Molecular Cloning,Expression And Functional Characterization Of Two Limonene Synthases From Rice (Oryza Sativa L.)

Terpenoids are the most abundant natural compounds produced by plant secondary metabolism.Several dozen thousand of terpenes have been found in the plant kingdom.However,each species is capable of synthesizing only a small fraction of this total.As secondary metabolites,terpenoids serve as both constitutive and inducible defense chemicals in many plant species,and volatile terpenes participate in plant indirect defense by attracting natural enemies of the herbivores.Terpene synthase(TPS)is a group of enzymes that is responsible to the production of various terpene molecules in plant.Two isomeric 5-carbon precursor 'building blocks',IPP(isopentenyl diphosphate)and DMAPP(dimethyallyl diphosphate),bond to each other followed a head-to-tail manner to form geranyl diphosphate(GPP),farnesyl diphosphate(FPP)or geranylgeranyl diphosphate(GGPP)under the action of prenyltransferases.Then,TPSs accept GPP,FPP or GGPP as substrates to synthesize diverse terpene compounds.An increasing number of TPSs have been cloned and characterized after years of researches.The understanding of the structure and reaction mechanisms became comprehensive.However,there are only limited reports on rice(Oryza sativa L.)which is the most important grain crop in the world.Based on the whole genome sequence,we found that the rice genome contains 51 genes and homologs of TPS.Expect the 17 genes that have been characterized,we studied the rest 34 genes and got 8 clones:OsTPS04(Os02g26014),OsTPS18(Os04g01810),OsTPS22(Os04g27190),OsTPS23(Os04g27340),OsTPS24(Os04g27400),OsTPS29(Os04g27670),OsTPS32(Os04g27790)and OsTPS39(Os08g07080).The soluble recombinant proteins of OsTPS22 and OsTPS23 were obtained by prokaryotic expression in Escherichia coli,and the enzymatic assays were carried out.OsTPS22 and OsTPS23 could both accept GPP as substrate to form limonene in different conformations.Transient expression of GFP-tagged OsTPS22 and OsTPS23 revealed that these two proteins were both localized in plastids.The transcript abundances of these two TPSs exhibited distinct tissue specificity.OsTPS22 was mainly expressed in stems and roots,while OsTPS23 in roots.Both TPSs could be induced by mechanical wounding which indicated their potential roles in plant defense against herbivores.