies were picked for further culture, and the extracted plasmid was identified by restriction endonuclease BglII. The cut identification results are shown in Figure 22 (lane M: 1Kb DNA Marker, lane 1-2: recombinant RNA interference expression plasmid, lane 3: pBI121), the recombinant plasmid shown in lane 1-2 was cut into three fragments, of which One fragment has the same size as the 7948bp fragment of the control pBI121, and the other two fragments are about 3700bp (the electrophoresis results show that the two fragments overlap). Using the recombinant plasmid as a template, specific primers ttom3-g: 5'-CCGTCTGTCCTCACACTAGGCAG-3' and Under the guidance of primer 35S-F, further PCR identification was carried out. The PCR identification results are shown in Figure 23 (lane M: pUCMix8 DNA Marker, lane 1-2: recombinant RNA interference expression plasmid), PCR amplified a specific DNA marker with a length of about 440bp. Sexual bands indicated that the RNA interference fragments had been connected to the expression vector pBI121, and the RNA interference vector of ToTOM3 was named pBIToT3R-B.

2. Genetic transformation of tomato

1. Transformation of plant expression plasmids into Agrobacterium by three-parent combination method

The three plant expression plasmids pBIToT3-A, pBIToT3R-A and pBIToT3R-B (Rogers S.G., Horsch R.B., and Fraley R.T. 1986. Gene transfer inplant: production of transformed plants using Ti-plasmid vectors.MethodsEnzymol., 118:627-640) were transformed into Agrobacterium EHA105, and screened on LA plates containing 50mg/mL rifampicin (Rif) and 50mg/mL kanamycin to obtain expression plasmids containing the above three kinds of plants Agrobacterium transformants.

2. Genetic transformation of tomato

The Agrobacterium transformants transformed with pBIToT3-A, pBIToT3R-A and pBIToT3R-B were respectively introduced into tomato by the following method, and the following medium for tomato tissue culture and transformation was MS minimal medium.

1) Preparation of Flamingo Bill explants

L.，et al.2001.Enhanced regeneration of tomato and pepper seedlingexplants for Agrobactereium-mediated transformation.Plant cell，Tissue andorgan culture.，67：173-180)制备Flamingo Bill外植体，具体方法为：将番茄种子一个个地分开，用蒸馏水洗3次，然后用80％乙醇浸泡并不停地搅动2分钟，加入4-6滴100％的Tween-80，倒去乙醇溶液，再用无菌水洗涤2-3次。加入有效氯为1％的NaClO溶液与4-6滴100％Tween-80在搅拌器上不停地搅动15分钟，倒去NaClO溶液，用无菌水洗2次，再重复二次，直至番茄种子的颜色由褐色变为金黄色，至此，番茄种子基本无菌，再用无菌水冲洗6-10次，将其置于无菌滤纸上晾干。晾干后把种子接入倒有1/2MS培养基(含3％的蔗糖)的培养瓶中，封口，在25-28℃的光照培养箱中培养7天，再把培养瓶放在超净台中，打开盖子，补充培养瓶中的水分，使无菌苗在开放的环境下培养15个小时，使其直立生长，子叶平展，以利下一步操作。将培养的无菌苗的一侧子叶从叶柄基部连同顶芽及周围分生组织全部切去，只留一侧子叶，另外将无菌苗的大部分根系切去只保留长度为2-3cm的胚根。Referring to the methods of Javier Pozueta-Romero et al. (Pozueta-romero, J., Houlnè, G.,

L., et al.2001.Enhanced regeneration of tomato and pepper seedling explants for Agrobactereium-mediated transformation.Plant cell, Tissue andorgan culture., 67:173-180) prepare Flamingo Bill explants, the specific method is: a tomato seed Separate them one by one, wash them three times with distilled water, then soak them in 80% ethanol and stir them continuously for 2 minutes, add 4-6 drops of 100% Tween-80, pour off the ethanol solution, and wash them with sterile water for 2-3 minutes. Second-rate. Add NaClO solution with 1% available chlorine and 4-6 drops of 100% Tween-80, stir continuously on the stirrer for 15 minutes, pour off the NaClO solution, wash 2 times with sterile water, and repeat twice until the tomato seeds The color of the tomato is changed from brown to golden yellow. So far, the tomato seeds are basically sterile, and then rinsed with sterile water for 6-10 times, and placed on sterile filter paper to dry. After drying, insert the seeds into a culture bottle poured with 1/2MS medium (containing 3% sucrose), seal it, and cultivate it in a light incubator at 25-28°C for 7 days, then put the culture bottle in an ultra-clean In Taichung, open the lid, supplement the water in the culture bottle, and cultivate the sterile seedlings in an open environment for 15 hours to make them grow upright and the cotyledons flatten to facilitate the next operation. Cut off one cotyledon of the cultured aseptic seedling from the base of the petiole together with the terminal bud and the surrounding meristem, leaving only one side of the cotyledon, and cut off most of the root system of the aseptic seedling, leaving only the 2-3cm in length. Radicle.

