CN100432101C - Tomato RNA virus host factor and its coding gene and use thereof - Google Patents

Abstract

The invention discloses a method for cultivating virus-resistant tomato, which is to introduce the RNA interference expression vector of tomato ToTOM1 shown in SEQ ID No.: 2 in the sequence table into tomato to obtain transgenic plants; the method specifically includes the following steps: tomato ToTOM1 Construction of RNAi interference expression plasmids, genetic transformation of tomato, differentiation induction, and finally virus-resistant tomato plants. The method of the invention has great practical significance and broad application prospects in the cultivation and breeding of anti-virus plants.

Description

A kind of method of cultivating virus-resistant tomato

technical field

The invention relates to a protein and its coding gene in the field of biotechnology and a method for cultivating virus-resistant tomato by using the gene.

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 Tomato mosaic virus (ToMV), Potato virus Y (PVY), Tomato bushy stunt virus (TBSV), Tomato aspermy virus (TAV), and tomato chlorosis Spot virus (Tomatochlorotic spot virus, TCSV).

Since viruses are strictly obligate parasites, no strictly selective chemical agents can be effectively used for the prevention and treatment of plant virus diseases. In production practice, the hazards of viral diseases are mainly controlled by preventive measures such as removing the source of the virus and cutting off the transmission route of the virus, killing vector insects with chemical agents, and cultivating new disease-resistant varieties. Among them, cultivating new disease-resistant varieties is the most economical efficient. 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 its application in conventional breeding. The application of plant genetic engineering in breeding has opened up new ways for the prevention and treatment of plant virus diseases (Powell A.P., N elson R.S., HoffmanN., et al.Delay of disease development in transgenic plants that express thetobacco mosaic virus coat protein gene.Science , 1986, 232:738-743). 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), but the virus-resistant plants obtained by this method generally show vertical disease resistance, and there is a certain potential danger in this method Factors, that is, the viral sequences used to transform plants may recombine with viruses existing in the field to produce new viruses; another method is to use the plant's own disease resistance genes (such as N genes) to obtain virus-resistant plants (Goldbach R., Bucher E., and Prins M. 2003. Resistance mechanisms to plant viruses: an review. Virus Research., 92: 207-212).

The study of virus host factor genes provides a new way for antiviral genetic engineering. Host factors refer to all the convenient conditions provided by the host cell to the virus during the virus infection process (including virus invasion, viral gene expression, virion assembly and release, etc.), also known as host proteins or cell protein (cellular proteins). If these host factors are lacking in the host cell, the virus will not be able to replicate or the efficiency of replication will be 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.

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 ToTOM1, which is derived from Lycopersicon esculentum Miller and is a protein having 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 288 amino acid residues. Containing multiple highly hydrophobic regions, it is speculated that the protein is a transmembrane protein with 6-7 transmembrane domains, and the homology with Arabidopsis AToTOM1 gene at the amino acid level is 74.9%.

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 (ToTOM1) 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 6727 bases, has 11 exons and 10 introns, and the 1st-158th base from the 5' end is the first exon of the genome gene The 838-928th base from the 5' end is the second exon of the genome gene, and the 1015-1067th base from the 5' end is the third exon of the genome gene, from 5' The 1941-2033 bases at the 'end are the fourth exon of the genome gene, the 2109-2180 bases from the 5' end are the fifth exon of the genome gene, and the 3052nd bases from the 5' end Base -3131 is the sixth exon of the genome gene, bases 3236-3274 from the 5' end are the seventh exon of the genome gene, bases 3498-3561 from the 5' end The base is the eighth exon of the genome gene, the 5842-5895th base from the 5' end is the ninth exon of the genome gene, and the 6342-6419th base from the 5' end is the genome The tenth exon of the gene, the 6497-6727th base from the 5' end is the eleventh exon of the genome gene, and the 9th-11th base from the 5' end is the beginning of the genome gene The start codon ATG, the 6587-6589th base from the 5' end is the stop codon TGA of the genomic gene; the 159th-837th base from the 5' end is the first intron of the genomic gene, from Bases 929-1014 at the 5' end are the second intron of the genome gene, bases 1068-1940 at the 5' end are the third intron of the genome gene, bases at the 5' end are the third intron Bases 2032-2108 are the fourth intron of the genome gene, bases 2181-3051 from the 5' end are the fifth intron of the genome gene, bases 3132-3235 from the 5' end The base is the sixth intron of the genome gene, the 3273-3497 base from the 5' end is the seventh intron of the genome gene, and the 3562-5841 base from the 5' end is the The eighth intron of the genome gene, bases 5996-6341 from the 5' end are the ninth intron of the genome gene, bases 6420-6496 from the 5' end are the bases 6420-6496 of the genome gene Ten introns.

Its cDNA gene 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 31st-897th 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 virus-resistant plants provided by the present invention is to introduce the antisense gene fragment of ToTOM1 or the RNA interference expression vector of ToTOM1 into plants to obtain transgenic plants.

According to conventional methods in the field of plant genetic engineering, the antisense gene fragment of ToTOM1 or the RNA interference expression vector of ToTOM1 can be introduced into transformed plant cells or tissues, such as Agrobacterium-mediated transformation, gene gun-mediated transformation or pollen tube passage law etc. The transformed plant host can be a plant of Solanaceae, Brassicaceae or Cucurbitaceae.

The tomato RNA virus host factor ToTOM1 provided by the present invention is a related protein for the survival of RNA viruses in plants, and the virus-resistant plants can be obtained by silencing the gene encoding the protein in plants by transfecting antisense gene fragments or RNA interference . The results of the disease resistance experiment of transgenic plants showed that the transgenic tomato transfected with ToTOM1 antisense RNA significantly inhibited the accumulation of CMV in tomato. The accumulation of TMV in the transgenic tomato plants with the RNA interference plasmid of ToTOM1 was also significantly reduced compared with the control non-transgenic tomato, indicating that tomato ToTOM1 is a host factor gene of CMV and TMV, and tomato varieties resistant to CMV and TMV can be obtained by inhibiting or silencing ToTOM1. The tomato RNA virus host factor gene ToTOM1 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 is the agarose gel electrophoresis profile of the 3' RACE product of ToTOM1

Figure 2 is the BamHI and Xho I enzyme digestion identification map of the recombinant plasmid containing the 3' RACE product of ToTOM1

Figure 3 is the agarose gel electrophoresis profile of the 5'RACE product of ToTOM1

Figure 4 is the EcoR I enzyme digestion identification map of the recombinant plasmid containing the 5' RACE product of ToTOM1

Figure 5 is the agarose gel electrophoresis profile of the genomic gene fragment of ToTOM1 amplified by PCR

Fig. 6 is the agarose gel electrophoresis pattern of the recombinant plasmid containing the genome gene fragment of ToTOM1

Figure 7 is the enzyme digestion identification map of the recombinant plasmid containing the ToTOM1 gene fragment

Figure 8 is the plant expression vector pBI121 for transgenic use

Figure 9 is the agarose gel electrophoresis pattern of the ToTOM1 genome fragment amplified by PCR

Figure 10 is the enzyme digestion identification map of the connection direction of the exogenous fragments in pUCm-T

Figure 11 is the enzyme digestion identification map of the plant transformation plasmid pBIT1-2 of ToTOM1

Figure 12 is the agarose gel electrophoresis profile of the ToTOM1 cDNA fragment used to construct the RNA interference expression plasmid for PCR amplification

Figure 13 is the enzyme digestion identification map of the recombinant plasmid pUCGAT1 (-370)

Figure 14 is the enzyme digestion identification map of the RNA interference intermediate vector pUCGAToTOM1 (RNAi-370)

Figure 15 is the Pst I enzyme digestion identification map of RNA interference expression plasmid pBIToTOM1 (RNAi-370)

