油葵油脂合成关键酶基因DGAT2的克隆及烟草遗传转化

Cloning of a lipid synthesis-related gene DGAT2 from sunflower and transformation into tobacco

为了研究油葵DGAT2基因的功能,利用RT-PCR结合RACE技术,从"新葵杂5号"未成熟种子中克隆了1个二酰基甘油酰基转移酶(diacylglycerol acyltransferase,DGAT)基因HaDGAT2。分析表明该基因cDNA全长1387 bp(GenBank登录号为HQ248185),包含993 bp的开放阅读框,编码330个氨基酸;氨基酸比对表明该基因编码产物与其他植物DGAT2蛋白序列的相似性较高(66%-71%);进化树分析表明HaDGAT2与油桐和蓖麻DGAT2亲缘关系较近,并与蒺藜苜蓿、拟南芥、油菜等植物聚为一支;半定量RT-PCR分析显示,HaDGAT2在油葵根、叶、花和未成熟种子中均有表达,且在未成熟种子中表达量最高。在烟草中过量表达HaDGAT2基因能提高烟草叶片含油量以及油酸(C18:1)、亚油酸(C18:2)的含量,推测HaDGAT2在调控TAG的积累过程中起关键作用。

The purpose of this study is to study the function of sunflower DGAT2 gene.A gene(named HaDGAT2) encoding diacylglycerol acyltransferase was amplified from the tissue of developing seeds of Helianthus annuus L.Xinza 5 by reverse transcription polymerase chain reaction(RT-PCR) and(rapid amplification of the cDNA end) RACE methods.The full-length cDNA of HaDGAT2 was 1387 bp containing a 993 bp open reading frame(ORF) which encoded a polypeptide of 330 amino acids.The amino acids alignment indicated that HaDGAT2 shared high identity with plant DGAT2 protein(66%-71%),and phylogenetic tree analysis showed that HaDGAT2 has a close kinship with the Ve.rnicia fordii and Ricinus communis and belongs to the same branch with Medicago truncatula,Arabidopsis thaliana and Brassica napus.Semi-quantitative RT-PCR analysis showed that HaDGAT2 transcript was detectable in the roots,leaves,flowers and developing seeds,but was highest in the developing seeds.Compared with wild type plants,the transgenic tobacco plants exhibited a significant increase in leaf crude lipid,oleinic acid and linoleic acid contents.