2) Infect the explant Flamingo-Bill with Agrobacterium EHA105

Streak the Agrobacterium transformed with pBIToT3-A, pBIToT3R-A and pBIToT3R-B on the YEB plate containing 50 mg/mL kanamycin and 50 mg/mL rifampicin, culture at 28°C for about 24 hours, and grow After colonization, pick a single colony and inoculate it in YEB liquid medium containing corresponding antibiotics, culture it on a constant temperature shaker (200 rpm) at 28°C for about 24 hours until the OD ₆₀₀ is 04-0.6, transfer it to a sterile centrifuge tube, and centrifuge at 3000rpm After 10 minutes, the supernatant was discarded, and the collected cells were washed once with MS culture medium and resuspended, diluted 50 times and then used for transformation of tomato.

Put the explant Flamingo-Bill prepared in step 1) into the MS liquid medium containing the Agrobacterium activated in step 2) (plus acetosyringone with a final concentration of 100 μmol/L), and shake it continuously for 10 minutes , the explants were taken out and rinsed twice with sterile water, and then one root was put into MS co-cultivation medium (adding 6-BA 2.0mg/L on the basis of MS basic medium, indole acetic acid 0.1mg/L, AS 100μmol/L) in a 15cm-diameter large petri dish, sealed, and cultivated in a light incubator for 2 days.

3) Differentiation induction

After cultivating for 2 days, it was transferred into the differentiation screening medium (added ZT1.0mg/L on the basis of MS basic medium, IAA 0.1mg/L, Km 50mg/L and Cef 200mg/L), and continued to Cultivate in a light incubator for 2-3 weeks, the culture condition is 25-28°C, 15 hours of light/9 hours of darkness per day.

4) Rooting, hardening and transplanting of transgenic plants

Plants growing normally (about 2-3 cm long) in the differentiation selection medium were excised from the callus (be careful not to attach the callus). Insert in the rooting medium after excising (on the basis of MS basic medium, added Km 50mg/L, Cef 150mg/L and NAA 0.2mg/L), continue to cultivate and make it take root. After rooting, open the lid of the culture bottle and add water to make the plant gradually adapt to the external environment. After 3-4 days, slowly pull out the plant, wash the agar on the root, transplant it into the soil, and keep the soil humidity. The culture temperature was set at 25-30°C.

3. Molecular identification of transgenic plants

The kanamycin-resistant plants were potted in the insect-proof net room, and the leaves were taken after one month, and the total DNA was extracted according to the conventional method. Using this as a template, the primer Kam-R: 5'-GATCTGGATCGTTTCGCATG-3' and the primer Under the guidance of Kam-F: 5'-AAGGCGATAGAAGGCGATGC-3', PCR detection was carried out, and a total of 26 transgenic plants that could amplify a specific band of 800bp were obtained, of which 13 were transgenic antisense RNA plasmid pBIToT3-A, Ten strains were transformed into RNA interference plasmid pBIToT3R-A, and 3 strains were transformed into pBIToT3-B. The 26 transgenic plants were further detected by PCR and Southern hybridization using 35S-F and corresponding specific primers.

1. Molecular identification of antisense RNA transgenic plants

The total DNA of 13 transgenic plants transgenic to pBIToT3-A was extracted and used as a template to carry out PCR detection under the guidance of primer 35S-F and primer ttom3-G (5'-CAATTGAATTCATTTTTTCTCC-3'). Tomato was used as a control, and the results are shown in Figure 24 (lane M: pUCMix8 DNA Marker, lane CK: non-transgenic tomato, lanes 1-13: transgenic tomato), and the amplified specific band with a length of about 400bp is the transgenic plant. The total DNA of the transgenic plants was digested with the restriction endonuclease BamH I, and the RNAi (B) fragment labeled with ^P32 was used for Southern hybridization. The hybridization results are shown in Figure 25 (swimming lane M: 1kb DNA Marker, swimming lane CK: Non-transgenic tomato, lanes 1-13: transgenic tomato transformed with pBIToT3(A), all plants have an endogenous 6.2kb endogenous hybridization band, while transgenic plants can also have a different size in addition to the endogenous hybridization band Hybridization bands, indicating that the antisense RNA fragments of ToTOM3 have been integrated into different positions of the tomato genome.

2. Identification of RNA interference transgenic plants

Using the total DNA of the transgenic plants transformed with the RNA interference expression plasmid pBIToT3R-A as a template, under the guidance of primer 35S-F and primer ttom3-RNAi-1F, PCR detection was performed, and the detection results are shown in Figure 26 (lane M: pUCMix8 DNA Marker, lane CK: non-transgenic tomato, lane 1-10: transgenic tomato), the transgene-positive plants that can amplify a specific band with a length of about 600bp; Under the guidance of g, PCR detection was performed on the transgenic plants transformed with pBIToT3R-B, and the detection results are shown in Figure 28 (lane M: 1kb DNA Marker, lane CK: non-transgenic tomato, lanes 1-10: transgenic pBIToT3(A) Tomato), the transgene-positive plants that can amplify a specific band with a length of about 400bp. The total DNA of the above-mentioned plants was digested with the restriction endonuclease EcoRI, and the RNAi (A) fragment transfected with pBIToT3R-A and ^P32 was subjected to Southern hybridization, and the hybridization results were shown in Figure 27 (swimming lane M: 1kb DNA Marker, lane CK: non-transgenic tomato, lanes 1-10: transgenic tomato with pBIToT3 (A), Southern hybridization was performed on the transgenic pBIToT3R-B and ^P32- labeled RNAi (B), and the hybridization results are shown in Figure 29 (Lane M: 1kb DNA Marker, Lane CK: Non-transgenic tomato, Lanes 1-3: Transgenic tomato). All plants have an endogenous 5.8kb endogenous hybridization band, and the transgenic plants have a hybridization band of different sizes except for the endogenous hybridization band, indicating that the two RNA interference fragments have been integrated into different positions of the tomato genome superior.