Figure 16 is the PCR identification map of the Kn-resistant tomato plant transformed with antisense ToTOM1

Figure 17 is the Southern hybridization detection result of positive plants transformed with antisense ToTOM1 by PCR

Figure 18 is the PCR detection result of 35S, NOS, endogenous ToTOM1 gene and NPTII gene of the transgenic plant expressing RNA interference fragment resistant to kanamycin

Figure 19 is the Southern hybridization pattern of independent RNA interference transgenic plants with positive PCR reaction

Figure 20 shows the non-transgenic plants after 10 days of inoculation with CMV

Figure 21 is antisense RNA transgenic plant after inoculating CMV 10 days

Figure 22 is non-transgenic plants after inoculation with CMV 25 days

Figure 23 is the transgenic plant expressing antisense RNA 25 days after inoculation with CMV

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 ToTOM1

1. Cloning of ToTOM1 3' end cDNA

According to the tomato EST sequence BE458681 (528nt)[gi:9502983] found in GenBank that has a certain homology with Arabidopsis AtTOM1[gi:9967414], two forward primers were designed to amplify the cDNA sequence at the 3' end of ToTOM1. The primer sequences are as follows :

Tomato Tom1-A: 5'CTCCCTTTGTGCTTCC-TATGGTCT 3';

Tomato Tom1-1: 5'GGGTACCCGAGTATGGCTGGACAAC 3'

The cDNA sequence at the 3' end of ToTOM1 was synthesized using the 3'RACE System for Rapid Aplication of cDNA Ends kit (Invitrogen, catalog number 18373-027). The total RNA of tomato was extracted, and its first-strand cDNA was synthesized by reverse transcription. Using the first-strand cDNA as a template, under the guidance of the primer Tomato Tom1-A and the primer AUAP provided by the above kit: 5'GGCCACGCGTCGACTAGTAC3', the first The first PCR, and then use the first PCR product as a template, under the guidance of primer Tomato Tom1-1 and primer AUAP, carry out the second PCR, after the reaction, carry out 1.2% agarose gel electrophoresis detection to PCR, the detection result is as follows As shown in Figure 1 (lane M is GeneRuler 1kb DNA Ladder, and lane 1 is the 3'RACE product of ToTOM1), it shows that a specific band with a length of about 1000bp was amplified. Recover this specific PCR product, connect it with carrier pCR2.1-TOPO (Invitrogen Company), transform Escherichia coli DH5α with the connected product, select positive clone to extract plasmid after screening, use restriction endonuclease BamH I and Xho I Double enzyme digestion identification was carried out, and the results of enzyme digestion identification are shown in Figure 2 (lane M is GeneRuler 1kb DNA ladder, lane 1 is the 3'RACE PCR fragment of ToTOM1, and lanes 2-5 are the recombination of the 3'RACE PCR fragment containing ToTOM1 BamH I and Xho I digested products of the plasmid), indicating that the cloned 3'RACE PCR fragment of ToTOM1 with a length of about 1000 bp had been correctly inserted into the vector TOPO TA, and the recombinant vector was named pCToT1-3. The DNA sequence of pCToT1-3 was determined, and the sequencing results showed that the inserted DNA fragment was 1077bp in length, and had the DNA sequence from 206-1282 at 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 ToTOM15' end cDNA

According to the tomato EST sequence BE458681 (528nt) [gi:9502983] found in GenBank, which has a certain homology with Arabidopsis ATOM1, two forward primers were designed to amplify the cDNA sequence at the 5' end of ToTOM1. The primer sequences are as follows:

Tomato Tom1-2: 5'CCACCATATAGCAGAAAGCCCAAGG-3';

Tomato Tom1-B: 5'TCCTGAGCTTATCTGTTGGTAAACTCCTAGCCT-3'

The cDNA sequence at the 5' end of ToTOM1 was synthesized using SMART ^TM RACE cDNA Amplification Kit (Clontech Company, catalog number K1881-1). The total RNA of tomato was extracted, and its first-strand cDNA was synthesized by reverse transcription. Using this first-strand cDNA as a template, the first-strand cDNA was used as a template under the guidance of primer Tomato Tom1-2 and the primer UPM provided by the above kit: 5'CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3'. One PCR, then use the first PCR product (diluted 100 times) as a template, and carry out the second PCR under the guidance of primer Tomato Tom1-B and primer NUP; 5'-AAGCAGTGGTATCAACGCAGAGT-3', after the reaction, PCR The product was detected by 1.2% agarose gel electrophoresis, and the detection result is shown in Figure 3 (lane M is 100bp DNA Ladder Plus lane 1 is the 5'RACE product of ToTOM1), indicating that a specific band with a length of about 500bp was amplified . Recover the specific PCR product, connect it with the carrier TOPO TA, transform the ligated product into Escherichia coli DH5α, select the positive clone to extract the plasmid after screening, and use the restriction endonuclease EcoR I for enzyme digestion identification. The enzyme digestion identification results are as follows: As shown in Figure 4 (swimming lane M is GeneRuler 1kb DNA ladder, swimming lane 1 is the 5' RACE PCR fragment of ToTOM1, the 3' RACE PCR fragment of ToTOM1, and swimming lane 2-5 is the EcoR I of the recombinant plasmid containing the 5' RACE PCR fragment of ToTOM1 Digestion product), indicating that the cloned target fragment with a length of about 500 bp had been correctly inserted into the vector TOPO TA, and the recombinant vector was named pCToT1-5. DNA sequence determination was carried out on pCToT1-5, and the sequencing results showed that the inserted DNA fragment was 448 bp in length, and had the DNA sequence at positions 1-448 from the 5' end of SEQ ID No.: 3 in the sequence listing.

3. Obtaining the full-length cDNA of ToTOM1

Sequence analysis of the 5'RACE PCR fragment and 3'RACE PCR fragment obtained in step 1 and step 2 shows that the two fragments have an overlapping region with a length of 243nt, and the 5'RACE PCR fragment obtained in step 2 There is a restriction endonuclease Mun I cutting site at the 279th base at the 5' end of the vector, and there is a restriction endonuclease Xba I cutting site in the sequence of the vector pCToT1-3 and pCToT1-5 point. The cDNAs of 3'RACE and 5'RACE were connected by Mun I and Mba I restriction sites to obtain the full-length cDNA of ToTOM1. 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 ToTOM1 with Vector NTI software, and the analysis results showed that its open reading frame (ORF) was from the 31st to 897th bases at the 5' end, and the length of the 5' non-coding region was 30 bases base, the length of the 3' non-coding region is 385 bases, the amino acid residue sequence of SEQ ID №: 1 in the coding sequence list, the homology comparison between ToTOM1 and the amino acid residue sequence encoded by Arabidopsis AtTOM1, ToTOM1 The amino acid homology with Arabidopsis AtTOM1 is 74.9%. The method of Kyte&Doolittle (Kyte&Doolittle, A simple method for displaying the hydropathic character of a protein, J Mol Biol, 157 (1): 105-132) was used to analyze the hydrophobicity of the protein encoded by ToTOM1, and the results showed that the protein encoded by ToTOM1 has high Hydrophobic region, inferred that the protein is a transmembrane protein with 6-7 transmembrane domains.

