牡丹DGAT基因的克隆及表达分析

Cloning and Expression Analysis of DGAT Gene from Paeonia suffruticosa

利用RT-PCR和RACE技术,从牡丹种子中克隆得到1个二酰甘油酰基转移酶基因(DGAT),命名为PaDGAT1(GenBank登录号为MG214258)。PaDGAT1基因cDNA全长为2 028bp,包含1 554bp开放阅读框,编码517个氨基酸。PaDGAT1蛋白属于疏水性碱性蛋白,分子量为58.86kD,理论等电点为8.62,二级结构预测表明,无规则卷曲和延伸链是该蛋白的主要结构元件。氨基酸序列对比分析表明,PaDGAT1基因编码的蛋白属于DGAT1亚家族,与油橄榄(Olea europaea)的DGAT1蛋白亲缘关系最近。实时荧光定量分析结果表明,PaDGAT1基因在花芽中表达水平较高,在叶、茎和未发育子房中表达水平较低;在种子发育过程中,PaDGAT1基因表达水平呈现出升高-降低-升高的趋势,其中在发育28d时表达水平最高,随后其表达水平逐渐减低,在种子发育70d时又升高,至发育末期的85d时表达水平升高至较高水平;在种子收获后,常温存放7d时,PaDGAT1基因表达水平最高,随后其表达水平逐渐下降。结果推测,DGAT基因在牡丹种子的油脂合成中起重要的调控作用。

The diacylglycerol acyltransferase gene PaDGAT 1(The accession number in GenBank:MG214258)was cloned from seed of Paeonia suffruticosa by the method of RT-PCR and RACE.The full-length cDNA of PaDGAT 1 gene was 2 028 bp,with an open reading frame of 1 554 bp encoding 517 amino acids.The PaDGAT 1 gene encoding protein was a hydrophobic alkaline protein with molecular weight of 58.86 kD and the academic isoelectric point of 8.62.Prediction of secondary structure showed that the random coil and extended strand were the main structural elements of the protein.Amino acids sequences alignment analyses indicated that the amino acid sequence of PaDGAT 1 belongs to DGAT 1 subfamily,which was closed to DGAT1 from Olea europaea.The qRT-PCR analyses showed that the expression level of the PaDGAT 1 gene was the highest in bud,and was lower in leaf,stem and undeveloped ovary.During the seed developing,the expression level of PaDGAT 1 gene in developing seed showed a trend of increasing firstly,then decreasing and increasing again.The expression level of the PaDGAT 1 gene was the highest at 28 d in developing seed,then the level was decreased gradually,and was increased again at 70 d with a higher level at 85 d in developing seed.After harvest,the expression of the PaDGAT 1 gene was increased with the highest level in seed under room temperature after harvest for 7 d,then it was decreased gradually.The results implied that the PaDGAT 1 gene play an important role in the lipid synthesis during seed developing and maturing.