Research On The Specificity Determining Region Of Sesquiterpene Synthase In Artemisia Annua L By Gene Mutations

The terpene refers to the hydrocarbons and their oxygenated derivatives which have thegeneral formula of (C5H8)n, that can be considered as a class of natural compounds that linkedby the isoprene or isopentane in various ways. So far,55,000a variety of terpene compoundshas been isolated and identified from the nature organism. With a large number of valuableterpene was found, the biosynthesis and regulation of terpene synthase is becoming the focusof the research.Using the method of overlap extension PCR, we interchanged the the C-terminalstructure in Amorpha-4,11-diene Synthase and Epi-cedrol Synthase which of the two kind ofsesquiterpene synthase in Artemisia annua L. The hybrid molecule we obtained has lost itscatalytic activity. This proves the important role of the C-terminal structure on the enzymeactivity.Using the site-directed mutagenesis to introduce mutations to Amorpha-4,11-dieneSynthase, we studied the catalytic specificity changes of the enzyme. The protein expressedby this method obtain a new activity to produce β-Bisabolene. The mutation of amino acidresidues has a different nature, can make the enzyme changes its catalytic activity. Thisresearch can provide a basis for further research.Using the method of reverse transcription, we constructed the expression vector pETGwith the His tag by inserted the cDNA of germacrene into the pET-21vector. Transform thepETG into Rosetta to induced expression of protein, then purified the product bynickel-agarose column. By GC-MS analysis, the purified protein which adding to theenzymatic reaction system showed the activity of catalyze FPP to farnesol.