Embodiment 2, the cloning of tomato ToTOM1 genome fragment

According to the obtained full-length cDNA sequence of tomato ToTOM1 and the 23-45 nucleotide sequence from the 5' end and the 1035-1012 nucleotide sequence from the 5' end of the obtained full-length cDNA sequence of tomato ToTOM1 and its coding region, primers were designed to amplify the genome sequence of ToTOM1, The primer sequences are as follows:

ToTOM1-D: 5'-GAGCTGAAATGGCTAGGTTGCCG-3';

ToTOM1-E: 5'-CGTTGAATCTTTGCCTTTCCGCAG-3'

With the total DNA of tomato as template, under the guidance of primer ToTOM1-D and primer ToTOM1-E, 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 ( Lane M is the GeneRuler 1kb DNA ladder, and lane 1 is the genomic fragment of ToTOM1 amplified by PCR), indicating that a specific band with a length of about 6.7kb was amplified. The specific amplification product was recovered, cloned into the vector pCR2.1-TOPO (Invitrogen Company), and detected by 0.8% agarose gel electrophoresis, and the detection results were shown in Figure 6 (swimming lane 1 is CK: pCR2 .1-TOPO, swimming lanes 1-6 are recombinant plasmids containing ToTOM1 genome fragments), then carry out enzyme digestion identification with restriction endonuclease Sac I and Xba I, the enzyme digestion identification results are as shown in Figure 7 (swimming lane M is GeneRuler 1kb DNA ladder, lane 1 is the Sac I and Xba I digestion product of the recombinant plasmid containing the ToTOM1 genome gene fragment), indicating that the cloned target fragment of about 6.7kb in length has been correctly inserted into the vector pCR2.1TOPO, and will contain the The recombinant plasmid of the target fragment was named pT1DE-9. The DNA sequence of pT1DE-9 was determined, and the sequencing results showed that the genomic gene of ToTOM1 had the polynucleotide sequence of SEQ ID No. 2 in the sequence table, which consisted of 6727 bases, and it was compared with the full-length cDNA sequence of ToTOM1 Alignment, the comparison results show that the genomic gene of ToTOM1 has 11 exons and 10 introns, and the 1st-158th base from the 5' end is the first exon of the genomic gene, from the 5' The 838-928th base at the end is the second exon of the genome gene, the 1015-1067th base from the 5' end is the third exon of the genome gene, and the 1941-1067th base from the 5' end is the third exon of the genome gene Base 2033 is the fourth exon of the genome gene, bases 2109-2180 from the 5' end are the fifth exon of the genome gene, bases 3052-3131 from the 5' end It is the sixth exon of the genome gene, the 3236-3274 base from the 5' end is the seventh exon of the genome gene, and the 3498-3561 base from the 5' end is the genome gene The eighth exon of , the 5842-5895th base from the 5' end is the ninth exon of the genome gene, and the 6342-6419th base from the 5' end is the tenth exon of the genome gene Exons, bases 6497-6727 from the 5' end are the eleventh exon of the genome gene, bases 9-11 from the 5' end are the start codon ATG of the genome gene, The 6587-6589th base from the 5' end is the stop codon TGA of the genomic gene; the 159th-837th base from the 5' end is the first intron of the genomic gene, and the 929th base from the 5' end The -1014th base is the second intron of the genome gene, and the 1068-1940th base from the 5' end is the third intron of the genome gene, and the 2032-2108th base from the 5' end The base is the fourth intron of the genome gene, the 2181-3051 base from the 5' end is the fifth intron of the genome gene, and the 3132-3235 base from the 5' end is the genome The sixth intron of the gene, the 3273-3497 base from the 5' end is the seventh intron of the genome gene, and the 3562-5841 base from the 5' end is the eighth intron of the genome gene The 5996-6341 base from the 5' end is the ninth intron of the genome gene, and the 6420-6496 base from the 5' end is the tenth intron of the genome gene .

Embodiment 3, the acquisition of transgenic tomato plants

1. Construction of plant expression vectors

The binary expression vector used in this example is pBI121 (Clontech), its physical map is shown in Figure 8, it has the necessary sequence for T-DNA integration, and the kanamycin resistance gene is used as the selection marker gene. The foreign gene inserted into the site is controlled by the CaMV 35S promoter.

1. Construction of a plant expression vector containing an antisense fragment of the ToTOM1 genome gene

According to the cDNA sequence of the tomato ToTOM1 cloned in Example 2, a pair of specific primers Tomato Tom1-1 (5'-GGGTACCCGAGTATGGCTGGACAAC-3') and Tomato Tom1-2 (5'-CCACCATATAGCAGAAAGCCCAAGG-3') were designed. Using the total DNA of tomato as a template, under the guidance of primer Tomato Tom1-1 and primer Tomato Tom1-2, PCR amplification was carried out. After the reaction, the PCR product was detected by 0.8% agarose gel electrophoresis. The detection result is shown in Figure 9 As shown (swimming lane M is 1Kb DNA Marker, and swimming lane 1 is PCR amplification product), indicating that a specific band with a length of about 2.6kb was amplified. The specific amplification product was recovered and sequenced at both ends, and the sequencing result showed that the DNA fragment was a nucleotide fragment of ToTOM1. The specific amplification product of recovered ToTOM1 was purified and cloned into the vector pUCm-T (Shanghai Shenggong), and was identified by restriction endonuclease Kpn I, and the results of the identification were shown in Figure 10 (swimming lane M is 1Kb DNA Marker, lane 1 is the forward ligation product of the recovered fragment and pUCm-T, lane 2 is the Kpn I digestion product of the reverse ligation product of the recovered fragment and pUCm-T, and lane 3 is the recovered fragment and pUCm- The Kpn I digested product of the forward connection product of T) indicates that the gene fragment is connected into the vector pUCm-T in the forward and reverse forms respectively, and the recombinant plasmid connected in reverse is named pUT1-2. The ToTOM1 fragment cloned in pUT1-2 was excised with restriction endonucleases EcoR V and Xba I, and the DNA fragment was recovered after gel electrophoresis, and connected to the vector pBI121 double digested with Sma I and Xba I Above, the plant transformation plasmid pBIT1-2 of ToTOM1 was obtained, and it was digested and identified with restriction endonuclease Hind III. The cut recombinant plasmid pBIT1-2, lane 2 is the HindIII digestion product of pBIT1-2), indicating that the DNA fragment of ToTOM1 has been correctly connected to the vector pBI121, and the plant expression vector of ToTOM1 was constructed correctly.

2. Construction of tomato ToTOM1 RNAi interference expression plasmid

Two primers were designed according to the full-length cDNA sequence of tomato ToTOM1, and the primer sequences are as follows:

ToTOM1-RNAi-2F (879-899): CCTC AGATCT GCACAATACCACCCAAT (the underlined base is the Bgl II recognition site);

ToTOM1-RNAi-2R (1258-1227): CCTC ACTAGT AAAGGAGATCCAGAACATC (underlined bases are Spe I recognition sites)

