Strain Identification Of Potato Virus Y In Shandong And The Coat Protein Gene Cloning

During their growth, potatoes are attacked by various viruses, which will cause serious degradation in quality. The most economical and most effective way to control plant virus diseases is to breed genetically engineered anti-virus varieties. Among all the genes used to transform host plants are the viral coat protein genes whose transformation has become a standard strategy in anti-virus breeding. The analysis of resistance of plants transformed viral CP genes showed that RNAs mediate the resistance of transgenic plants. Due to its advantage of high bio-safety and effect, the RNA-mediated resistance now becomes a hot spot in the domain of plant anti-virus gene engineering. In this research program the isolate of P V Y from Shandong was extracted and purified for future use in anti-PVY breeding. RT-PCR was used to clone the CP gene of PVY-SD. Meanwhile, experiments were carried out to compare the resistance to PVY and PVY-SD of an immune line of transgenic Plants. We also conducted a survey of potatoes plant carrying PVY in fields in Shandong. The results are as the following:1 PVY- SD was extracted from potato plants grown in Shandong. PVY-SD formed pine-wheel inclusion bodies in plant cells. The subunits' molecular weight of the coat protean was: 33kD. The shape of virus particles is filament-like: 680-71 OX 1 Inm. In Vitro resistance tests showed that the longevity was 72 hours, the dilution end point is 10'3~10"*-and the inactivation temperature is 55~65'C. The research of different hosts' response suggested that PVY-SD could infect plants of 23 species from 8 families. Thus PVY-SD was tentatively attributed to Strain PVY?2 An up-dated method of extracting and purifying PVY was applied in this research. Using the Protease K method, we acquired the viral genome-RNA. Then RT-PCR and specific primers were used to amplify the gene encoding the coat protein. This sequence was cloned on to vector pUC19 to transform E.coli DH5ct through heat shock.. Sequence analysis showed that the CP gene of PVY-SD included 804bp encoding 267 amino acids; the coding sequence had the highest homology (99%) to that of PVY?whose registration number in GENEBANK is AJ390306: AJ223595. So PVY-SD is placed in the strain of PVY?on molecular biology level.3 The immune lines Mil and M13 of tobaccos transformed with untranslatble CPgene of PVYN were inoculated with PVY-SD, which has a 97.8% homology to PVYNand showed immune response, too. This indicated: RNA-mediated resistance in tobacco had a wide range of targets.4 Using random sampling and ELISA, we conducted a preliminary survey of plants carrying PVY in fields Results were: the rate of samples from Liu Qiao, Jie He, Ping Yin and An Qiu were 64%, 60%, 56%and 52% respectively. PVY diseases were rather severe.