Cloning And Expression Of α-Farnesene Synthase Gene From Peel Tissue Of Apple (White Pearmain)

α-Farnesene is a secondary metabolite in plant. This compound, among many other components in apple volatiles, has been proven to play major roles in host plant searching and reproduction of codling moth, to be correlated to the degree of plant sufferred from cold, and to the development of superficial scald which is one kind of serious storage disorder of several apple cultivars. The accumulation of α-farnesene in the skin of apple fruit during storage appears to be predominantly through classical mevalonate (MVA) pathway. 3-Hydroxy-3-methylglutaryl CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS) and α-farnesene synthase (AFS) are the rate-limiting enzymes in the mevalonate pathway. AFS is the final, rate-limiting enzyme that converts farnesyl diphosphate (FPP) to α-farnesene in the mevalonic acid (MVA) pathway of terpene biosynthesis. Studies have been focused on the control of α-farnesene synthesis and molecular bases of scald development by gene knockout or antisense RNA of AFS. To elucidate the relationship between AFS expression and α-farnesene synthesis during low temperature storage and after apples warming to room temperature, the cDNA encoding α-farnesene synthase gene (AFS) was cloned from 'White Pearmain'apple. Specific primers for PCR designed from AFS-1 had been registered in GenBank (AY182241). The results were as followed. 1 According to the specific primer a sequence about 1800bp concluding the ORF of AFS gene was cloned by RT-PCR, the clone contains an open reading frame (ORF) of 1728 bp comprising 576 amino acid residues and was registered in GenBank (Accession No.AY563622). 2 The deduced amino acid sequence shows high identities with other α-farnesene synthase gene from other apple and pear varieties and low identities with crops such as cucumber, pinus and picea. Southern blot analysis indicated that AFS gene is only one copy in genome of apple. 3 Northern blot analysis for elucidating the expression characteristic of AFS gene in cold storage indicated that the expression of AFS gene in control was low at beginning of cold storage, and reached a peak at 12th weeks, and decreased in followed cold storage. The expression of AFS gene in apple peel was inhibited by 1-MCP, and there is no detection from 8th to 20th weeks. The expression of AFS in apple peel treated with DPA was postponed and reached maximal in 16th weeks, and dropped during continued storage. 4 Northern blot analysis showed that AFS expression level of non-treated apple kept increasing gradually when cold storage apple was shifted to room temperature for 10 days. AFS expression in apple peel treated with DPA reached a higher level at first day and dropped gradually then after; there was no AFS expression in fruit treated with 1-MCP at the first day, less expression was detected at the fifth day, and more at the tenth day. 5 Quantification of α-farnesene was performed with HPLC system. The content of α-farnesene in apple peel tissue treated with DPA decreased at 10 days after warming up to room temperature, and the content in apples treated with 1-MCP and in control increased. But the content of α-farnesene in apple peel treated with DPA was higher than that of non-treated apple at the first day.