Elimination And Detection Technique Of Grapevine Fanleaf Virus

The elimination of Garpevine fanleaf virus was achieved by the methods of heat treatment, heat treatment+meristem culture, heat treatment+ Radix Isatidis+ meristem culture and cryopreservation+ meristem culture. The plants after virus elimination were detected by the methods of Dot-ELISA, dsRNA, RT-PCR and IC-RT-PCR. The results are as follows:1. Thirty-seven grapevines were detected by Dot-ELISA. 78.4% of plants were infected with GLFV.2.The plants infected GFLV and GLRV-â…¢ were detected by dsRNA. By Agarose gel electrophoreses ,two belts were found,one that was about 3700bp showed GFLV ,another that was about 17000bp showed GLRV possibly. But by the RT-PCR,the former was proved to be GFLV and the latter wasn't GLRV-â…¢.3.The way of improved CTAB,SDS-acid phenol and several steps centrifugal method were taken to extract RNA from the leaves of grapevines.The result showed that the method of improved CTAB was the best. The way of SDS-acid phenol was better than the several step centrifugal method.4.The effectiveess of reverse transcription polymerase chain reaction(RT-PCR) and immune capture reverse transcripion polymerase chain reaction(IC-RT-PCR) in detecting GFLV in grapevine was evaluated.We found that IC-RT-PCR was more sensitive than RT-PCR, The percent of detection were 69.2%by RT-PCR was and 92.3% by IC-RT-PCR.5.The six partial RNA secquences of GFLV were cloned from leaves of Vitis vinifera 'Wink'. Three nucleotide secquences showed 100% indentity with the accessed GFLV sequence in GENBANK(accession number: NC-003623), while the other three show 99% indentity.The three sequences were submitted in GENBANK,the accession number are DQ672565,DQ672566,DQ672567 respectively.6 In the method of heat treatment with shoot tip culture, the optimal time of heat treatment began after the plantlets cultured for 20 days, the ratio of shoot tip survival was...