Applying Cucumber mosaic virus 2b gene to construct transgenic resistance and producing specific antiserum against 2b protein

• Main Authors: De-Shiu Chen, 陳德旭
• Other Authors: 陳煜焜
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/91938891288408225827

碩士 === 國立中興大學 === 植物病理學系所 === 96 === Cucumber mosaic virus (CMV) causes severe damage and economical loss to many commercial crops. Many approaches, including conventional resistance breeding and transgenic resistance, have been developed to confer resistance in plants against Cucumber mosaic virus. Comparing to the time-consuming conventional measures, transgenic approaches are currently the main stream of building resistance against viruses in plants. CMV contains three major RNAs, most genes on these RNAs have been applied to generate transgenic resistance in the literature. The 2b gene has applied to generate transgenic resistance with expressing artificial RNA in plants, however, never applied in the form of full length. In this study, the 2b gene of a tomato isolate of CMV (CMV-peets) was amplified with a degenerated primer pair and the product was cloned. Sequence analysis showed that the nucleotide of the 2b gene of CMV-peets is highly homologous to that of CMV isolates belong to subgroup IB. The 2b open reading frame was introduced into protein expression vector to express 2b recombinant protein in E.coli. The expressed CMV 2b protein was purified and applied to produce a polyclonal antiserum. The produced antiserum was applicable to detect homologous antigen in vitro, but failed to detect CMV 2b protein for immunoblot tests. In the mean time, five constructs consist of 2b ORF in sense or anti-sense orientations (pCMV2b-S and pCMV2b-AS, respectively), 2b ORF with flanking sequences in sense or anti-sense orientations (pCMV2b-EXT-S and pCMV2b-EXT-AS, respectively), and 2b ORF in inverted repeat form (pCMV2b-IR), were transferred into tobacco (Nicotiana benthamiana) mediated by Agrobacterium tumefaciens. Our results indicated all five constructs confer transgenic resistance to plants and the inverted repeat construct has the best efficiency among the five constructs.