Functional analysis of horned melon Cm4 and Cm9 transgenic tobacco (Nicotiana benthamiana)

• Main Authors: Che-Hsien Yu, 余哲賢
• Other Authors: 古新梅
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/96747659292677981322

碩士 === 國立中興大學 === 農藝學系所 === 104 === Previous studies was performed to cloned partial Cm9 and Cm4 gene form Papaya ringspot virus (PRSV) infected Cucumis metuliferus. Previous researcher cloned full length Cm9 and Cm4 by genome block walking and rapid amplification of cDNA end (RACE). Cm9 gene function was predicted as DEAD-box RNA helicase. In this studies, Nicotiana benthamiana transgenic plant that carried Cm9 genome full length was generated. Cm9 transgenic plant shown same phenotype with wild-type tobacco, confirmed that Cm9 gene may not conferred function of plant growth and development. To further understand that Cm9 conferred virus resistance or not, the virus inoculation experiment was performed. Southern blot and polymerase chain reaction was performed to select homozygote one copy Cm9 and Cm4 transgenic tobacco. Virus inoculation experiment result showed that all Cm9 and Cm4 transgenic tobacco was susceptible to Potato virus Y (PVY), Pepper veinal mottle virus (PVMV), Chilli veinal mottle virus (ChiVMV) and Turnip mosaic virus (TuMV). This result suggested that Cm9 and Cm4 gene does not conferred virus resistance to transgenic tobacco. To understand Cm9 gene conferred abiotic stress resistance or not, the transgenic tobacco leaf discs was treated with salt, drought, heavy metal and cold stress. The result showed that transgenic tobacco harboring Cm9 gene show significant less total protein and chlorophyll that wild-type tobacco, but transgenic plant also showed significant less hydrogen peroxide than wild-type plant, suggest that Cm9 gene may conferred reactive oxygen species (ROS) scavenging function. This research suggested that Cm9 and Cm4 gene didn’t conferred virus resistance function to various virus above, but may conferred ROS scavenging mechanism. However, its exact role of Cm9 and Cm4 in against to virus need to be further confirm by Cm9 and Cm4 gene silencing Cucumis metuliferus in the future.