Molecular Cloning, Expression and Characterization of Pathogenesis Related Protein-1 Isoforms in Jelly Fig Achene

• Main Authors: I-Chun Tsai, 蔡宜錞
• Other Authors: 周文敏
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/j5n8jd

碩士 === 國立虎尾科技大學 === 生物科技研究所 === 100 === Jelly fig (Ficus awkeotasang Makino) is one of the endemic plants in Taiwan. Water extract from jelly fig achenes is utilized to produce jelly curd for making a popular drink in local markets. MALDI-TOF analysis suggested that there is pathogenesis related-1 protein (PR-1) in jelly curd. Plant PR-1s may play a role in host defense system, and are classified into acidic and basic types based on pI value. Here, four cDNA fragments encoding three full-length (PR1-1, PR1-2 and PR1-4) and one partial-length of jelly fig PR-1 isoforms (PR1-3) were obtained by PCR cloning. The pI values of these predicted mature proteins are 6.44, 7.12, 8.25 and 8.92, and their predicted mature molecular mass were 14.8, 21.3, 16.0 and 16.0 kDa, respectively. PR1-1, PR1-2 and PR1-3 over-expressed as insoluble proteins in Escherichia coli were used to raise antibodies. Western blot analyses suggested that PR-1 isoforms may presence as minor pericardial proteins extracted from jelly fig achenes. PR1-1, PR1-3 and PR1-4 were successfully expressed in yeast Pichia. The purified PR1-1 exhibited antifungal activity toward Glomerella cingulata. In addition, we found that PR1-3 and PR1-4 may form dimers structure. Moreover, PR1-3 probably exhibits weak serine protease activity using azocasein as substrate.