Heterogenous Expression and Characterization of Different Domain of Jelly Fig Pathogenesis Related-4 Protein

• Main Authors: Yi-Hung Lin, 林一弘
• Other Authors: Wing-Ming Chou
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/k4e3w6

碩士 === 國立虎尾科技大學 === 生物科技研究所 === 102 === Jelly fig (Ficus awkeotasang Makino) is one of the endemic plants in Taiwan. The water extract from jelly fig achenes forms jelly curd, a common summer beverage. Some pericarpial proteins extracted from the jelly curd have been identified as a pectin methylesterase (PME), chitinase, thaumatin-like protein (TLP), two PR proteins, pathogenesis related-1(PR-1) and pathogenesis related-4 (PR-4). PR-4 was classified into two groups, Class I contains extra chitin binding domain (CBD) in N-ternimus and Class II has no such domain. cDNA fragment coding for Jelly fig PR4 (FaPR4) has been cloned and its deduced protein has CBD in N-terminus and a vacuolar signal in C-terminus. Using designed primers and PCR technology obtained cDNA fragnmemt encoding FaPR4(noCt), a jelly fig PR-4 without vacuolar signal. FaPR4(noCt) and FaPR4(noCt/mutant) were successfully expressed in yeast Pichia pastoris. FaPR4(noCt/mutant) is a point-mutant of FaPR4(noCt). The purified r-FaPR4, r-FaPR4(noCt), r-FaPR4(noCt/mutant), r-FaPR4(NO) and r-FaPR4(NO/mutant) were subjected for RNase activity assay. The recombinant proteins eluted from different salt concentrate showed different RNase activity. Recombinantion r-FaPR, r-FaPR4(noCt)and r-FaPR4(noCt/mutant) exhibited antifungal activity toward Glomerella cingulata and Fusarium oxysporum. After heating at 100oC for 10 min, the recombination proteins remained some activity, and r-FaPR4 had the highest antifungal activity. In addition, recombination proteins could cause ROS response in Rhizoctonia solani hyphae, and r-FaPR4 has the most obvious effect on ROS response.