月季花瓣乙烯生物合成酶活性对乙烯的响应及其中ACC合成酶基因的克隆和原核表达

Ethylene is a crucial factor in regulating flower opening and petal expansion in roses. In this work, we determined the ethylene production and ethylene biosynthetic enzymes activities of rose petals in response to exogenous ethylene and its action inhibitor, 1-MCP. Our results showed that ethylene production was enhanced obviously by ethylene and longer treatment time caused stronger production, while 1-MCP failed to decrease the ethylene production. The changes of ACS (ACC synthase) and ACO (ACC oxidase)　activities well supported the results of ethylene production. We also isolated a member which is regarded as the key ethylene-responsible ACS gene based on our previous work. The entire Rh-ACS3 gene is 1734 bp in length, and contains a 1614 bp ORF which encodes a putative ACS protein with 537 aa and flanking by a 100bp 5’ UTR (untranslated region) and a 20bp 3’ UTR. The ORF sequence of Rh-ACS3 gene was inserted into a protein expressed vector pET-30a(+) and expressed in E. coli BL21 strain. A 60 kD recombinant protein was greatly induced and expressed by isopropyl β-D-thiogalacto-pyranoside (IPTG) at a final concentration of 0.6 mM for 3 h at 37˚C, which is identical to predict MW value of the putative Rh-ACS3 protein, indicating that we successfully obtained the Rh-ACS3 protein.