Embodiment 4, detection of disease resistance of transgenic plants

Adopt rubbing inoculation method, use CMV and TMV to carry out inoculation experiment to the transgenic tomato of all 5-6 leaf stage obtained in embodiment 3 respectively, take the non-transgenic tomato of the same growth period as contrast, carry out symptom observation regularly, and after inoculation 75 days The virus content in the transgenic tomato plants was determined by conventional biological assay methods and DAS-ELISA.

1. Symptoms

10 days after inoculation, non-transgenic tomato seedlings inoculated with TMV showed mottled symptoms (Fig. 20B), non-transgenic tomato seedlings inoculated with CMV showed typical mosaic symptoms (Fig. 20D), and transgenic seedlings showed no symptoms (Fig. 20A, Fig. 20C). After 30 days of inoculation, except for a few transgenic seedlings showing slight mottled symptoms, most of the transgenic seedlings still had no symptoms. 90 days after inoculation, the non-transgenic plants inoculated with TMV still showed mottled symptoms (Fig. 21B), and the transgenic plants showed no symptoms (Fig. 21A). The leaves of tomato seedlings inoculated with CMV non-transgenic plants were severely yellowed, forming uneven blisters (Fig. 21D), the transgenic plants showed mild mottled symptoms and formed a small amount of blisters (Fig. 21C).

2. Relative content of TMV in transgenic plants

Seventy-five days after inoculation with TMV, take the new leaves (the penultimate new leaves) of the transgenic plants and the control plants respectively, cut the same area (about 2×2cm ² ), and wash them with 1mL phosphate buffer solution (0.02M _Na2HPO4 , 0.001M KH2PO4 , pH7.2) was ground into a homogenate, diluted 100 times with phosphate buffer, inoculated on the leaves of Amaranthus quinoa, and counted the number of dead spots after five days. The statistical results are shown in Table 1. With the transgenic tomato as the inoculation source, the number of dead spots formed on the amaranthus quinoa was significantly lower than that of the control tomato plants, and the abbreviation of the single plant transfected with the antisense RNA expression plasmid was An (n is The number of individual plants), and the individual plants transfected with RNA interference plasmids are abbreviated as R(A)n and R(B)n respectively.

Take another 0.1g leaves, add 1mL coating buffer (0.015M Na ₂ CO ₃ , 0.035M NaHCO ₃ , pH9.6) to grind into a homogenate, dilute 1000 times and use for DAS-ELISA detection, the detection results are shown in Table 2 As shown, it shows that the concentration of TMV in all transgenic plants is significantly lower than that in non-transgenic plants, which is consistent with the results of the scab test. The content of TMV in all transgenic plants is significantly reduced, and they have strong resistance to TMV.

The biological assay result (inoculation 75 days) of TMV relative content in the transgenic plant of table 1

strain Average number of dead spots Standard Deviation (S.D.) Standard Error (S.E.) significant Non-transgenic plants (control) 171.00 3.61 2.08 A6 20.33 3.22 1.86 significant A25 25.67 1.53 0.88 significant A36 40.67 7.02 4.06 significant A52 52.67 3.22 1.86 significant A53 12.00 2.65 1.53 significant A59 51.67 4.04 2.33 significant A94 24.00 3.61 2.08 significant A99 24.33 2.52 1.45 significant A119 22.00 3.61 2.08 significant A121 34.33 3.06 1.76 significant A123 20.67 2.52 1.45 significant A134 90.67 5.13 2.96 significant A155 58.67 4.51 2.60 significant R(A)6 31.00 4.58 2.65 significant R(A)8 26.00 2.65 1.53 significant R(A)12 37.00 7.55 4.36 significant R(A)55 86.67 5.86 3.38 significant R(A)66 22.33 3.06 1.76 significant R(A)69 25.00 2.65 1.53 significant R(A)71 38.33 3.06 1.76 significant R(A)111 23.67 2.31 1.33 significant R(A)114 90.67 6.43 3.71 significant R(A)139 64.67 3.79 2.19 significant R(B)16 17.00 2.00 1.16 significant

