四倍体小麦Langdon HMT1基因的克隆及原核表达

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。

Cloning and Prokaryotic Expression Analysis of HMT1 Gene in Tetraploid Wheat Langdon

Selenium is one of the most important essential trace elements for humans and enough selenium must be supplemented to ensure body health. Homocysteine-S-methyltransferase(HMT) is homologous to selenocysteine methyltransferase(SMT),which is the key enzyme to enrich selenium for plants. In order to explore and use the key enzyme genes of selenium-rich in wheat crop,we designed especial primers H1-F/H1-R to clone the open reading frame(ORF) of the HMT1 in tetraploid wheat Langdon,based on the sequence of homocysteine-S-methyltransferase 1 gene from Aegilops tauschii Coss.( AetHMT1) from NCBI and finally verified through the method of prokaryotic expression. The results showed that two sequences with the same size of 975 bp from Langdon(LDN) were cloned,respectively,named LDNHMT1-1 and LDNHMT1-2. Like the AetHMT1,the LDNHMT1-1 and LDNHMT1-2 genes both encode 342 amino acids,with the molecular weight of 35.19 kDa and 35.18 kDa,respectively. The LDNHMT1-1 and LDNHMT1-2 have high similarity with other HMTs and SMTs,based on amino acid sequence alignment and phylogenetic tree analysis. We successfully gained the prokaryotic expression strains of LDNHMT1-1 and LDNHMT1-2 by the way of constructing the prokaryotic expression vector and SDS-PAGE. The protein molecular weight of prokaryotic expression was consistent with the predicted one.