CLONING AND CHARACTERIZATION OF HOMOGENTISATE PHYTYLTRANSFERASE GENES FROM CLITORIA TERNATEA AND ARTOCARPUS LAKOOCHA

Abstract

Prenylated aromatic compounds are secondary metabolites found to be distributed in various plant families. The group of key enzymes catalyzing the prenylation reaction to produce these prenylated aromatic compounds is called aromatic prenyltransferases (PTases). Each of these enzymes transfers a prenyl group in different lengths (C5, C10, C15 or C20) to an aromatic substrate at a specific carbon position to form a prenylated aromatic product. In this study, two genes encoding similar homogentisate phytyltransferases (HPT), a member of aromatic PTases were isolated from Clitoria ternatea L. (clt) and Artocarpus lakoocha Rox. (alc2). The full-length cDNAs of clt and alrc2 were 1,495 and 1,625 bp in size, containing 1,224 bp and 1,233 bp ORF, respectively. The clt and alrc2 genes encoded CLT and ALRC2 proteins of 407 and 410 amino acids with predicted MWs of 45.58 and 45.59 kDa, respectively. Both proteins contained important characteristics of aromatic PTase structures, including a signal transit peptide at N-terminal, Asp-rich regions of substrate binding site (NQXXDXXXD and KDXXDXD), and nine trans-membrane α-helixes. According to the results from phytogenetic analysis, both were closely related to the HPT family members. The functional study of clt and alrc2 was then carried out in tomato by transient expression using agroinfiltration method, and evaluated by RT-PCR. For their enzyme activities, these were indirectly evaluated by detection of the accumulation of the intermediate 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ) and the pathway product α-tocopherol by TLC and GC-MS. The results revealed that the isolated clt and alrc2 could enhance the α-tocopherol accumulation in tomato leaves after 3 days agroinfiltration by 2.4 + 0.38 and 1.4 + 0.05 fold higher than control. Taken together, both genes were possibly functioned as HPT enzyme.