Expression of Gene Related to Oil Content in Transgenic Tobacco

Diacylglycerol acyltransferase(DGAT,EC 2.3.1.20) is a key enzyme in triacylglycerol(TAG) biosynthesis.In this work,the sequence of DGAT from sunflower(Helianthus annuus L.) was cloned by reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE) which named HaD1(GenBank accession No.HM 015632).The expression plasmid pBI-HaD1 was constructed by fusing the cDNA of HaD1 with the constitutive promoter CaMV 35S and introduced into tobacco(Nictiana tabacum) by Agrobacterium tumefaciens mediated transformation.Transgenic assays were performed using GUS activity and PCR analysis.At the same time,the composition and contents of fatty acids in leaves of transgenic tobacco were determined by GC-MS.The results showed that the length of HaD1 cDNA was 1 936 bp,which include an open reading frame(ORF) with 1 524 bp encoding 507 amino acids.The deduced amino acids sequence of HaD1 had 70%~80% identity with the reported DGAT1 proteins of other plants.A putative diacylglycerol-binding motif 'HKWIVRHLYFP' wihich is unique to DGAT1,was observed in HaD1 protein indicated that HaD1 belongs to the DGAT1 gene family.The HaD1 gene was confirmed to be integrated into the genome of tobacco by GUS and PCR analysis.The leaves of transgenic tobacco contained higher amount of oleic acid(C18∶1),palmitic acid(C16∶0) and stearic acid(C18∶0) than those of the untransformed plants.HaD1 gene is important in plant oil biosynthesis.