R(B)28 25.33 2.52 1.45 significant R(B)46 14.67 3.06 1.76 significant

DAS-ELISA assay result of relative content of TMV in table 2 transgenic plants (inoculation 75 days)

strain Average OD Standard Deviation (S.D.) Standard Error (S.E.) significant   Non-transgenic plants (control) 1.982 0.003 0.002 A6 0.235 0.002 0.001 significant A25 0.229 0.003 0.001 significant A36 0.362 0.003 0.002 significant A52 0.527 0.003 0.002 significant A53 0.235 0.004 0.002 significant A59 0.512 0.003 0.001 significant A94 0.222 0.003 0.002 significant A99 0.220 0.003 0.002 significant A119 0.220 0.001 0.001 significant A121 0.405 0.004 0.002 significant A123 0.216 0.003 0.002 significant A134 0.948 0.003 0.002 significant A155 0.669 0.002 0.001 significant R(A)6 0.275 0.003 0.002 significant R(A)8 0.211 0.003 0.002 significant R(A)12 0.433 0.003 0.002 significant R(A)55 0.770 0.002 0.001 significant R(A)66 0.213 0.002 0.001 significant R(A)69 0.281 0.003 0.001 significant R(A)71 0.381 0.003 0.001 significant R(A)111 0.204 0.003 0.001 significant R(A)114 0.748 0.003 0.002 significant R(A)139 0.504 0.003 0.001 significant R(B)16 0.254 0.003 0.002 significant R(B)28 0.313 0.003 0.002 significant R(B)46 0.215 0.005 0.003 significant

3. Relative content of CMV in transgenic plants

70 days after inoculation with CMV, take the new leaves (the third last new leaf) of the transgenic plants and the control plants respectively, cut out the same area (about 2×2cm ² ), grind them into a homogenate with 1mL phosphate buffer solution, and inoculate them directly into amaranth On the leaves of quinoa quinoa, the number of dead spots was counted after five days. The statistical results are shown in Table 3. Using the transgenic tomato as the inoculation source, the number of dead spots formed on the amaranthus quinoa was significantly lower than that of the control non-transgenic plants.

Take another 0.1g leaf, add 1mL coating buffer to grind into a homogenate, dilute it 10 times and use it for DAS-ELISA detection. The detection results are shown in Table 4. The concentration of TMV in all transgenic plants was significantly lower than that of non-transgenic plants. Plants, consistent with the results of the dead spot assay, showed that all transgenic plants had certain resistance to CMV.

The biological assay result (inoculation 70 days) of CMV relative content in the transgenic plant of table 3

strain Average number of dead spots Standard Deviation (S.D.) Standard Error (S.E.) significant Non-transgenic plants (control) 148.00 2.65 1.53 A6 22.00 4.36 2.52 significant A25 18.33 2.08 1.20 significant A36 22.67 3.79 2.19 significant A52 23.67 8.02 4.63 significant A53 28.33 2.52 1.45 significant A59 22.00 2.65 1.53 significant A94 21.33 3.22 1.87 significant A99 26.67 3.79 2.19 significant A119 30.67 4.04 2.33 significant A121 74.33 5.13 2.96 significant A123 31.00 6.00 3.46 significant A134 78.00 2.65 1.53 significant A155 60.00 7.00 4.04 significant R(A)6 25.00 4.00 2.31 significant R(A)8 85.67 5.69 3.28 significant R(A)12 62.00 5.57 3.22 significant R(A)55 76.00 5.57 3.22 significant R(A)66 25.33 4.51 2.60 significant R(A)69 28.33 3.06 1.76 significant R(A)71 58.33 5.51 3.18 significant R(A)111 28.00 3.61 2.08 significant R(A)114 74.67 5.69 3.28 significant R(A)139 46.33 5.86 3.38 significant R(B)16 21.67 3.06 1.76 significant R(B)28 29.00 2.65 1.53 significant R(B)46 20.00 2.65 1.53 significant

The DAS-ELISA assay result (inoculation 70 days) of CMV relative content in table 4 transgenic plants

strain Average OD value Standard Deviation (S.D.) Standard Error (S.E.) significant Non-transgenic plants (control) 1.512 0.003 0.001 A6 0.192 0.003 0.002 significant A25 0.213 0.003 0.001 significant A36 0.231 0.003 0.002 significant A52 0.243 0.003 0.002 significant A53 0.224 0.003 0.001 significant A59 0.251 0.003 0.002 significant A94 0.213 0.003 0.002 significant A99 0.201 0.002 0.001 significant A119 0.209 0.002 0.001 significant A121 0.384 0.002 0.001 significant A123 0.195 0.003 0.002 significant A134 0.892 0.003 0.002 significant A155 0.625 0.002 0.001 significant

R(A)6 0.209 0.001 0.001 significant R(A)8 0.608 0.003 0.001 significant R(A)12 0.503 0.002 0.001 significant R(A)55 0.572 0.001 0.001 significant R(A)66 0.201 0.003 0.002 significant R(A)69 0.259 0.002 0.001 significant R(A)71 0.363 0.003 0.002 significant R(A)111 0.184 0.002 0.001 significant R(A)114 0.403 0.003 0.001 significant R(A)139 0.334 0.003 0.002 significant R(B)16 0.203 0.002 0.001 significant R(B)28 0.284 0.003 0.001 significant R(B)46 0.195 0.003 0.002 significant