Using the plasmid containing the full-length cDNA of tomato ToTOM1 as a template, PCR amplification was carried out under the guidance of primers ToTOM1-RNAi-2F (879-899) and primers ToTOM1-RNAi-2R (1258-1227), and the two ends contained respectively The cDNA fragment of ToTOM1 of restriction endonuclease Bgl II and Spe I recognition site, named after TT1, the 1.2% agarose gel electrophoresis detection result of this DNA fragment is as shown in Figure 12 (swimming lane M is GeneRuler 100bp DNAladder, swimming lane 1 is TT1), indicating that a 370bp DNA fragment was amplified, consistent with the expected result. The fragment was recovered, and cloned into the RNA interference vector pUCGA (construction method: using the total potato DNA as a template, after primer GA-F was double digested with restriction endonucleases Bgl II and Spe I) : 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-3' (the underlined part of the base is the recognition site of the restriction endonuclease BamHI) under the guidance of PCR amplification to obtain the first intron fragment (199bp) of the potato GA20 oxidase gene , the fragment was digested with Sac I and BamH I, and connected with the carrier pUC18 (TaKaRa Company) that was double digested with the same enzyme, and the recombinant vector obtained was named pUCGA), and the recombinant vector containing the recovered fragment was obtained, named It is pUCGAT1 (-370), and the recombinant vector is identified by restriction endonuclease Pst I, and the results are shown in Figure 13 (swimming lane M is GeneRuler 100bp DNA ladder, swimming lane 1 is the Pst I digestion product of pUCGA, swimming lane 2 It is shown as the Pst I digestion product of pUCGAT1 (-370), indicating that the recovered DNA fragment has been correctly ligated into the vector pUCGA. pUCGAT1(-370) was double-digested with restriction endonucleases Xba I and BamHI, and then ligated with TT1 double-digested with Bgl II and Spe I to obtain the recombinant RNA interference intermediate plasmid of ToTOM1, which was named pUCGAToTOM1( RNAi-370). The recombinant RNA interference intermediate vector was identified by restriction endonuclease Pst I, and the results are shown in Figure 14 (swimming lane M is GeneRuler 100bp DNA ladder, and swimming lane 1 is the Pst I digestion product of pUCGAT1 (-370), Lane 2 is the PstI digested product of pUCGAToTOM1(RNAi-370)). The RNA interference plasmid pUCGAToTOM1 (RNAi-370) connected with the RNA interference fragment TT1 of ToTOM1 was digested with the restriction endonuclease Sal I and made up, then digested with the restriction endonuclease Spe I, and passed through 1.2% agar After sugar gel electrophoresis, a small fragment with a length of about 900bp was recovered, and the recovered fragment was connected to the carrier pBI121 double-digested with restriction endonuclease EcoRV and Xba I to obtain the RNAi interference plant expression plasmid of tomato ToTOM1, named pBI121ToTOM1( RNAi-370), it was identified by restriction endonuclease Pst I, and the results are shown in Figure 15 (swimming lane M1 is GeneRuler 100bp DNA ladder, swimming lane M2 is GeneRuler 1kb DNA ladder, and swimming lane 1 is Pst I of pBI121 The digested product, lane 2 is the Pst I digested product of pBIToTOM1 (RNAi-370), indicating that the RNAi interference plant expression plasmid of tomato ToTOM1 was constructed correctly.

2. Genetic transformation of tomato

1. Three-parent combination to introduce plant expression plasmid pBIT1-2 into Agrobacterium

E. coli containing the plant expression plasmid pBIT1-2 was combined with E. coli containing the helper plasmid pRK2073 and Agrobacterium EHA105 respectively (Rogers S.G., Horsch R.B., and Fraley R.T.1986. Gene transfer in plant: production of transformed plants using Ti-plasmidvectors.Methods Enzymol., 118: 627-640), screened on the LA plate containing 50mg/L rifampicin (Rif) and 50mg/L kanamycin, obtained the agricultural agent containing the plant expression plasmid pBIT1-2 Bacillus transformants.

2. Genetic transformation of tomato

1) Infect the explants with Agrobacterium EHA105 containing the expression plasmid pBIT1-2

Streak the Agrobacterium containing the plant expression plasmid pBIT1-2 obtained in step 1 on a YEB plate containing 50mg/L rifampicin (Rif) and 50mg/L kanamycin, and culture it at a constant temperature of 28°C for about 24h After the colony grows, pick a single colony and inoculate it in the YEB liquid medium containing the same antibiotic, cultivate it on a constant temperature shaker (200 rpm) at 28°C for about 24 hours until the OD ₆₀₀ is 04-0.6, and transfer it to a sterile centrifuge tube , centrifuged at 3000rpm for 10min, discarded the supernatant, washed the collected bacteria once with MS culture medium and resuspended, diluted 50 times and used for transformation of tomato. 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 and organ culture., 67:173-180) prepare Flamingo Bill explants, the specific method is: the tomato Separate the seeds one by one, wash 3 times with distilled water, then soak in 80% ethanol and stir continuously for 2 minutes, add 4-6 drops of 100% Tween-80, pour off the ethanol solution, and wash with sterile water 2-3 times; Add NaClO solution with 1% available chlorine and 4-6 drops of 100% Tween-80 and stir on the stirrer for 15 minutes, pour out the NaClO solution, wash 2 times with sterile water, and repeat twice. Until the color of the tomato seeds changes from brown to golden yellow, rinse them with sterile water for 6-10 times, and dry them on sterile filter paper. After drying, insert the seeds into a culture bottle equipped with 1/2MS medium (containing 1.5% sucrose), cultivate them in a light incubator at 25-28°C for 7 days, and then put the culture bottle in a super-clean bench. Open the cover, and replenish the water in the culture bottle, cultivate the sterile seedlings in an open environment for 15 hours, and then cut off one side of the cultured sterile seedlings from the base of the petiole together with the terminal buds and the surrounding meristems. , only one side of the cotyledon is left, and a large part of the root system of the aseptic seedling is cut off in addition and only the radicle with a length of 2-3cm is kept as an explant.

Put the explants into the activated MS liquid medium of Agrobacterium EHA105 containing the expression plasmid pBIT1-2 (additionally acetosyringone with a final concentration of 100 μmol/L), shake it continuously for 10 minutes, take it out and use a sterile Bacteria water was washed twice, and then one by one was put into a 15cm-diameter large tube filled with MS co-cultivation medium (adding 0.15 mg/L zeatin and 0.05 mg/L indole acetic acid on the basis of MS basic medium). Seal the petri dish and culture it in a light incubator for 2 days.

2) Differentiation induction

After culturing for 2 days, the explants transformed with Agrobacterium EHA105 containing the expression plasmid pBIT1-2 were transferred into the differentiation and selection medium (adding ZT 0.15 mg/L, IAA 0.05 mg/L on the basis of MS basic medium). L, Km 50mg/L and Cef 200mg/L), continue to cultivate in the light incubator for 2-3 weeks, the culture condition is 25-28 ℃, 15 hours of light, 9 hours of darkness.

3) Rooting, hardening and transplanting of transgenic plants

The normal plant (grow up to about 2-3cm in height) in the differentiation screening medium was excised from the callus and inserted into the rooting medium (adding Km 50mg/L on the basis of MS basic medium, Cef 150mg/L, NAA 0.2mg/L), 25-28°C, 15 hours of light, 9 hours of darkness until rooted. When the root system of the regenerated seedlings is about 3cm long, open the lid of the culture bottle and add water. Place it for 3-4 days at 25-28°C and light conditions of 15 hours/day, then slowly pull out the plants and wash them. The agar carried by the root is transplanted into the soil, the humidity of the soil is kept, and the temperature is controlled at 25-30°C.

The same method as above was used to transform tomato plants with the RNAi interference expression plasmid of tomato ToTOM1.

3. Molecular identification of transgenic plants

1. Molecular identification of antisense RNA transgenic plants

A total of 19 kanamycin (Kn) resistant antisense RNA transgenic plants were obtained. The PCR detection of exogenous genes was carried out on the seedlings before potting. The specific method was: take the Kn-resistant transgenic tomato plant leaves, extract the total DNA of the tomato plant leaves with the CTAB method, and use this as a template, and use the primer Tomato-ToTOM1-1 And a primer sequence designed according to the 35S promoter sequence: under the guidance of 5'-AATCTTCGTCAACATGGTGGAGCAC-3', PCR detection was carried out, and the results are shown in Figure 16 (swimming lane M is lambda/HindIII+EcoRI, and swimming lanes 1-19 are transformed with The total DNA of different transgenic plants with antisense ToTOM1 is the PCR amplification product of the template, and swimming lane 20 is the positive control, and the PCR amplification product using the plasmid pBIT1-2 as the template), showing that 12 of the 19 transgenic plants can An exogenous target gene fragment with a size of about 3300bp was amplified. The positive transgenic tomato plants identified by PCR were named T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and the numbers corresponded to lanes 1, 2, 3, 4, 7, 8, 9, 10, 13, 14, 17 and 19.

