Identification and functional characterization of ent-kaurene synthase gene in Ilex latifolia

Diterpenes are the most complex and abundant plant metabolites,some of which play significant roles in both primary and secondary metabolism.Ent-kaurene synthase is the key enzyme for gibberellins(GAs)biosynthesis,which may act as a catalyst in the formation of entkaurene,the precursor for GAs,by cyclization or rearrangement of the substrate ent-copalyl diphosphate(ent-CPP).Ilex latifolia Thunb(Aquifoliaceae)is a widely distributed Chinese plant whose leaves are used to process a popular Chinese bitter tea named'Kudingcha',which has anti-microbial,anti-oxidant and anti-inflammatory properties.Here,we isolated a diterpene synthase gene from the leaf transcriptome of I.latifolia,and further identified its biosynthesis activity by in vitro enzymatic testing.The heterologous expressed ent-kaurene synthase of I.latifolia(IlKS)in E.coli could catalyze ent-copalyl diphosphate(ent-CPP)to form ent-kaurene.Tissue-specific expression indicated that IlKS had the highest transcript level in roots which is maybe the major location of ent-kaurene biosynthesis.This study would help us to determine diterpenoid metabolism and GAs biosynthesis in I.latifolia,to better understand the regulation function of GAs in growth and development.