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谷胱甘肽-S-转移酶(glutathione S-transferases,GSTs)是植物抗氧化系统的重要成员,在初生代谢、次生代谢、细胞信号转导及逆境响应等生物学过程中发挥着重要作用。本研究以Gen Bank中公布的玉米谷胱甘肽-S-转移酶23基因(ZmGST23)m RNA序列(Gen Bank登录号:NM_001111524)为依据,采用RT-PCR方法从玉米(Zea mays)自交系F83中克隆得到ZmGST23基因669 bp的完整开放阅读框(open reading frame,ORF),编码222个氨基酸,蛋白分子量为24.84 k D,理论等电点为5.68。保守结构域分析表明,该基因具有GSTs所特有的N端及C端结构域,并含有典型的G位点及H位点,属于Tau类谷胱甘肽硫转移酶。系统进化分析表明,ZmGST23编码蛋白与高粱(Sorghum bicolor)GST蛋白亲缘关系最近,且在不同物种间存在明显的种属特性。启动子序列分析结果显示,ZmGST23基因启动子区含有多个响应逆境及植物激素的作用元件,包括脱落酸(abscisic acid,ABA)响应元件、生长素(auxin,IAA)响应元件、赤霉素(gibberellin,GA)响应元件、厌氧响应元件、防御及逆境响应元件以及干旱诱导的MYB转录因子(v-myb avian myeloblastosis viral oncogene homolog,MYB)结合位点。胁迫诱导表达分析表明,ZmGST23的表达显著受干旱脱水、水涝、盐、ABA、IAA、GA、低温及高温等非生物胁迫的诱导。组织特异性表达分析表明,ZmGST23基因在幼芽和成熟叶片中表达量较高,在幼根、花丝、苞叶、雌穗及雄穗中表达量较低,存在明显的组织特异性。将ZmGST23基因片段连接至原核表达载体p EASY-E1中,转化大肠杆菌(Escherichia coli)BL21,用0.5 mmol/L的异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达1~4 h后获得30 k D的诱导蛋白。本研究结果为进一步利用该基因进行玉米抗性改良提供了一定的理论依据。
Glutathione-S-transferases (GSTs) are important members of antioxidant systems in plants and play an important role in biological processes such as primary metabolism, secondary metabolism, cell signaling and stress response . In this study, the maize glutathione-S-transferase 23 (ZmGST23) m RNA sequence (Gen Bank Accession No. NM_001111524) published in Gen Bank was used as a marker for selfing from Zea mays The complete open reading frame (ORF) of ZmGST23 gene was cloned from F83, encoding a 222 amino acid protein with a molecular weight of 24.84 kD and a theoretical isoelectric point of 5.68. Conserved domain analysis showed that the gene has the N-terminal and C-terminal GTP-specific domains, and contains typical G sites and H sites, belonging to the Tau glutathione S-transferase. Phylogenetic analysis showed that the ZmGST23 protein was the closest to Sorghum bicolor GST protein and had obvious species characteristics among different species. Promoter sequence analysis showed that the promoter region of ZmGST23 gene contained multiple response elements and plant hormones, including abscisic acid (ABA) response element, auxin (IAA) response element, gibberellin gibberellin, GA) response elements, anaerobic response elements, defense and stress response elements, and the v-myb avian myeloblastosis viral oncogene homolog (MYB) binding sites. Stress induced expression analysis showed that the expression of ZmGST23 was significantly induced by abiotic dehydration, waterlogging, salt, ABA, IAA, GA, low temperature and high temperature. Tissue-specific expression analysis showed that the expression level of ZmGST23 gene was higher in young shoots and mature leaves, and lower in young shoots, filaments, bract, ear and tassel. There was obvious tissue specificity. The ZmGST23 gene fragment was ligated into prokaryotic expression vector pEASY-E1, transformed into Escherichia coli BL21 and induced with 0.5 mmol / L isopropyl β-D-1-thiogalactopyranoside (IPTG) After 1 to 4 h of expression, a 30 kD induced protein was obtained. The results of this study provide some theoretical basis for further utilization of the gene for improvement of maize resistance.