Molecular Cloning And Characterization Of Hevea Brasiliensis HbSQS

Squalene synthase (SQS) is a key enzyme in plant terpenoid biosynthetic pathway. Squalene synthase catalyzesthe condensation of two molecules of farnesyldiphosphate(FPP) to produce squalene(SQ), the first committed precursorf or sterol, brassinosteroid, and triterpene biosynthesis. At present, The Hevea brasiliensis of terpenes anabolic path and regulation mechanism is not entirely clear. Research on terpene anabolic key enzyme SQS genes and their expression patterns will study its impact on natural rubber synthetic and terpene anabolism mechanism.In this research, a squalene synthase gene was isolated from Reyan7-33-97rubber trees and designated HbSQS. Then HbSQS was analyzed by bioinformatics and its expression pattern was investigated. The main results were summarized as follows:1. We designed primers based on the SQS sequence obtained from Hevea brasiliensis EST data base and amplified a fragment of SQS gene. According to the sequence of this fragment, the full-length cDNA of HbSQS was isolated by RACE. The full-length cDNA of HbSQS contains a1239bp open reading frame and encodes a predicted protein of413amino acids. The molecular mass (MW) of the putative SQS protein is46967.54with a theoretical pI of7.95.2. Amino sequence alignment analyzed with SQS enzymes of Ricinus communis, Glycine max, Buxus hebecarpa, Vitis vinifera. Nicotiana tabacum, Arabidopsis thaliana, Oryza sativa and Panax ginseng reveals that the homology of HbSQS is96.37%、86.44%、85.96%、85.96%.83.54%、69.66%、76.63%、83.37%. There are6conserved domains in HbSQS.3of them are very conservative.3. The exprssion of HbSQS in latex was induced by ethylene. The expression level of HbSQS in bark is higher than that in leaf. But the exprssion of HbSQS in leaf was up-regulated as the development of leaf.4. A fragment of peptides of HbSQS was expressed in E. coli.5. HbSQS overexpression and silencing for fuction analysis in tobacco. The sense strand and antisense strand of HbSQS’ ORF were constructed into pCXSN vector. The two constructs were transformed into tobacco by the leaf disc transformation method.