Fabrication And Application Of Genechip Detecting Virus And Viroid Infecting Potato

Potato is a kind of popular and important economic plant that grows in most of countries of the world. The diseases caused by viruses or viroids bring about substantial losses in agricultural production. There are more than 30 viruses and viroids can infect potato. The proper diagnosis of causative viruses and viroids is the first requirement for the control of such diseases. So developing a high throughput, rapid, sensitive and specific method is necessary. Genechip, giving a large amount of sequence information directly and simultaneously, has become one of the best solutions to detect virus genes.In this study, using RT-PCR and cloning method, we got the 3( end sequences of genome of three kind of virus isolates (PVA, PVY and TMV), and all sequence of genome of two PSTVd isolates which were found in potato. The above sequence data obtained in our study were then compared with previously reported sequences of other isolates obtained from other parts of the world. The PVA isolates were then found belonging to three groups according to nucleic acid similarity of coat protein, and PVY isolates can be classified into four strains, and TMV isolates can be classified into three groups. PSTVd genome sequence is very conservative, the isolates belonging to two sub-groups. No genomic adoption of the genomic variation to geological origination was found when compared with genomic similarity among reports of PVA. In certain degree, similarity of CP gene of TMV and PSTVd and isolates of each PVY strain was found related with the geological origination.In this research, we fabricate an oligonucleotide microarray , which can simultaneously detect ten kinds of potato virus and viroid (TMV,AlMV,CMV,PVA,PVX,PVY,PMTV,PVM,PLRV,PSTVd).The virus-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the target regions that are highly conserved. These Cy5-labeled target cDNAs of different viruses were amplified by Reverse transcriptase PCR(RT-PCR) respectively, using specific primers. These fluorescent cDNAs were then hybridized to their specific oligonucleotide probe-coated glass slides. After optimizing the hybridization protocols, the fabricated gene-chip can detect seven kinds of virus and viroid at one time, with absence of cross-hybridization between the unrelated oligonucleotide probes and each virus produced a unique hybridization pattern. To simplify the operation, we try to label target by reverse transcriptase. Four kinds of virus (CMV, PVX, PVY and TMV) were detected by this multiplex RT labeling and hybridization method.We also first established a new method for the diagnosis of plant RNA viruses and viroid using dot blot hybridization to glass slide and using a fluorescently labeled probe by directly spotting large-scale extracted RNA samples onto a silanated glass slide and then hybridization with CY5-labeled probe of specific virus or viroid prepared by PCR. Factors affecting the result of hybridization were demonstrated by performing large numbers of conditional experiment in the course of detecting CMV. By the testing of 40 potato samples singly with PSTVd-specific probes, this method was demonstrated more accurate and reliable than nylon membrane hybridization with 32P-labelled probes.