Functional Identification of 13 Brassica napus GPATs Encoding Genes by Genetic Complementation in Yeast (Saccharomyces cerevisiae)
DUAN Qian-Qian1, LIN Yi-Xin1, DING Shuo2, WEI Lin-Yan1, GAN Yi1, ZHENG Yue-Ping1, LIU Hong-Bo1,*
1 School of Agriculture and Food Science, Zhejiang A&F University, Hangzhou 311300, China; 2 School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, China;
Abstract：Glycerol-3-phosphate acyltransferases (GPAT) are rate limited enzymes that catalyze the initial step of de novo glycerolipid biosynthesis in plants. Thus, isolation and identification of GPAT encoding genes from Brassica napus will help to in-depth uncover the molecular mechanisms of glycerolipid biosynthesis and metabolism in oilseeds. In this study, 13 candidate genes, possessing conserved acyltransferase domains, were amplified by reverse transcription-PCR (RT-PCR) from B. napus, and constructed into yeast (Saccharomyces cerevisiae) heterologous expression vector yADH1-pYES2-Kan V2 (exogenous gene expression was induced by glucose, inhibited by galactose). Verification of digestion and sequencing of the recombinant yeast expression vector showed that the sizes and sequences of 13 gene fragments were as expected. According to alignment of amino acid sequences, 1~2 transmembrane domains and 4 conserved acyltransferase domains were in these BnGPATs, respectively. Then, yeast conditional lethal double knockout mutant strain ZAFU1 (BY4742, gat1Δgat2Δ+[pGAL1::AtGPAT1 LEU2]) was used to conduct genetic complementation verification. It was found that the growth of ZAFU1 could be rescued with heterologous expression of BnGPAT1-1, BnGPAT4-1, BnGPAT4-2 and BnGPAT9-1 induced by glucose, which indicated that the enzymes encoded by these genes might mimic the function of the yeast acyltransferase encoding gene YKR067w (named GAT1). The results of this study could provide reference data for the genetic improvement of oil content in B. napus.
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