四倍体小麦Langdon HMT1基因的克隆及原核表达

发布时间：2018-05-17 20:05

本文选题：四倍体小麦Langdon + 硒　；参考：《麦类作物学报》2017年09期

【摘要】：硒是人类不可缺少的重要微量元素之一,人类必须补充足够的硒才能保证身体健康。同型半胱氨酸甲基转移酶(homocysteine-S-methyltransferase,HMT)基因与植物富硒关键酶硒代半胱氨酸甲基转移酶(selenocysteine methyltransferase,SMT)基因同源。为了发掘并利用麦类作物中的富硒关键酶基因,基于NCBI已公布的节节麦(Aegilops tauschii Coss.)同型半胱氨酸甲基转移酶1基因(AetHMT1)的序列设计HMT1基因的特异性引物H1-F/H1-R,克隆四倍体小麦Langdon HMT1的开放阅读框(open reading frame,ORF)后进行序列分析,并通过原核表达验证该基因。结果表明,利用H1-F/H1-R从四倍体小麦Langdon(LDN)克隆出两条长度均为975bp的序列,分别命名为LDNHMT1-1和LDNHMT1-2。LDNHMT1-1、LDNHMT1-2与AetHMT1基因一样,均编码342个氨基酸,分子量大小分别为35.19kDa和35.18kDa。通过氨基酸序列比对和进化树分析发现,LDNHMT1-1、LDNHMT1-2与其他HMT、SMT有较高的相似性。构建原核表达载体,并通过SDS-PAGE鉴定,成功获得LDNHMT1-1、LDNHMT1-2的原核表达菌株,且原核表达的蛋白质与预测分子量大小一致。
[Abstract]:Selenium is one of the most important trace elements in human being. Homocysteine methyltransferase homocysteine-S-methyltransferase (HMT) gene is homologous to the selenocysteine methyltransferase (SMT) gene of plant selenocysteine methyltransferase. In order to explore and utilize selenium-rich key enzyme genes in wheat crops, Aegilops tauschii Coss.of NCBI has been published. The sequence of homocysteine methyltransferase 1 gene (AetHMT1) was designed as the specific primer of HMT1 gene H1-F / H1-R. the open reading frame of tetraploid wheat Langdon HMT1 was cloned and sequenced. The gene was confirmed by prokaryotic expression. The results showed that H1-F/H1-R was used to clone two 975bp sequences from tetraploid wheat Langdonn.It was named LDNHMT1-1 and LDNHMT1-2.LDNHMT1-1 LDNHMT1-2 encoded 342 amino acids, and their molecular weights were 35.19kDa and 35.18kDa, respectively. Amino acid sequence alignment and phylogenetic tree analysis showed that LDNHMT1-1 and LDNHMT1-2 were similar to other HMT-SMT. The prokaryotic expression vector was constructed and identified by SDS-PAGE. The prokaryotic expression strain of LDNHMT1-1 and LDNHMT1-2 was successfully obtained, and the prokaryotic expression protein was consistent with the predicted molecular weight.
【作者单位】：中国科学院西北高原生物研究所;中国科学院大学;青海省作物分子育种重点实验室;四川农业大学小麦研究所;
【基金】：国家自然科学基金项目(31101140) 中国科学院战略性A类先导科技专项子课题(XDA08030106);中国科学院西部之光一般项目青海省重点研发与转化计划项目(2016-HZ-808) 青海省高原作物种质资源创新与利用国家重点实验室培育基地开放课题(2014-09)
【分类号】：S512.1

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