Cloning And Construction Of Plant Expression Vector Of α-Farnesene Synthase Gene From Yali Pear

α-farnesene, an acyclic sesquiterpene, is an important secondary metabolite, which has many important biologic effects. Many evidences had revealed that α-farnesene is one of aroma related volatile in pear fruit. E, E-farnesene has been proven to play an important role in searching and reproduction of codling moth.α-farnesene, as a constituent of pear skin cell layers, has been associated with the occurrence of scald. The accumulation ofα-farnesene in the skin of apple fruit during storage appears to be predominantly through classical mevalonate pathway. 3-Hydroxy-3- methylglutaryl CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS) andα-farnesene synthase (PFS) are the rate-limiting enzyme in the mevalonate pathway. PFS is the final, rate-limiting enzyme that converts farnesyl diphosphate (FPP) toα- farnesene in the mevalonic acid (MVA) pathway of terpene biosynthesis. The role ofα-farnesene in Yali pear fruit requires further study.China is an important producer of the Yali pear (Pyrus bertschneideri Reld). Yali pear belongs to super white pear. To elucidate function ofα-farnesene synthase gene, specific primers for PCR were designed from AFS1 registered in GenBank (accession number: AY182241). The coding region and partial 3'untranslated region ofα-farnesene gene were obtained, then the antisense RNA expressing vector and RNAi mediated expression vector were constructed by digestion and ligation, which were used as genetic transformation. The main results were as follow:1 RNA was isolated from peel tissue of Yali pear andα-farnesene synthase gene (PFS;GenBank accession number DQ364626) with the coding region of 1824bp in length was cloned by a similarity-based cloning strategy using RT-PCR with primers based on appleα-farnesene synthase.2 Sequence analysis showed that the 1728bp open reading frame encodes a deduced protein of 576 amino acids(aa) long with a molecular mass of approx 66 KDa. The nucleotides sequence was highly homologous to PFS and AFS1. The deduced amino acid sequence for PFS cDNA showed low similarity with farnesene synthases from cucumber, fir and pine. The cDNA appeared to encode a typical N-terminal RRX8W motif (amino acids 33–43), and the expected DDXXD (amino acids 326–330) element in the C-terminal domain. The result of sequence alignment was phylogenetically grouped within angiosperm monoterpene synthases subfamily (terpene synthase b). Two domains are critical to enzymatic function.3 Northern blot analysis indicated that the expression of PFS in pericarp andα-farnesene content were the highest, the next was in leaf, and relatively lower in stem, flower and root. HPLC measurement was performed. Expression pattern of PFS gene was closely correlated withα-farnesene accumulation in pear.4 In order to suppress the expression of PFS gene after its transcription in Yali pear, a double-stranded RNAi plasmid was constructed by ligating the 515 bp fragment from PFS cDNA gene to 396 bp fragment in inverted directions, the results confirmed by restriction enzymes digestion and sequencing that the constructed RNAi plasmid contained the designed structure.5 A recombinant plant expression vector PBI-PFS-L was also constructed and used as genetic transformation system.