The positive transgenic plants identified by PCR were planted in pots, and the total DNA was extracted after 4 weeks, digested with SalI, separated by 0.7% agarose gel electrophoresis, transferred to the hybridization membrane by the down capillary method, and labeled with P ³² Use Tomato-ToTOM1-1 and a primer on the 35S promoter: 5'-AATCTTCGTCAACATGGTGGAGCAC-3' to perform Southern hybridization on the PCR fragment obtained, the results are shown in Figure 17 (lane M is 1kb DNA marker, lane CK is non-transgenic Plants, lanes T ₁ -T ₁₂ are different transgenic lines), indicating that in addition to the hybridization band of endogenous ToTOM1 (shown by the arrow in the figure), there is also a hybridization band of exogenous ToTOM1, and all plants that were positive for PCR detection were There is an insertion of an exogenous ToTOM1 fragment. Judging from the size difference of the SalI fragments, more than half of the transgenic plants had exogenous gene fragments inserted into different sites on the chromosome, that is, they belonged to different transgenic lines. The insert fragments in some plants are similar in size (such as T1, T3), so it is not easy to judge, and it needs to be cut with other enzymes and then hybridized to confirm. However, the number of chromosomal bases in tomato is huge, and the possibility of exogenous genes being inserted into the same position is very small, so the obtained transgenic plants may all be different lines. Figure 17 also shows that most of the transgenic plants are single copy, but 3 plants are double copy (T4, T9, T10).

2. Identification of RNA interference transgenic plants

A total of 93 kanamycin-resistant RNA interference transgenic plants were obtained. Do a preliminary PCR test before potting, amplify NPTII under the guidance of primer Kam-R: 5'-GATCTGGATCGTTTCGCATG-3' and primer Kam-F: 5'-AAGGCGATAGAAGGCGATGC-3', and amplify NPTII under the guidance of primer NOS-F: 5'-TGCTACCHAHCTCHAATTTC -3' and NOS-R: Amplify NOS under the guidance of 5'-ACGACGGCCAGTGAATTCCC-3', amplify 35S under the guidance of primer 35S-F: 5'-AATCTTCGTCAACATGGTGGAGCAC-3' and ToTOM1-RNAi-2F (, in Under the guidance of primers TA-TOM1-1: 5'-GGGTACCCGAGTATGGCTGGACAAC-3' and TA-TOM1-2: 5'-CCACCATATAGCAGAAAGCCCAAGG-3', ToTOM1 was amplified, and the NPTII, NOS, 35S and endogenous The ToTOM1 gene test was positive, and part of the test results are shown in Figure 18 (lane M is the GeneRuler 100bp DNA ladder, and lanes 1-5 are the 35S detection of resistant plants (lanes 1-5 are MLC-ToTOM1-RNAi-370-11 in sequence) , -49, -71, -79, -80), lanes 6-10 are NOS detection (lanes 6-10 are MLC-ToTOM1-RNAi-370-11, -49, -71, -79, -80) , lanes 11-15 are endogenous ToTOM1 detection (lanes 11-15 are followed by MLC-ToTOM1-RNAi-370-11, -49, -71, -79, -80), lanes 16-20 are NPTII gene detection (lanes 16-20 are successively MLC-ToTOM1-RNAi-370-11,-49,-71,-79,-80). Positive plants are carried out potted. Detect with the same method after one month, the result is only in 32 strains There are 3 strains (MLC-TOTOM1-RNAi370-71, -79 and -80) still have positive signals. Extract the total DNA of these 3 positive transgenic plants, and go through 0.7% agarose gel electrophoresis after complete digestion with EcoR I Separation, transfer to the hybridization membrane by downward capillary method, carry out Southern hybridization with the RNA interference TT1 fragment of ^P32 mark, the result is shown in Figure 19 (swimming lane M is GeneRuler 1kb DNAladder, swimming lane ck (-) is non-transgenic tomato EcoRI digestion product, ck(+) is the EcoR I digestion product of pBITOTOM1RNAi-370, lane 1 is MLC-TOTOM1-RNAi370-80, lane 2 is MLC-TOTOM1-RNAi370-79, lane 3 is MLC-TOTOM1-RNAi370 -71), indicating that in addition to the 4.2kb endogenous hybridization band, all transgenic plants had a second hybridization band of different sizes, proving that the RNA interference fragment had been integrated into the tomato genome.

Embodiment 4, detection of disease resistance of transgenic plants

1. Disease resistance experiment of antisense RNA transgenic plants

Using the rubbing inoculation method, all the transgenic tomatoes at the 5-6 leaf stage obtained in Example 3 were rubbed inoculated with cucumber mosaic virus (CMV), and non-transgenic tomatoes at the same growth stage were used as controls.

1. Symptoms

After 10 days of inoculation with CMV, the non-transgenic tomato seedling control began to show mosaic symptoms, and the newly grown leaves were obviously chlorotic (Figure 20), while the transgenic plants were normal (Figure 21); at 25 days, the control non-transgenic plants The symptoms of the disease are very serious, and the newly grown leaves are almost completely chlorotic, and the new leaves are deformed (Figure 22). Although the transgenic plants at this time also showed symptoms, they were significantly lighter than those of the control, and the leaves were only slightly chlorotic (Fig. 23).

2. Determination of the Relative Quantity of Viruses in Transgenic Tomato Plants

The relative content of virus in transgenic tomato plants was determined by bioquantitative method using Chenopodium amaranthus as the host of dead spot. Twenty days after inoculation, take some new leaves (about 0.5g) of the tomato plants and grind them into a homogenate, and inoculate them into the leaves of the host plant Amaranthus amaranthus, respectively. Each treatment receives 2 leaves, and 2 replicates are set. After five days, The number of dead spots was counted and a significant difference analysis was performed. The results are shown in Table 1, which indicated that the number of dead spots formed on Amaranthus quinoa was significantly lower than that of control tomato plants when the transgenic tomato was used as the inoculation source. The number of dead spots formed by the transgenic plant T9 (34.5/leaf) was only 29% of that of the control (119/leaf). The number of dead spots formed by different transgenic lines on Chenopodium amaranthus was significantly different. For example, the number of dead spots of T1 plants (68.5/leaf) was nearly 1 times higher than that of T9 plants (34.5/leaf). Blight test showed that the CMV concentration of antisense ToTOM1 tomato was significantly lower than that of non-transgenic tomato.

Table 1 Bioquantification of antisense ToTOM1 gene-transferred tomato on the host Amaranthus spp.

strain Average number of dead spots t_value t_0.05 t_0.01 Significance CK 119.5 T1 68.5 7.408 2.365 3.499 significant T2 45.5 7.300 2.365 3.499 significant T3 63.5 7.743 2.365 3.499 significant T4 37.5 11.62 2.365 3.499 significant T5 37.5 9.133 2.365 3.499 significant T6 38.5 11.38 2.365 3.499 significant T9 34.5 8.933 2.365 3.499 significant T10 38.0 8.759 2.447 3.707 significant

2. Disease resistance experiment of transgenic plants expressing RNA interference fragments

Adopt rubbing inoculation method, use tobacco mosaic virus (TMV) to carry out inoculation experiment to 3 RNA interference transgenic plants (5-6 leaf stage) that embodiment 3 obtains, take the non-transgenic tomato that is in the same growth period as contrast, observe The results were recorded, and the results showed that 10 days after inoculation, the non-transgenic tomato seedlings showed mottled symptoms, while the transgenic plants showed no symptoms. 70 days after inoculation, the non-transgenic plants still showed mottled symptoms, and the transgenic plants still had no symptoms. After 75 days, take by weighing 0.1 g of new leaves (the third last new leaf) of transgenic plants and control plants, and use the DAS-ELISA method to measure the virus content in the transgenic plants. The specific method is: add 1ml of coating buffer (0.015M Na ₂ CO ₃ , 0.035M NaHCO ₃ , pH 9.6) was ground into a homogenate, diluted 1000 times and tested. The test results are shown in Table 2, indicating that the concentration of TMV in all transgenic plants was significantly lower than that of non-transgenic plants .

