The Preliminary Research Of Processing Tomato Interaction With ToMV And CMV Relatives Proteins

The ability of plant viruses to infect host plants and cause diseases is determined by molecular interactions between host and virus factors. These interactions directly affect virus replication, cell-to-cell movement, systemic movement, and symptom development, as well as the elicitation of host defense responses. Nowdays, many host factors have been identified to interact specifically with some viral gene products, including coat protein (CP), movement protein (MP) and replicase protein. However, the explicit roles of these host factors and their interactions with virus factors in viral pathogenicity and host plant resistance remain to be investigated.The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. With the characteristics of convenience and high sensitivity, which had been used in many aspects of plant virology.In recent years, plant virus spreading in many areas of Xinjiang, especially the spreading of ToMV and CMV becomes more and more serious, which infects decrease the production of tomatoes or the worse is nothing yield. Furthermore, with the expansion of planting area and adjustment of cultivation techniques, the adaptability variation of the virus and other factors are increasing the processing tomato virus diseases and leading a large area of the spread trend. Thus serious impact on processing tomato fruit quality and tomato production, that becomes a restricted factor to the development of Xinjiang Tomato Industry. Then in order to resolve this problem, ToMV CP, MP and CMV 2b genes were amplified by RT-PCR from CMV and ToMV Xinjiang separations, which were cleaved with restrict endonuclase, ligation, transformation, recombinant plasmids'restrict identification and gene sequencing, yeast expression vectors pSos-cp, pSos-MP, pSos-2b were constructed successfully.The cdc25H transformed with bait plasmids grew well on SD/Glutose (-UL), without toxicity and could not activate the transcription of reporter gene alone in yeast two-hybrid system.Meanwhile, a processing tomato cDNA library was constructed in the yeast pMyr expression vector with RNA extracted from the processing tomato seedlings leaves that were inoculated with ToMV and CMV. The first and second strand cDNA were synthesized by using CytoTrap XR library construction kit. The blunted cDNAs were added with EcoR I adaptor and then digested by Xho I, and ligated into pMyr yeast expression vector. The recombinant plasmids were transformed into the host strain XL10-Gold. The result showed the primary cDNA library had a total clones of 1.5×106cfu/mL. Libraries recombination-rate are larger than 98%, insert cDNA fragments mostly distributed in 700bp-2000bp between, while the titer of amplified was 3.0×109cfu/mL. This indicates that this two-hybrid library is qualified enough to further research. The pMyr cDNA library and pSos-CP, pSos-MP and pSos-2b cotransformant colonies are selected at permissive temperature and amino acids defects, "Putative positives"are identified by transferring the conransformants testing for galactose-dependent growth at 37℃. In different bait screening cDNA library obtained a different number of positive cloning. Using the pSos-CP,pSos-MP and pSos-2b as bait respectively, we gained 9,28,7 positive clones from the library. Positive clones were identified by PCR in the cDNA insert size and restriction enzyme digestion verified and was sent to sequencing. Sequence in the NCBI database, the results of the Blast alignment. Positive clones according to the database corresponding to homologous sequences information, related to the same cloning were classified on the same merger. The putative positives are subjected to further analysis to verify the interaction.In summary, we had used the yeast two-hybrid system to screened processing tomatoes cDNA library, it was found that host proteins could interaction with CP, MP, and 2b. This study for further clarify the ToMV and CMV related protein function, ToMV and CMV pathogenesis and host resistance against mechanism and development disease-resistant germplasm resources of new material processing tomato laid a foundation.