sequence listing

<160>3

<210>1

<211>295

<212>PRT

<213>Lycopersicon esculentum Miller

<400>1

Met Gly Arg Ala Glu Met Val Val Gly Pro Ser Glu Lys Val Ala Val

1 5 10 15

Val Ala Tyr His Leu Asn Asp Ala Ile Asn Trp Trp Asp Asp Val Asn

20 25 30

Arg Ser Leu Asp Trp Gln Asn Arg Ile Phe His Val Leu Ala Val Leu

35 40 45

Tyr Gly Val Val Ala Val Val Ala Leu Val Gln Leu Ile Arg Ile Gln

50 55 60

Met Arg Val Pro Glu Tyr Gly Trp Thr Thr Gln Lys Val Phe His Phe

65 70 75 80

Leu Asn Phe Phe Val Asn Gly Val Arg Ser Leu Val Phe Thr Phe Arg

85 90 95

Arg Asp Val Gln Lys Leu His Pro Glu Ile Val Gln His Ile Met Leu

100 105 110

Asp Met Pro Ser Leu Ala Phe Phe Thr Thr Tyr Ala Leu Leu Val Leu

115 120 125

Phe Trp Ala Glu Ile Tyr Tyr Gln Ala Arg Ala Val Ser Thr Asp Gly

130 135 140

Leu Arg Pro Ser Phe Phe Thr Ile Asn Gly Val Val Tyr Ala Ile Gln

145 150 155 160

Ile Ile Leu Trp Leu Ile Met Trp Trp Lys Pro Ile Arg Val Leu Phe

165 170 175

lle Leu Ser Lys Met Phe Phe Ala Gly Val Ser Leu Phe Ala Ala Leu

180 185 190

Gly Phe Leu Leu Tyr Gly Gly Arg Leu Phe Leu Met Leu Gln Arg Phe

195 200 205

Pro Val Glu Ser Arg Gly Arg Arg Lys Lys Leu Gln Glu Val Gly Tyr

210 215 220

Val Thr Thr Ile Cys Phe Ser Cys Phe Leu Ile Arg Cys Val Met Met

225 230 235 240

Cys Phe Asn Ala Phe Asp Lys Ala Ala Asp Leu Asp Val Leu Tyr His

245 250 255

Pro Ile Leu Asn Leu Ile Tyr Tyr Leu Leu Val Glu Ile Leu Pro Ser

260 265 270

Ser Leu Val Leu Phe Ile Leu Arg Lys Leu Pro Pro Lys Arg Gly Ile

275 280 285

Thr Gln Tyr His Pro Ile His

290 295

<210>2

<211>8129

<212>DNA

<213>Lycopersicon esculentum Miller

<400>2

aaatgttaga ggttttacca acatccgttc cccaaaagcc ccaaaaatcc aacaagacac 60

cacgtataac gtgatcttag aattattcat atgaacgttc aacattttct ttattataaa 120

gaaagatatt ttactctcta ctgtttctcg taatagaaaa tagctaaaga attatctata 180

tttacaacaa tgcgaaatcc taatcctata ttattatagact aaagttttta ttagttgatt 240

tcttctccta atgcatgaag gacatgggtt taaaaaaatc gaacataccg ataaatcgaa 300

tcgaaaaaat gttattgggg tattgttatt gtgttattga tttatgggg tttaatgagt 360

ttataaaaat aatttattag gttattggtt cggcatcagt ttttactatt ggattattgg 420

taaatcgata acccaataaa atggtaataa tttattatt tgcccttcct aattattaat 480

attaatactt taatatataa ttaaacacta taactatatc aaattattag aactctacca 540

acttcaccgt atgccatttt actagctttt actatttact caaaacctaa agattaaagt 600

aaggggttca caattgaagc tatttgattt tagttttagt tttagcttta gtgttggact 660

agtttatttt agttttagtt tgtaattgta ggttgtagca tttgcaagtt tgtaaaggtt 720

cataatttga ttaacataaa aaattacata ctatgttttg gcgataacat gtaattataa 780

ccttcgaact atatattctc ttattgatgt aatttctcat tatctttctt gtttcactat 840

atcaaaacat ctagagaagt gaaaagacat ataatatttc acgggcattt tcttattgga 900

taaatcgaaa tcgaactgat aatgattaaa aatcggtaca tcgaaaaccg ataaagaata 960

tcttattggt ttgttattgg attagcatat ttaaaaacca aaaaccgata atccgaaccg 1020

aaccgaccgg tgcacacccc taatcaagga tatatatata tatcggaca cattacccct 1080

ctaaacgttt tttttaatat tgtatttgtt tttatatata ataaaataat accttttttt 1140

tctagccgtc cattttgaaa aacaaaagaa aaaaaagtac aaatttaaat aaacaaaaat 1200

agtaaatgaa tttttttaaa aattaatttc tgaaaaaggt agttgccgat tttggtgttt 1260

gatttttttt tggggggatt ttgaaattgg tgagtttgat tttggaatct ccggtgatgg 1320

gacgggcgga gatggttgta ggcccgtcgg agaaggtggc ggtggtggca tatcatctga 1380

atgatgcaat caattggtgg gacgatgtga acagatctct tgattggcaa aaccgtatat 1440

tccatgtcct tgctgttctc tacggcgttg tcgccgtcgt tgctcttgta aattctgtct 1500

ctctatcttt acgtctcagc gtttgagttt tgaattttat gagttaattt taacataatt 1560

gttatgttga ttgggataag agaggttcca tcaggtggta agcatccttc cttgtgaact 1620