Table 2 DAS-ELISA quantification of TMV in transgenic plants expressing ToTOM1 RNA interference fragment

strain Average OD value Standard Error (S.E.) Standard Deviation (S.D.) Significance Non-transgenic plants (control) 1.982 0.0015 0.0026 MLC-TOTOM1-RNAi-370-71 0.328 0.0012 0.0020 significant MLC-TOTOM1-RNAi-370-79 0.231 0.0012 0.0026 significant MLC-TOTOM1-RNAi-370-80 0.491 0.0015 0.0021 significant

sequence listing

<160>3

<210>1

<211>288

<212>PRT

<213>Lycopersicon esculentum Miller

<400>1

Met Ala Arg Leu Pro Leu Gly Ser Ser Ser Pro Ile Asp Ile Ala Gly Pro

1 5 10 15

Val Thr Asn Trp Trp Asp His Val Asn Glu Ser Val Gln Trp Gln Asp

20 25 30

Gly Ile Phe Tyr Ser Leu Cys Ala Ser Tyr Gly Leu Val Ser Ala Val

35 40 45

Ala Leu Ile Gln Leu Ile Arg Ile Asp Leu Arg Val Pro Glu Tyr Gly

50 55 60

Trp Thr Thr Gln Lys Val Phe His Leu Met Asn Phe Val Val Asn Gly

65 70 75 80

Val Arg Ala Ile Val Phe Gly Phe His Lys His Val Phe Leu Leu His

85 90 95

Tyr Lys Val Leu Thr Leu Ala Ile Leu Asp Leu Pro Gly Leu Leu Phe

100 105 110

Phe Ser Thr Phe Thr Leu Leu Val Leu Phe Trp Ala Glu Ile Tyr His

115 120 125

Gln Ala Arg Ser Leu Pro Thr Asp Lys Leu Arg Ile Ser Tyr Ile Ala

130 135 140

Ile Asn Gly Ala Ile Tyr Phe Ile Gln Ala Cys Ile Trp Val Tyr Leu

145 150 155 160

Trp Ile Asn Asp Asn Ser Thr Val Glu Phe Ile Gly Lys Ile Phe Met

165 170 175

Ala Val Val Ser Val Ile Ala Ala Leu Gly Phe Leu Leu Tyr Gly Gly

180 185 190

Arg Leu Phe Leu Met Leu Arg Arg Phe Pro Ile Glu Ser Lys Gly Arg

195 200 205

Arg Lys Lys Leu His Glu Val Gly Ser Val Thr Ala Ile Cys Phe Thr

210 215 220

Cys Phe Leu Ile Arg Cys Phe Val Val Val Leu Ser Ala Phe Asp Ser

225 230 235 240

Asp Ala Ser Leu Asp Val Leu Asp His Pro Val Leu Asn Leu Ile Tyr

245 250 255

Tyr Leu Leu Val Glu Ile Leu Pro Ser Ala Leu Val Leu Tyr Ile Leu

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Arg Lys Leu Pro Pro Lys Arg Val Ser Ala Gln Tyr His Pro Ile Ser

