忽地笑酌-生育酚甲基转移酶基因LaTMT的克隆与表达分析

摘要

采用RACE技术从忽地笑也Lycoris aurea ( L'Hér.) Herb.页叶片中克隆获得γ-生育酚甲基转移酶(γ-TMT)基因,命名为LaTMT。序列分析结果显示：该基因cDNA全长1458 bp,其中开放阅读框( ORF)长1017 bp,编码338个氨基酸残基。 LaTMT基因编码蛋白质的理论相对分子质量37560,理论等电点pI 8.70,为亲水性蛋白,无跨膜结构但具有信号肽结构；并具有S-腺苷甲硫氨酸( SAM)甲基转移酶保守结构域,包含3个SAM结合位点；该蛋白的二级结构中包含44.08%的α-螺旋、32.84%的无规则卷曲、12.72%的延伸链和10.36%的β-转角。序列比对和系统进化树分析结果显示：LaTMT蛋白属于S-腺苷甲硫氨酸-依赖性γ-生育酚甲基转移酶家族,与其他植物γ-TMT蛋白的一致性为64%~75%；在 NJ系统树上, LaTMT蛋白与单子叶植物γ-TMT蛋白聚为同一大类,并与油棕( Elaeis guineensis Jacq.) EgTMT和美洲油棕也Elaeis oleifera ( Kunth) Cortés页EoTMT聚为同一类,亲缘关系最近。基因表达分析结果显示：LaTMT基因可在大肠杆菌中成功表达,且表达量随异丙基硫代半乳糖苷( IPTG)诱导时间的延长而增加；在忽地笑的根、叶片、花苞、子房、雄蕊、花瓣和鳞茎中LaTMT基因均可表达,其中在叶片中的相对表达量最高,在子房、雄蕊和鳞茎中的相对表达量相对较低,具有明显的组织特异性。研究结果表明：忽地笑LaTMT基因在进化过程中具有很高的保守性；该基因主要定位于叶绿体中,并与忽地笑对非生物逆境胁迫的抗性相关。%Using RACE method,γ-tocopherol methyltransferase (γ-TMT) gene was cloned from leaf of Lycoris aurea ( L'Hér.) Herb., which was named LaTMT. The result of sequence analysis shows that full-length cDNA of LaTMT gene is 1 458 bp, in which, length of open reading frame ( ORF) is 1 017 bp, and 338 amino acid residues are encoded. Theoretical relative molecular mass of the protein encoded by LaTMT gene is 37 560 , theoretical isoelectric point is pI 8 . 70 , which is a hydrophilic protein without transmembrane structure but with signal peptide structure. And this protein has S-adenosylmethionine ( SAM) methyltransferase conserved domain and includes three SAM binding sites. Its tertiary structure includes 44. 08% of α-helix, 32. 84% of random coil, 12. 72% of extended strand and 10. 36% of β-turn. The analysis results of sequence alignment and phylogenetic tree show that LaTMT protein belongs to S-adenosylmethionine-dependent γ-tocopherol methyltransferase family, and its identity with γ-TMT protein from other plants is 64%-75%. In NJ phylogenetic tree, LaTMT protein andγ-TMT protein from monocotyledon are clustered into the same large category, and this protein with EgTMT of Elaeis guineensis Jacq. and EoTMT of Elaeis oleifera ( Kunth) Cortés is clustered into the same category, their relationship is close. The analysis result of genetic expression shows that LaTMT gene can express successfully in Escherichia coli, and its expression increases with prolonging of inducing time of isopropyl thiogalactoside ( IPTG) . Also, LaTMT gene can express in root, leaf, bud, ovary, stamen, petal and bulb of L. aurea, in which, relative expression is the highest in leaf, and that is relative low in ovary, stamen and bulb, with obvious tissue specificity. It is suggested that LaTMT gene from L. aurea has high conservation in evolutionary process, the gene is mainly located in chloroplast and is related to resistance of abiotic adversity stress.