越橘谷胱甘肽巯基转移酶基因的克隆及表达

Cloning and Expression Analysis of Glutathione S-transferase Gene in Blueberry

以越橘(Vaccinium L.)品种"北陆"为试材,采用反转录PCR克隆技术成功克隆越橘谷胱甘肽巯基转移酶基因c DNA全长序列,获得一个完整的编码区c DNA序列,长为690 bp,编码229个氨基酸,将其命名为VcGSTU1(Gen Bank登录号KT601064)。经与谷胱甘肽巯基转移酶家族其他成员的氨基酸序列比对和系统进化分析,初步确定VcGSTU1属于谷胱甘肽巯基转移酶Tau家族。生物信息学分析表明,该蛋白不稳定指数为28.80,属于稳定蛋白。预测该蛋白分子量为25.499 ku,等电位点为5.68。蛋白质序列同源比对发现其含有完整的GST-N-Tau和GST-C-Tau结构域。利用反转录PCR和实时荧光定量PCR(qRT-PCR)2种方法分析谷胱甘肽巯基转移酶的表达模式,该基因在越橘的根、茎、叶芽、叶、花芽、绿果、粉果、蓝果、蓝果果皮、蓝果种子中均有表达,且在蓝果种子中相对表达量最高。

A full length of c DNA encoding glutathione S-transferase（ GST） gene was cloned from blueberry cultivar ＂Northland＂by reverse transcription PCR（ RT-PCR） technology. It was named VcGSTU1. The accession number of VcGSTU1 in Gen Bank is KT601064. Sequence analysis showed that the length of VcGSTU1 was 690 bp and contained a single open reading frame of 690 bp. The predicted VcGSTU1 protein had 229 amino acids with an estimated molecular mass of 25. 499 ku and an isoelectric point of 5. 68. The instability index（ Ⅱ） of VcGSTU1 was computed to be 28. 80.This classifies the protein as stable. Protein alignment showed that they contained the complete GST_N_Tau and GST_C_Tau domains. Expression analysis by RT-PCR and quantificational RT-PCR（ qRT-PCR） showed that VcGSTU1 expressed in different organs of blueberry,including root,stem,leaf bud,leaf,flower bud,green fruit,pink fruit,blue fruit peel and seed. Its expression level in mature seed was the highest.