agaagaaagg aggtctagga ggtaagttgt tgtcatgtta gtatgatgtg acaggttcga 1680

gcggtgtaaa taggcttttt gcatccaata gaccaatgtg gttcagtgca tagtaggagc 1740

ttagtataga agaaaggagg tcgtctagac gggaaagatg ttgtcatgtt agtaggatgt 1800

gacaggtttg agctgtgtaa ataggctttt tgcatccaat agagctttgt ggttcagtgc 1860

atagcaggag cttagtatac tgggctgctc tttttatatt gttatgttga ttggtaaagt 1920

aagattgggc tcatatatgt gttggagatg tatgaagaat ctgaatattt gttttatgtt 1980

gcttgttagt tgtttgaatt gaatgtcatg tgcatcatgt aactccaaac tttgatgttt 2040

tcgtcaaggg aagaattcta ggtggaactt ttgaatttga ggcattttag acttgttttt 2100

gatggtcctt gaaacgggga tagataactc atagagtatg catcaagctt tcaaggggcg 2160

cttactagaa ctatcactca gcagagcttt agtttcggct catatcagtg ttttagacgg 2220

caaaaggcga caagggtctg ccttgccata aggcgaggcg accggcgagg cactcacctt 2280

tttgaagtgt ggcaccaatt aataccaaaa aattaaaata ttgaattgca tatgtaaata 2340

ataaaaatct caatagcaat aacatattag caaatagttt aattcaaaac taaaaataga 2400

tagtacatac tataattcta gaacatgaat gagaaaaaaa caaaacaaaa gtcaaaacag 2460

aggtagaagt gactctgaag tccgaagaag aaggaaaaag aaacccaaaa aagaagaagt 2520

ttgaagaaga aaagaagaaa catgtcttgg ttggactttc gagtagccgg aaaagtttgc 2580

ctggtcgtcg aaggagtcta atcgtaagcc ctaaaattac cttttaactt ttaaaatcct 2640

aaattggtag tcttttttta agaatataca aaaggtgacg cctttcttgc ttttcttgca 2700

tctcacctct cgccttttgt cgccatggct cgcttcgcca caaagttaaa aggcgatgag 2760

gggtcgcctc gcctctcgcc cattggcgac gaggcgatcg cctttcgcaa cacttggctc 2820

atatggatag ggataggaaa aagagagatt ttcgtcgttt tgggatcatt cggataaaca 2880

cagtaatttg cggaagtgga gtattctata gttatagtag attaatacac aatttgttca 2940

agagacaatt gcttcttaac cggaaaatac ttgcttaaat agatatatca aataggaatt 3000

gtctttat gatttcatat agacacttga aagtgattaa aactgaaatt tgaaactaaa 3060

aagggaagtt gttgaagtag gtaaaacttt taaaggattg catcttttcc tccctgaaag 3120

ttcatgatta ttaatttccgg tatggcgggt gttgttgttg gtgttcaggt gtttagtcat 3180

ggcaaaacta ttatatgttt ccagggagaa tatgcactta tagtatgtat atttttggca 3240

aaagttgaaa ttgtctaatt tccactaatt ggtttatgta tgcaaaaaag aggggggcaca 3300

tggcctggag tttgaaattt aaattttgcc aattggtttg tttgtgtctc attgattgat 3360

tttggtatac ttgtttaaat tttcatgcat gttcttgcgc attggatgat catattgtta 3420

ttcagttatt gctctacaaa atttattatta ccagtaccaa tacttcaatt tatgtgcggc 3480

tctggaattt gtttcttact ttgcaacttg gcaatcaggt acaattaatt cgcattcaaa 3540

tgagagttcc tgaatatggc tggaccactc aaaaagtctt ccactttctc aatttctttg 3600

tgaatggagg tcagtatctt tcttttcttt acttctaatt attttttgaa catcctgatg 3660

ggaagttcca ttacacatgc ttttcaaatg attgttcctg tacactccac tttgttttga 3720

cgtgttgtct ttgttattta actgtgtctt catgttcttg gcagttcgct cgctagtttt 3780

tacatttcgt cgggatgttc agaagttgca cccggaggtt aagcaacaga gtatcttgat 3840

gttttttgtc ttacactcct ttaattttta acgctttata ctacattttt gcagattgtg 3900

caacatatta tgcttgatat gccaagtctt gcattcttca caacttatgc tctgctagta 3960

ttattctggg ctgagatata ctaccaggtt cttgtggtct gtctcaattt ttgtgtagat 4020

atatatcttc tcaaggttaa catatttgtt ttttccatga caaggcacgt gctgtgtcca 4080

cggatgggct tagaccctagt ttcttcacaa tcaacggagt ggtttatgct attcaggtaa 4140

atatgtgaca gttatgcaat acttatataa actgcttgtt tcaatttatt cgtctttctt 4200

tcctttttgg tccatttcaa aagaatccct tggcaggcgc atcaatttat tttggtgctt 4260

gctagaaata aactctggtc tgggagtttt gacatcaata tatttctaaa agtgacaaca 4320

agtttcatct gatcagaatt tttctcaaca ttcttaaaca tgtcgttcat agggtacttc 4380

actcagtacc ccggccatcc tctccaactc ccaaacgtta aggataccct ccacctgagc 4440

atcagcaatt actacaattt tcccttctta agggtattct attagcagaa atgagagcac 4500

agtgcctaat atttcacaaa atgctgagac cactcataaa tcttagggat atatcaaacg 4560

