Cloning and characterization of the putative virus-resistant 14-3-3 family genes of cucurbits

• Main Authors: Chia-Wei Mou, 牟家緯
• Other Authors: Chung-Chi Hu
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/89943518894453868653

碩士 === 國立中興大學 === 生物科技學研究所 === 97 === Production of cucurbits is often severely limited by virus diseases which cause serious economical losses. The use of virus-resistant cucurbit varieties is one of the most effective and economical method for the prevention of virus disease. However, suitable resistant gene(s) and molecular markers, which are difficult to obtain, are required for the breeding of virus-resistant varieties. Previously, the TVI4204 wax gourd line has been shown to be highly resistant to common RNA viruses by Dr. Deng and colleagues in Taiwan Arigultural Research Institute. A distinct protein highly expressed in the virus-resistant line was identified in our laboratory to be a 14-3-3 family protein through two-dimensional electrophoresis (2-DE) and LC/MS/MS analysis. The 14-3-3 family contains important regulatory proteins for cell metabolism which maybe have important function in virus resistance. Therefore, the objectives of the current study are to clone and characterize the wax gourd 14-3-3 genes, and to test whether the 14-3-3 protein can serve as a molecular marker for disease-resistance breeding programs. To confirm the the effect of 14-3-3 family protein on symptom expression in wax gourds, leaf samples were collected from side-grafted resistant and susceptible wax grourds and analyzed by 2-DE. The results showed that the expression of 14-3-3 family protein in leaves proximal to the side-grafting point in susceptible variety was increased, compared to that in the distal positions, resulting in attenuated symptom. Therefore, further cloning and characterization of 14-3-3 family genes were performed. Currently, two isoforms of the 14-3-3 gene family have been cloned from resistant and susceptible varieties of wax gourds. Sequence analyses revealed that the 14-3-3 isoforms share high similarity. Specific antiserum was therefore raised against the bacterially expressed 14-3-3 protein. The higher expression level of 14-3-3 protein was revealed in virus-resistant waxgourds than that in the susceptible ones, using western blot analysis with the above antiserum. The results suggested that the 14-3-3 family proteins, together with the specific antiserum, possess high potential to serve as molecular markers and analysis tools in future breeding programs and researches for virus-resistance in cucurbits.