Construction of Infectious clones of Squash leaf curl virus from Lagenaria siceraria and Analysis of factors involved in leaf curl symptoms

• Main Authors: Ya-Wen Yang, 楊雅文
• Other Authors: 胡仲祺
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/98381819775155032801

碩士 === 國立中興大學 === 生物科技學研究所 === 101 === Squash leaf curl virus (SqLCV) is a species of the which harbors single-stranded, circular DNA (sscDNA) genomes. The genetic information of SqLCV is divided into two sscDNAs, designated DNA A and DNA B. Cucurbit plants infected by SqLCV exhibit various degrees of leaf curling symptoms which impair the growth and fruiting of the crops and cause severe losses to agricultural economy. In this study, leaf samples of the bottle gourd (Lagenaria siceraria) showing very severe curling symptoms were collected from Peitou township in Changhua County, which provided convenient source for the study of factors involved in leaf curling symptom in cucurbits. Therefore, the specific aims of this study are the isolation and sequence analyses of the SqLCV from the bottle gourd, the construction of infectious clones for the study of symptom expression, and the production of specific antibodies against SqLCV. Genomic DNAs of SqLCV were amplificed by Rolling Circle Amplification (RCA), followed by cloning and sequence analyses. To construct the infectious clones of SqLCV, tandem dimmers of DNA A and B were generated by partial restriction enzyme digestion of the RCA products and subcloned into the binary vector pBin19. Two independent SqLCV infectious clones of each DNA A and DNA B, designated BGSQ1A, BGSQ5A, BGSQ1B and BGSQ2B, have been constructed. Infectivity assays showed that the combination of BGSQ5A and BGSQ1B or BGSQ2B could elicit leaf curl symptoms on L. siceraria following the vacuum inoculation method developed in this study. Furthermore, SqLCV coat protein gene has been cloned in the pCyCP vector, over-expressed and purified as a fusion protein, and subjected to the production of specific antibodies. The results in this study provided useful tools for further study of the mechanism underlying the control of leaf curl symptoms.