aagtgtcatt gataaggaaa tatgtttctg tcatttggag atactttatc aagactacat 4620

tttgtttgga tgagatgttc agtgcccgca gttagacatg gtgactttta tgttttctca 4680

tggaaactgt tgcatgaaac atgtgagaca agggtttact cttttaaatt gctgtaccta 4740

tttggtggat gttcattgct gattttcttt gtttcttata tatcttgcag attatattat 4800

ggctgataat gtggtggaaa cctattcgag tactcttcat cttatccaag atgttttttg 4860

caggttcttg ttttagattt aacttatttt tagtattgca tggatctgtt gcgaagtatt 4920

aacataatgt acttgcaggt gtatccctat ttgcagcatt gggatttctc ctctacggtg 4980

gaaggtaaaa tcttgatttt tagtatcagt cactttgtca ccgacaggaa ttaccgaatg 5040

tacatttgtt aaatcgcata cagaatgggt aaaatcaag cacataagca taaagcttag 5100

cacttgtttc tatcgttcgt taaaggatgt tttatcttct gattcgttgc tattcttaac 5160

acttctttag aaggtagaga tggatatgta gtcaattgtt gtttgctttg atcaaaagcc 5220

aatttttttg gacgaaaagg tgatctgctt cctagcctat acatccacag atctaaacct 5280

tcacgagatg acttctgttg tgtcctaagc gtcaccattt tctgaaatta agtatcttgt 5340

atattggcat gtatcttatg cgatcatcgg ttactttcag taacctgtac ttcattagtt 5400

tgatatttga taccaggagc acacattgac aaagaggaca taagtaagaa gaagatgtag 5460

aaggaaggag ggaaaagaag gagggggcgg gagtgaagaa ggaaaatgaa agaaacatac 5520

cctaatatgg tgaattagag aagagaggag caaaagattg aaaattggaa ccttcgagaa 5580

atgaatggag agctcattag gaggagagat gagtggagga gtttcccatt cttgaccaac 5640

tattttgaca atgcaaccat taacctctgt tttgcccttg tcgcattcga cacctgctat 5700

atcgatcatt tttatcctcc tagctaaaaa gtggaagatt ccccattgaa gtcttccctc 5760

atctctaaag agtactctag agtgaagttc ctaatacata ggaaaagaaa ttggcaggtg 5820

ctagattgaa tgaacgcagc aaccacggca gtaatagatg acaaacatat aaaaaagtaa 5880

aatatcttca attacactta cttttagtga aagttcatgt gaccttttat tgccctgcct 5940

aatgaaagtt caaaagtctt ctcaagcatt atcttagttt gtcggcacgg tcgtacaaag 6000

atgcatagtg ttgttgaacg tgctgcttaa aaataaaatt agtaatttaa atattaagat 6060

actgcaaaaa aagttcttac agcactgcgt atagcacatg tttgtttagt ttgtggattg 6120

acctttgcta tatctcgctt gagtaccttg tgtactctta tgtttgaata attgggtcca 6180

tgcaacttgt atgtcattca tattcatcct tcaatatcac cttactgttt ggcttgatat 6240

tctttcactt tcttccaggc tttttcttat gttacagcgg tttccagtag aatcaagagg 6300

gagacgcaag aagctgcagg aggtatgtta ttagtacttc tctttcttgg tatttcattc 6360

ttattagggtc ttttgacttc tccggcattt tggcttctgt aggttggtta tgtcacgaca 6420

atatgttttt catgcttcct cattagatgc gttatggtaa gtccatattc ctcgtgaact 6480

aaacattttt tagctgatca taactaaccc atgtttgtag tgtgtgaatg atatctatat 6540

tttgctttag atggtttctt ttttgtctaa aacctttctt gatacaataa gttactctta 6600

gctgagatgc gtgcaagctg ggtcagacac ccttttttcc ggaaaaaaaa agtcttagct 6660

gatgcattga ttttctttct atagggcatc tttctcaaat tttttccttt ctatctatag 6720

ggcataaagc ctctttggtt attctatatg gcttaatggt tagtcactct attattatct 6780

ttgctacaga tgtgcttcaa tgcatttgat aaagctgcag atcttgatgt tttgtatcat 6840

cctattttga atttgatata ttacctggtg agttccatat cactgaactt ctatagcac 6900

ttctttaggt aaaatattgg ttgctgcgcc ataatcaaaa gaagtattgg cagtcagtat 6960

attttatata attattgcca aatcacgata aattggataa ttcacatgat atttcagctt 7020

gtggcatctt acaatcagtt ttatgcgcca aaattctgtt tggctccttt tctatgtagt 7080

cttttgagtt gggatggggg tttcttactt gtgccatgaa ctcaggtttc tttctcttcg 7140

tttgacattt tgtttaaatt gaaaattgga ttcaaaatct taagattatg gtgaaatatt 7200

tgagacacag ggagtggaaa tcatgttctg aaattatagc atggttatta ttttggcaca 7260

tacataaaca gacacttaaa cttggcctca acgggcatat aagcattgca actttgagaa 7320

agcacatcta gacacctcaa ctaaacattc caactcatcc ccactctata tcttgtggac 7380

actctgatgt ggctcataaa ttttggaggt gtctagatga tcactttgta agtcggagtg 7440

ttcaactgac acaatggaga cgagttgagg tgtcgtgtgt gcatactcaa agttggagtg 7500

