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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。
Selenium is one of the important trace elements indispensable to mankind. Mankind must be supplemented with enough selenium to ensure good health. Homocysteine-S-methyltransferase (HMT) gene is homologous to selenocysteine methyltransferase (SMT), a plant-rich selenocysteine methyltransferase (SMT) gene. In order to explore and utilize the key enzyme genes related to selenium enrichment in wheat crops, the HMT1 gene was designed based on the published sequence of homology cysteine methyltransferase 1 (AetHMT1) from NCBI published Aegilops tauschii Coss. The sequence was analyzed after the open reading frame (ORF) of the cloned tetraploid Wheat H1-F / H1-R, and verified by prokaryotic expression. The results showed that two sequences of 975 bp in length were obtained from the tetraploid wheat Langdon (LDN) by H1-F / H1-R and named LDNHMT1-1 and LDNHMT1-2, respectively. LDNHMT1-1 and LDNHMT1-2, like the AetHMT1 gene, all encoded 342 amino acids with molecular weights of 35.19 kDa and 35.18 kDa, respectively. By amino acid sequence alignment and phylogenetic tree analysis, LDNHMT1-1, LDNHMT1-2 and other HMT, SMT have a higher similarity. The prokaryotic expression vector was constructed, and prokaryotic expression strains of LDNHMT1-1 and LDNHMT1-2 were successfully obtained by SDS-PAGE, and the prokaryotic expressed protein was consistent with the predicted molecular weight.