Construction of a Cucumber mosaic virus vector and its application on gene silencing and disease control

• Main Authors: Hsiao-Hsuan Jan, 詹孝軒
• Other Authors: Ting-Hsuan Hung
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/17729331146142691834

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 104 === Virus induced gene silencing (VIGS) is to knockdown gene(s) with interests through virus vector. VIGS can induce post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS), and can be applied in gene functional analysis. Cucumber mosaic virus (CMV) belongs to the genus Cucumovirus, and it is important plant virus widespread around the world. CMV has a wide host range of over 1200 species in more than 100 families. Thus, it has good potential to become a favorable virus vector which can be applied on various plants. In this research, we try to modify our previously constructed CMV infectious clone to a viral vector. We used overlapping polymerase chain reaction (overlapping PCR) to introduce an additional restriction site on the 2b open reading frame on CMV RNA2, and used this restriction enzyme site for the insertion of foreign sequences. To test the viral vector in silencing inducing, we inoculated the CMV carrying a fragment of 35S promoter (pCMV-35Spro) on a transgenic Nicotiana benthamiana (35S::GFP), line 16c. We observed that plant infected with pCMV-35Spro only showed mild symptoms, decreased expression of green florescence expression under UV illumination and decreased expression of GFP gene on the inoculated plants. We also tried our vector to induce VIGS of glutamate-1-semialdehyde aminotransferase (GSA) in N. benthamiana. GSA is an enzyme involved in early biosynthesis of chlorophyll (Matters and Beale, 1994). Plants induced GSA silencing by our vectors were expected to show chlorotic phenotypes and decrease GSA gene expression. We also inserted a fragment of Tobacco mosaic virus (TMV) into the CMV vector p(CMV-TMVCP) in an attempt to see whether the plants inoculated with pCMV-TMVCP can protect later wildtype CMV and TMV infection. The results showed that pCMV-TMVCP inoculated plants show mild symptoms, and 71% of inoculated plants did not show severe symptoms after inoculation with wildtype TMV or CMV. However, some pCMV-TMVCP inoculated plants showed severe symptoms and PCR analysis revealed that some TMV gene was disappeared from pCMV-TMVCP.