gttacttgcc agttgaggcc gattttgagt gtctgtttat gtattatgcc tattatttca 7560

cttgatcgtg tcctatccaa accttagttt catccaaatt tcaaatcatg agtatatact 7620

tagacatggt gttttttact ttgcgctttg gggggtagaa agtggagaaa atggagtaat 7680

attttctgt atgttttgag actaagcaaa ctttttaacc tcttcgcagt tagtggagat 7740

actgccttct tctcttgtcc tttttatttt aaggaagttg cctccaaagc gagggatcac 7800

ccaataccac cctattcact aatacattaa gagggtagat aatgatgaaa atcaggctcc 7860

gggatcaggt attaagtaag ttggctttta cctggatgtg atttgcaagc aagaaatacg 7920

agaggagtga taatgtaaat tgaagtatgg ttcgtctata ctgaaatatc cgtctgtcct 7980

caactaggc agattgtagc tctgttttgt accactagtt atagatggaa ttgtgaagta 8040

tcttacgacc tttagtgtat tatttcgcct ttgtatgtgt cagatttcaa ttgaattcat 8100

tatttctcct gaaacttcac gttgttctc 8129

<210>3

<211>1282

<212> DNA

<213>Lycopersicon esculentum Miller

<400>3

gccgattttg gtgtttgatt tttttttggg gggattttga aattggtgag tttgattttg 60

gaatctccgg tgatgggacg ggcggagatg gttgtaggcc cgtcggagaa ggtggcggtg 120

gtggcatatc atctgaatga tgcaatcaat tggtgggacg atgtgaacag atctcttgat 180

tggcaaaacc gtatattcca tgtccttgct gttctctacg gcgttgtcgc cgtcgttgct 240

cttgtacaat taattcgcat tcaaatgaga gttcctgaat atggctggac cactcaaaaa 300

gtcttccact ttctcaattt ctttgtgaat ggagttcgct cgctagtttt tacatttcgt 360

cgggatgttc agaagttgca cccggagatt gtgcaacata ttatgcttga tatgccaagt 420

cttgcattct tcacaactta tgctctgcta gtattattct gggctgagat atactaccag 480

gcacgtgctg tgtccacgga tgggcttaga cctagtttct tcacaatcaa cggagtggtt 540

tatgctattc agattatatt atggctgata atgtggtgga aacctattcg agtactcttc 600

atcttatcca agatgttttt tgcaggtgta tccctatttg cagcattggg atttctcctc 660

tacggtggaa ggctttttct tatgttacag cggtttccag tagaatcaag agggagacgc 720

aagaagctgc aggaggttgg ttatgtcacg acaatatgtt tttcatgctt cctcattaga 780

tgcgttatga tgtgcttcaa tgcatttgat aaagctgcag atcttgatgt tttgtatcat 840

cctattttga atttgatata ttacctgtta gtggagatac tgccttcttc tcttgtcctt 900

tttattttaa ggaagttgcc tccaaagcga gggatcaccc aataccaccc tattcactaa 960

tacattaaga gggtagataa tgatgaaaat caggctccgg gatcaggtat taagtaagtt 1020

ggcttttacc tggatgtgat ttgcaagcaa gaaatacgag aggagtgata atgtaaattg 1080

aagtatggtt cgtctatact gaaatatccg tctgtcctca cactaggcag attgtagctc 1140

tgttttgtac cactagttat agatggaatt gtgaagtatc ttacgacctt tagtgtatta 1200

tttcgccttt gtatgtgtca gatttcaatt gaattcatta tttctcctga aacttcacgt 1260

tgttctcaaa aaaaaaaaa aa 1282

Claims (10)

1. A tomato RNA virus host factor, its amino acid residue sequence is as shown in SEQ ID NO: 1. 2. the coding gene of the tomato RNA virus host factor described in claim 1. 3. The coding gene according to claim 2, characterized in that: the base sequence of its genome gene is as shown in SEQ ID NO:2. 4. The coding gene according to claim 2, characterized in that: the base sequence of its cDNA gene is as shown in SEQID NO:3. . 5. An expression vector containing the coding gene of claim 2 or 3 or 4. 6. A transgenic cell line containing the coding gene of claim 2 or 3 or 4. 7. the host bacterium that contains the described coding gene of claim 2 or 3 or 4. 8. A method for cultivating anti-virus plants, comprising introducing the antisense RNA or RNA interference expression vector of the gene encoding the gene of claim 2 or 3 or 4 into the plant to obtain a transgenic plant. 9. The method according to claim 8, characterized in that: the transformed plant host is a plant of Solanaceae. 10. The application of the coding gene described in claim 2 or 3 or 4 in cultivating virus-resistant plants.

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