The Research On Processing Tomato Resist ToMV By Transgenic Technology

Tomato mosaic virus (ToMV) is a worldwide distributing plant virus. It can infect many plant species including the families of Solanaceae and Cruciferae and so on, and tomato is its major host. ToMV is a common plant virus spreading in many Xinjiang areas, which infects processing tomatoes causing complicated symptoms. The infecting percentage is as high as 100% in some lands resulting yield degradation。Recently, the bad effects of ToMV becomes more and more serious every year, and severely affect the yield and quantity of processing tomatoes.Tomato mosaic virus (ToMV), a member of the Tobamovirus genus, has a positive-sense single-stranded RNA genome that encodes at least four proteins:The 130/180-kDa replicase proteins, a 30-kDa protein and a coat protein (CP). The 130/180kDa replicase proteins are translated directly from the genomic RNA. The 30-kDa MP and 17-kDa CP are translated from the respective subgenomic mRNAs, which are synthesized during the replication cycle. It has been shown that both the replicase proteins are involved in viral RNA replication.MP is essential for cell-to-cell movement, and CP mainly plays a role in long distance movement of tobamoviruses in plants.In this research, RNAi technology is used to repress the homology target gene expression of Tomato mosaic virus by degrading mRNA of target gene, aiming to resist ToMV.Firstly, ToMV was detected from naturally infected processing tomatoes with symptoms of mosaic and malformation. After being separated and purified, according to the known bioinformatics, ToMV sequences amplification primers were designed from AF395128 sequence(Genebank accession number), then genome sequence was amplified by reverse transcription polymerase chain reaction (RT-PCR), ToMV isolate CJ-2 was obtained.Comparison of the nucleotide and amino acid sequences of ToMV CJ-2 isolate with the reported sequences of the part of the ToMV isolates reported showed that both of the homology is more than 99.8%. RNAi sequences were chosen and amplified from130/180kDa replicase proteins and coat protein encoding regions by PCR according to the conserved regions. RNAi vectors pBi35STo5, pBi35SToBK, pBi35ToMV3 were constructed by inserting the RNAi sequences into plant expression vector pBi35SG12 forward and reversely, respectively.Then the three recombinant plasmids were transformed into tobacco mediated by Agrobacterium tumefaciens GV3101. Transgenic plants were screened by Kanamycin resistant and verified by PCR analysis, which provides a foundation for further experiment of attacking virus.