Molecular Cloning And Sequence Analysis Of Complete Genome Of Tobacco Mosaic Virus And Construction Of Yeast Two-hybrid Cdna Library From Solanum Lycopersicum Infected By TMV

Tobacco mosaic virus(TMV)is one of the most serious viral pathogens of tomato(Solanum lycopersicum).The complete genome of TMV Hezhou strain was copied into cDNA clones,and the sequence was determined.Besides,yeast two-hybrid cDNA libraris from healthy and TMV-infected tomato leaves were constructed.These works paved ways to study on TMV-tomato interactions,since yeast two-hybrid assay is an effective system for identifying and analysing protein-protein interactions,which will provide insight into the molecular mechanisms of TMV pathogenicity on tomato,and help to introduce new srategy of tomato-TMV disease control.The full-length genome of TMV Hezhou strain was 6395 nts in length,including a 68 nts 5' UTR,and a 207 nts 3' UTR,coding 5 ORF:126 kDa protein,54 kDa(protein),183 kDa protein,movement protein and coat protein ORF.There was a 14 nts overlapping sequence between 183 kDa protein ORF and movement protein ORF.BLAST analysis showed that this strain shared the highest identity(99%)in nucleotide sequence with TMV-152(GenBank accession No.AF395129.1)?Yeast two-hybrid cDNA library from tomato leaves infected by TMV contained 1.54×106 CFU with a recombination rate of 100%,and was divided into three sub-libraries:0.5-1 kb,1-2 kb and ?2 kb sub-libraries.The 0.5-1 kb sub-library contained 4.2 × 105 CFU with an average insert size of 0.68 kb,the 1-2 kb sub-library contained 7.7 ×105 CFU with an average insert size of 1.21 kb,and the ?2 kb sub-library contained 3.5×105 CFU with an average insert size of 2.33 kb.The library from healthy tomato leaves contained 1.62 × 106 CFU with a recombination rate of 100%,was also divided into 0.5-1 kb,1-2 kb and ?2 kb sub-librarys,contained 4.7 × 105,7.8 × 105 and 3.7 × 105 CFU respectively,and with an average insert size of 0.77 kb,1.35 and 2.38 kb respectively.Analysis base on cloning capacity,average insert size and recombination rate indicated high quality of these two yeast two-hybrid cDNA libraries.These two libraries can be used to isolate tomato coded interactors with TMV coded proteins,or identify interactions between tomato proteins by yeast two-hybrid screening.These research works provide the foundation for further studies on the molecular mechanism of TMV-tomato interactions and functional analysis of interested tomato genes.