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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。
Using Vaccinium L. variety “Hokuriku” as test material, full-length cDNA of c-glutathione S-transferase gene was successfully cloned by reverse transcription PCR, and a complete coding region c DNA sequence, which is 690 bp long and encodes 229 amino acids. It is named as VcGSTU1 (Gen Bank accession number KT601064). Through amino acid sequence alignment and phylogenetic analysis with other members of the glutathione S-transferase family, VcGSTU1 was initially identified as a glutathione S-transferase Tau family. Bioinformatics analysis showed that the protein instability index was 28.80, belong to the stable protein. The predicted molecular weight of the protein was 25.499 ku and the isoelectric point was 5.68. Homology alignment of the protein sequence revealed that it contained the complete GST-N-Tau and GST-C-Tau domains. The expression patterns of glutathione S-transferase were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative PCR (qRT-PCR) Fruit, blue fruit, blue fruit pericarp, blue fruit seeds were expressed, and the relative expression of blue fruit seeds the highest.