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 ent-kaurene, 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.