Nucleotide sequence analysis of coat protein gene of alpinia mosaic virus and its application

• Main Authors: Hung, Chien-Lung, 洪建龍
• Other Authors: Liu Reuy-Fen
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/61877786448043794868

碩士 === 國立臺灣大學 === 植物病蟲害學系 === 85 === In order to characterize the coat protein (CP) gene of AlMV and also to develop a DNA probe for detection of this virus, a cDNA expression library was constructed using Uni-ZAP XR as a vector. Forty-five clones were selected from this library by immunoscreening and the inserts of two of these clones were further analyzed by DNA sequencing. The result indicated that the sequence contained 1,719 nucleotide residues, including part of the NIb gene, the complete CP gene, and the 3'-noncoding region of AlMV. Analysis of the deduced amino acid sequence of CP revealed neither NIa cleavage site consensus nor the DAG tripeptides, which are conserved in the CP gene of many aphid-transmitted potyviruses. Two putative NIa cleavage sites were proposed, nevertheless, to be located in YE/GLW or FQ/SQT. Cleavage by NIa at these site will generate coat protein with molecular mass of 44.6 kDa and 36.4 kDa, respectively. The deduced amino acid sequence of the CP gene of AlMV was also compared with those of fourteen viruses of Potyviridae. The sequence of AlMV was found to have 22~41% similarity with those of Potyvirus, lower than the similarity found among members of Potyvirus. Comparison of the coat protein sequences with all the other accepted genera within Potyviridae family indicated that the similarly is very low as well. (21~25%). The possibility that AlMV may represent a new genus of the Potyviridae family awaits further investigation. Besides, a fragment of the CP gene was amplified using primers P1 and P2 and used as a probe for slot hybridization analysis. The results indicated that this probe was very specific for AlMV and showed no signals with extracts from plants infected with potato virus Y (PVY), banana bract mosaic virus (BBMV), zucchini yellow mosaic virus (ZYMV), or papaya ringspot virus (PRSV). Sensitivity test indicated that it could detect as little as 0.5 pg of AlMV RNA. Moreover, an immunocapture/ RT-PCR method was established for the detection of AlMV as well. These methods provide fast, efficient, and accurate ways for the detection of AlMV and can be useful for virus diagnosis as well as generation of virus-free seedling.