Cloning and characterization of diacylglycerol acyltransferase (DGAT) cDNA sequence from Brassica juncea cv. Pusa Bold

Abstract

Diacylgycerol acyltransferase (DGAT: EC 2.3.1.20) is the only enzyme in the Kennedy pathway that is exclusively committed to the synthesis of storage oil in plants. In this study, cloning and characterization of DGAT gene sequence from Brassica juncea cv Pusa bold, an important oil seed crop of India is reported. A partial gene sequence of 2003 bp was PCR amplified and cloned from B. juncea. Sequence analysis showed that it has 10 exonic and 9 intronic sequences in the partial gene. Two cDNA sequences namely BjDGAT 1 and BjDGAT 2 (1.5 kb) encoding DGAT enzymes were amplified by RT-PCR from the developing seeds. The complete length of these two cDNAs as determined by RACE technique was 1768 bp, including 5’ and 3’-UTR. Comparative analysis of the sequences showed that BjDGAT1 was 85.1% and 96% identical to BjDGAT2 across 1512 coding region and 503 overlapping deduced amino acids respectively. These proteins were alkaline in nature (pI, 8.5-8.6), having similar molecular size (56-57 kD), an N-terminal hydrophilic segment and 9 transmembrane segments. Diacylglycerol/phorbol ester-binding motif (HKWXXRHXYXP) and acyl CoA binding motif (FYXDWWN) required for binding of substrates remained conserved in these proteins. Expression of the two transcripts of DGAT and their role in oil biosynthesis can further be studied.