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<210>2

<211>6727

<212>DNA

<213>Lycopersicon esculentum Miller

<400>2

gagctgaaat ggctaggttg ccgcttgggt cgtcgccgat tgacatcgcc ggtccggtga 60

ccaactggtg ggaccacgtc aacgaatccg ttcagtggca agatgggatt ttctactccc 120

tttgtgcttc ctatggtctt gtttcagcag ttgccctagt aagtttctct ttctcttcca 180

ttctcttttt ctgctctgta gatatgtaaa gattggtttt ttctttcctt tttgtgaatt 240

tcacgatcaa agtagagttt gggagctgaa atgccggaat tgatgttaga tgattatat 300

tttcaactcc tactaatttg atgttgagat gcagttgata gaattgcagt gtctagttca 360

tgtataaagt caagtacttg ctgttagttg tttttttttt cgtaacttct gttaaaaatg 420

gaaaatcttg gttggtcaac tgtaggaatg aaataatgga gtagggaatc agacgagaat 480

gtttgattac caagtgcaat ttaggggttt ccattaagac aaattacggt atattttgtg 540

cttttttatg tcgctaataa aagtttggag cagtggactg actggttaat catacttctg 600

gtaggttcgg ttagatggtt agtatttggg agtactcctt ttgtgtgtgc ttgtgaagct 660

tatgaccttt tccaataagt agataagttt cattgaaggg tgttttttca tactttgatt 720

aatatgtgtt tcagatacag tattaccta atactgcatg tgtagagaac tgttttcctc 780

gtggtgcttg cttagtcgca ggacttttgg ttgagtctat gagttaacta tgtgcagatt 840

caattaatac gaattgattt gagggtaccc gagtatggct ggacaacaca aaaggtgttc 900

catctgatga actttgttgt aaatggaggt aaagcggact cagactaaac aaaccgtcca 960

gctctttttc ttaatctgga attttgtttt aaatgcaaat cttttgtttt tcagttcgtg 1020

caattgtctt tggatttcac aaacatgttt ttctgctcca ttataaggta atattctttc 1080

ttaaaagtaa cttttcaatt gtcaatattt tttcttaatt atgttcagtt tgttttcaat 1140

agaacgcgac aatatatgca aacctgaata tattgccttt gttcttatca gccattaac 1200

ttttccccaa ataatggttc ttttcctata gtgcgtcaac ttacatagga actatcaaaa 1260

atcaaaatta ggtcagaact gtacctagaa aatttatcac ctgatgggac cattttcaag 1320

gcaagtgcat gcatgttact tttgctgatt ggagctgtag gctttcttat atctttgaca 1380

ctcttaactt tgagcttaat ttgacatttt agattcctta ccttctaatt tattattcg 1440

gaaggtcatc ctgctctctt acagctagta caatccaata gcagttcagc aatccttgtt 1500

tgttaatgtg tgagctaatt ggattgaaag cacattagta tatacattct gtctttcaga 1560

ttccagtttg aagcttatca cctagttact catcttttac ttgccctaaa attttggttt 1620

gactgtgatg ggccgatgac attgttcaag tgcttagttc ctctgcagcc agcaacaccg 1680

tagtgttacc atgtcaatcc tttagctttg actatcattt tattgagatg aattttctgt 1740

tagcaataac gaatctactg aaagcacctc ccctttacga cttccgtgat ctctttcctt 1800

catttgggtg ggctactaat ggccccgtcc ctttttagcc cgataactta agaagtagaa 1860

ataattaaca tgcccacaac tgcctcttaa actgctgcct aattgttaac ctgatgttag 1920

atttgcttca cgataaccag gtgctgactc tggcaatatt ggacctacca gggctccttt 1980

tcttttcaac attcacactc cttgttctat tttgggctga gatatatccc caggcaagtg 2040

aagttatagc atcttgcagg caaactgtaa atctattgat tctaaccctt gcatttatgt 2100

aatggcaggc taggagttta ccaacagata agctcaggat ttcttatatt gccattaatg 2160

gtgccatata cttcattcag gtttgctaca aatgtatccc ctgctcatac gctattggac 2220

atcatggttg ttcatatgga aatataatcg atcaagtatt attacttatt tttattttgt 2280

ctctagtaat atgtgaattc ctttaagatt cttttacgcc agtcaaaccc atctactttt 2340

gggttctatt tgataacccc ttgtattttg gtggtgacag gggtggatta gtgtagaagt 2400

tatgggttca actgaaaaag ggttcctgga acccaacatt agctcaaacc ctgtatatat 2460

gttaaaaatt ttgtaaacaa gtaaacataa tagattttga accactaaa tcaatcgggt 2520

tgtggtagaa tttctgagac acataaagtt cgaatcctgg agccgcctca ttgatctgtg 2580

ggcatatgtg tgcttctcaa tatgtggaat tctgagatat tcttctttct caaaatgtta 2640

tgccgggtta acctggggta acatgctagg agaaaagttt aattcggagc tctccatcta 2700

cttccattat caaatattca tagcttggtt ccggttatac ctttaattat tttttcgaga 2760

accgatggaa tatctttcca agaccttgca ataaattttt acagaaccat atgcaaattt 2820

agccttgaaa tagggctaga ttctcctaat ccccatcccc tgagaaccct atctcttccc 2880

tttcctaaag gatagagcat ttctaaataa ataaagatgt ccattgttct tgatttcttg 2940

gtgaacattt cctatcgacc cgcttgtctc aaaagctaag aatcttcttc agtcttcctg 3000

gttttatctc aggtaataaa ctaaatacaa ctgaaaaatc gtttttgtca ggcctgtatc 3060

tgggtttacc tctggatcaa tgacaatagc acagtggaat tcattgggaa gatatttatg 3120

gcagttgtat ctcttatata tcaattttct ccttttgccc tattttactg aaggttagat 3180

ctgctttcaa agttattgct ttctttaacc ttgcatgttt attgggcagt gtatcagtta 3240

ttgcagcctt gggctttctg ctatatggtg gaaggtgaat taagctctaa ttttctctgg 3300

gcattaatct tctaaagcat tattgagatt atcagttat gcagccttgg gctttctgct 3360

atatggtgga agggaattta agttctaatt ttctctgggc atttatcttc taaagcatta 3420

ttgatgattc ttagatgacc ttagacatga ttaaatttca ttgtttttct tggggaattg 3480

gtttttacaa tcatcaggtt atttctcatg ctgcggcgct tccctattga atctaaaggg 3540

aggagaaaga agcttcatga ggtttgtgta ctatacatct gtcttcctct ttctttttct 3600

ttttttcttt tcttttaact ggtaaagatt tttacacttt tatcttctta ctttatgtat 3660

acagaggtta gtgctgtagt ttggcataga atacgtttgc atgtaaggca taagatgtat 3720

tcctcacaaa cttaagttct ccctacagca ccgagaggga gaaacttttt aaacatgaag 3780

tttgattaat ttccttgtcc taagaagtcc tgctaattgt tctgctatat tataatctta 3840

acttgtgggt gaatgactgc tatatttagt ccagataaca tttaagtgga gagataagag 3900

taaggctatc tctgagattg tataatttaa ttctaaaatg ataattaaca agaacagatg 3960

gaaccttgtt atgacattct tattcagaca tttttatca gtgcgtgaag gggtaacctt 4020

ttggtcatgg atccactaaa ctgtcaaagg ctggtgcata cttctttttc tattagttt 4080

gtgtcacata atttgtttga cttacaccat aagctcaact ccagcaaatt attaatttct 4140

gtagctgcta tctgatggga ctgttcaaag ttgtttggat atacatagac agaagatgtc 4200

aagtaataaa gattgcctaa caaaatgagc aggaatgtaa caaattataa catacaacat 4260

tagggtacca actgttgatg tctcccttct ctagattcct cttttttgat gattaagtaa 4320

gattttatta aaccatctgc agcaccaaca aggtgtaaga gtgcccgata gtagctagaa 4380

atttattcct ttaacaatat aaggagtcaa ttaactccac tatatggtca aaatcactag 4440

cagcatcctg cttcaaaaat tcatgttctg taagcatttc aatgggcatt actaaaggta 4500

attaaaggaa cgtatgcttt aatcctgaaa gcagctgcag ttccctttct atattaacca 4560

tctgaaataa catagagaga ttctgtaagt tcctctgtat attttttaat aaggtcctct 4620

gtttcttctt tattccctgc attgtcatct ttgtactgcc tctaccaggc atcatccaaa 4680

caatcccaaa ccggtttaga aatatacacc aagttatcca tatgaatttg caacgtagga 4740

gtaactgtcc aaatgatta aagccctctt tgcagtagta ggatctggtg gagaagtgaa 4800

atcttccctc tgtgctgggc tatcataang tacttatgtt tcattaaggg taatacccaa 4860

aaancagcca ctttgggagt agctctggtc ctccaaagtc atcttccatg actattagag 4920

ccaatattac cacattcttc cttaacaact aactgtatca tgctttaggt gagaagttcg 4980

catcatccaa tgatccattt ccatcactcc ccctaattta acaactagca cacacagact 5040

gtacctagtg ttgatgattt ttttaagaaa aaaattgatg atttattcta cattggagat 5100

tccagcattc tcctttggag ggttctccaa tggtacgtcg aacactcttt gttgatgaca 5160

agattatatt aacttgggaa gagttatttc agagttctgg tttccagtca tttgtcttgc 5220

catagtttgt tcttctgctg ttatccactt tgacctttcc atctctgcaa actctcccca 5280

tggatttccg attcatcttc ctaatacaac tccataagat gtgactgctt tactgcatca 5340

ttgtccatca attccatatt aatctttgat catcccttcc acatcttctc tttgttttcc 5400

atggccattt gaacttaaaa gacaataaat agtctgaagc tcttcactac tagtggctga 5460

tttccttttc ttctctgttg atgaataaac aggaaaaaaa atatcttaga ttagtctgac 5520

tagatggaaa ctcaatatta acatatgatg tttctgaaga aagaatcttt tttatcatta 5580

tttgtgaacc taaggagttc ttttgttctt caatgatcat tagtgaccaa ctcaagaaaa 5640

tgatttattg gtctatctga ttgttctaac atcgtcttct acatcaccaa ccattaaatg 5700

atgctgctgt gaatatgttt gtgttatctg aagttctaat gaattgtttc taggcatatg 5760

catgcatgat aggtcatggc ttataattct taacatctct tactgtaaat catctgatct 5820

aatgtatgta tgtgaacaca ggttggatcg gtgactgcca tatgtttcac ctgtttcctc 5880

attagatgct ttgtggttgt aaggctttat gtacctgttt ctttctccct atgctggtca 5940

acctttttat catcccaagt ataacagtgt cctaagctcc atttattaagc ttttgcaaaa 6000

ctgggttaac caaaattaag ccacttctta agcttttttt acggggaagg tccaagcaag 6060

