冷胁迫下王百合GPAT基因保守区克隆及表达分析

Glycerol-3-Phosphate Acyltransferase Activity in Lilium regale and Cloning of Its Conserved cDNA Region under Cold Stress

以王百合为试验材料,通过同源克隆和巢式PCR方法从4℃低温诱导的王百合试管苗中分离得到了王百合GPAT基因的保守区序列,采用DNAman软件和BLASTN对该序列进行分析并分别从蛋白和基因角度分析了GPAT基因在4℃冷诱导情况下的表达情况.结果显示:(1)该保守区长744 bp,推测其编码247个氨基酸,氨基酸序列存在1个高度保守的区域(WIAPSGGRDRP),经过Blast比对分析发现,该保守区序列为LPLAT基因超级家族酶类的催化活性区,此家族多为催化酰基辅酶A(acylCoAs)或者酰基载体蛋白(acylACPs)中的酰基与受体蛋白结合的酰基转移酶类.(2)冷诱导促进GPAT基因的表达,随冷诱导时间延长,基因表达量不断增大,诱导4 h有大量表达,16 h表达量达到最高,16 h之后表达量随着冷诱导时间的延长逐渐下降,72 h时的表达量与0 h处理时基本一致.研究表明,GPAT在百合抵抗冷胁迫的过程中具有重要的作用.

With Lilium regale as material,a glycerol-3-phosphate（GPAT） gene conservative district sequence was cloned from the tissue culture regeneration plantlets of L.regale by the method of homology cloning and nested PCR.The sequence analysis and homology comparison of the cDNA were performed by using DNAman software and BLASTN program,then the expression of glycerol-3-phosphate（GPAT） gene under cold stresses at 4℃ was detected from protein and gene level.The results demonstrated that the conserved sequence was 744 bp in length,and coded 247 amino acids.There is a highly conserved domain（WIAPSGGRDRP） in the amino acid sequence of GPAT.Aligning the conserved domain with Blast,GPAT had a catalytic domain of LPLAT superfamily enzymes.This family mostly catalyzed acyl-coenzyme A（acylCoAs）or acyltransferase involved in the Acyl carrier protein（acylACPs） binding and receptor protein with acy1.The expression analysis of GPAT gene showed that low temperature could induce the expression of the gene in a short time.With the prolonged cold induction,the expression increased firstly and then decreased,a lot of expression were found after 4 h,and which peaked at 16 h,than down gradually,and at 72 h basically the same level in keeping with 0 h.The research showed that GPAT gene plays an important role in the process of lily resistance cold stress.