gaaattgagt ttttacccca tcgggtgctt ttagtttgta agaggtgaaa ccattaggct 6120

atctttatga aataaagggt tgcaacctat gccttccttc aggcttcagt caaaccataa 6180

ccttactcctg ggaacaaata ataggaaaag gtagactaga gaataggaac atgatcaaaa 6240

aaagagaaag cataggtta aagtaaagct aaaccggcct caaatctcta actctcatgc 6300

acatgagctt gttttctatg tctaatcttg gttcgctgca ggttgtgtta tctgcttttg 6360

atctctgacgc atctcttgac gtcttggatc atcctgtttt gaatctgata tactacctgg 6420

tatgtatcat tttcgttatt tcatgttcct tgcgcttact gctttgtctg agtgatcaca 6480

ttcttctctg tggcagctgg tagaaattct tccttcagct cttgtgctgt acatcctgcg 6540

aaaactgcct ccaaaaagag tgtctgcaca ataccaccca atcagttagc tgcagcagaa 6600

ttttatcgtt agtgatacac gttcccatgg tttctgttgc agaagctaac tggagttgtt 6660

caggaaaagt gaaactgcaa aaggatattc ggttgcaata attctgcgga aaggcaaaga 6720

ttcaacg 6727

<210>3

<211>1282

<212>DNA

<213>Lycopersicon esculentum Miller

<400>3

acaacaagat aagcggaggg gagagctgaa atggctaggt tgccgcttgg gtcgtcgccg 60

attgacatcg ccggtccggt gaccaactgg tgggaccacg tcaacgaatc cgttcagtgg 120

caagatggga ttttctactc cctttgtgct tcctatggtc ttgtttcagc agttgcccta 180

attcaattaa tacgaattga tttgagggta cccgagtatg gctggacaac acaaaaggtg 240

ttccatctga tgaactttgt tgtaaatgga gttcgtgcaa ttgtctttgg atttcacaaa 300

catgtttttc tgctccatta taaggtgctg actctggcaa tattggacct accagggctc 360

cttttctttt caacattcac actccttgtt ctattttggg ctgagatata tcaccaggct 420

aggagtttac caacagataa gctcaggatt tcttatattg ccattaatgg tgccatatac 480

ttcattcagg cctgtatctg ggtttacctc tggatcaatg acaatagcac agtggaattc 540

attgggaaga tattatggc agttgtatca gttattgcag ccttgggctt tctgctatat 600

ggtggaaggt tatttctcat gctgcggcgc ttccctattg aatctaaagg gaggagaaag 660

aagcttcatg aggttggatc ggtgactgcc atatgtttca cctgtttcct cattagatgc 720

tttgtggttg tgttatctgc ttttgattct gacgcatctc ttgacgtctt ggatcatcct 780

gttttgaatc tgatatacta cctgctggta gaaattcttc cttcagctct tgtgctgtac 840

atcctgcgaa aactgcctcc aaaaagagtg tctgcacaat accacccaat cagttagctg 900

cagcagaatt ttatcgttag tgatacacgt tcccatggtt tctgttgcag aagctaactg 960

gagttgttca ggaaaagtga aactgcaaaa ggatattcgg ttgcaataat tctgcggaaa 1020

ggcaaagatt caacgctttt ttggcagttg ttaaaacaga ggttaagctg ttttgcttac 1080

attatattgt ttctgtggtt ttagtgtgaa gcatgagaca aataagtgtt ccccacgtct 1140

gtgaaaaatc ctagtcatga tgtaatgacg cagagggtaa atctcagtat cgccattgta 1200

ctggcatgtt gtaactatga tgttctggat ctcctttact gcaatgactg atgtcctttg 1260

tttggtcaaa aaaaaaaaaa aa 1282

Claims (1)

1, a kind of method of cultivating antiviral tomato is with SEQ ID № in the sequence table: the rnai expression carrier of the tomato ToTOM1 shown in 2 imports in the tomato, obtains transfer-gen plant; May further comprise the steps:

1) structure of the RNAi interference expression plasmid of tomato ToTOM1

Design two primers, primer sequence is as follows:

ToTOM1-RNAi-2F：CCTCAGATCTGCACAATACCACCCAAT；

ToTOM1-RNAi-2R：CCTCACTAGTAAAGGAGATCCAGAACATC；

With the plasmid that contains tomato ToTOM1 full-length cDNA is template, under the guiding of primer ToTOM1-RNAi-2F and primer ToTOM1-RNAi-2R, carries out pcr amplification, obtains the cDNA fragment, called after TT1; Reclaim this fragment, use restriction enzyme Bgl II and Spe I double digestion rear clone on the rna interference vector pUCGA of same enzyme double digestion it, obtain containing this and reclaim segmental recombinant vectors, called after pUCGAT1 (370); Wherein, described rna interference vector pUCGA makes up in accordance with the following methods: with the total DNA of potato is template, under the guiding of primer GA-F:5 '-AGG GAGCTCCTCGAGACTAGTAGATCTGGTACGGACCGTACTACTCTATTCG-3 ' and primer GA-R:5 '-AGG GGATCCCTATATAATTTAAGTGGAAAAAAAGGTTAAC-3 ', carry out pcr amplification, obtain first intron fragment of potato GA20 oxidase gene, this fragment is cut with Sac I and BamH I enzyme, be connected with the carrier pUC18 through same enzyme double digestion, the recombinant vectors that obtains is pUCGA;

After with restriction enzyme Xba I and BamHI pUCGAT1 (370) being carried out double digestion, be connected interstitial granules during the recombinant RNA that obtains ToTOM1 disturbs, called after pUCGAToTOM1 (RNAi-370) again with through Bgl II with the TT1 of Spe I double digestion;

PUCGAToTOM1 (RNAi-370) is cut the back with restriction enzyme Sal I enzyme to be mended flat, carrying out enzyme with restriction enzyme Spe I again cuts, behind 1.2% agarose gel electrophoresis, reclaim the small segment of the about 900bp of length, be connected on the carrier pBI121 of restriction enzyme EcoRV and Xba I double digestion reclaiming fragment, the RNAi that obtains tomato ToTOM1 disturbs plant expression plasmid, called after pBI121ToTOM1 (RNAi-370);

2) genetic transformation of tomato

The intestinal bacteria that will contain plant expression plasmid pBI121ToTOM1 (RNAi-370) respectively carry out three parents with intestinal bacteria that contain helper plasmid pRK2073 and Agrobacterium EHA105 and combine, at the dull and stereotyped enterprising row filter of the LA that contains 50mg/L Rifampin and 50mg/L kantlex, obtain containing Agrobacterium-mediated Transformation of plant expression plasmid pBI121ToTOM1 (RNAi-370); This Agrobacterium-mediated Transformation is rule on the YEB flat board that contains 50mg/L Rifampin and 50mg/L kantlex, place 28 ℃ of constant temperature to cultivate about 24h, picking list colony inoculation is in containing identical antibiotic YEB liquid nutrient medium after growing bacterium colony, and 200 rev/mins, 28 ℃ constant temperature shaking tables are cultivated about 24h to OD ₆₀₀Be 04-0.6, change in the aseptic centrifuge tube that the centrifugal 10min of 3000rpm abandons supernatant liquor, the thalline of collecting is cleaned once with the MS nutrient solution and suspend again, be used for the conversion of tomato after diluting 50 times;

To keep length is that the aseptic seedling of radicle of 2-3cm is as explant, explant is put into activatory be added with the MS liquid nutrient medium of Agrobacterium EHA105 that final concentration is the Syringylethanone of 100 μ mol/L, ceaselessly shook 10 minutes, explant takes out twice of aseptic water washing in back, put into the MS that is added with zeatin 0.15mg/L and indolylacetic acid 0.05mg/L then and be total to culture medium, in illumination box, cultivated 2 days;

3) induction

Cultivate after 2 days, explant is transferred in the differentiation screening culture medium, continue to cultivate 2-3 week in illumination box, culture condition is 25-28 ℃, illumination in 15 hours, 9 hours dark;

4) the taking root of transfer-gen plant, hardening and transplanting

To in the differentiation screening culture medium, downcut from callus by the normal plant of growth, insert in the root media, 25-28 ℃, illumination in 15 hours, dark condition was cultivated until taking root down in 9 hours;

When 5) root system for the treatment of regrowth is about 3cm with the covered opening of culturing bottle and keep the skin wet, at 25-28 ℃, under 15 hours/day the illumination condition, placed 3-4 days, and then plant extracted at leisure, clean root with agar, be transplanted in the soil, the humidity that keeps soil, and temperature is controlled at 25-30 ℃, obtain antiviral tomato plant.

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