黄花草木樨MoSOS1基因克隆及表达分析

Cloning and Expression Analysis of MoSOS1 Gene in Melilotus officinalis

植物质膜Na+/H+逆向转运蛋白基因SOS1是植物耐盐性必需的基因之一,在抵御盐胁迫过程中发挥十分重要的作用。以黄花草木樨叶片总RNA为模板,通过RT-PCR结合RACE方法克隆得到黄花草木樨MoSOS1基因全长序列,命名为MoSOS1。序列分析表明该基因全长为3 931 bp,开放阅读框(ORF)为2 874 bp,编码957个氨基酸,分子量为112.8 k D,等电点为5.31。TMHAM软件跨膜区的预测分析表明,黄花草木樨MoSOS1蛋白具有8个跨膜结构区域,N端和C端都位于细胞外。氨基酸序列分析表明,MoSOS1蛋白含有1个Na+/H+Exchanger superfamily和一个c NMP(Cyclic nucleotide-monophosphate)结合位点以及1个CAP_ED(Catabolite gene activator protein-effector domain)superfamily结构域。生物信息预测显示,MoSOS1的编码蛋白为不稳定酸性蛋白,不存在信号肽,二级结构多为α-螺旋和无规则卷曲。荧光实时定量RT-PCR分析表明:随着Na Cl浓度的增加,黄花草木樨地上部和根中MoSOS1基因表达水平呈增加趋势,根中表达量大于地上部,表明MoSOS1基因的表达受盐胁迫诱导和调节。

Plant salt overly sensitive 1（SOS1）gene,encoding a Na＋/H＋ anti-port protein,plays an important role in biologicalprocesses of plants against salt stress. Using the total RNA extracted from the young leaves of Melilotus officinalis（L.）Lam. as the template,the full-length sequence of SOS1 gene was amplified by the reverse transcription polymerase chain reaction（RT-PCR）method and rapidamplification of c DNA ends（RACE）,named MoSOS1. The bioinformatics analysis showed that MoSOS1 was in a length of 3931 bp with acomplete open reading frame（ORF 2 874 bp）,encoding a 957 amino acid residues of MoSOS1 protein with an estimated molecular weight of112.8 k D and a calculated isoelectric point（p I）of 5.31. The prediction of transmembrane region of MoSOS1 by TMHAM software indicatedthat MoSOS1 had 8 transmembrane regions and N-terminal and C-terminal were located outside the cell. Amino acid alignment analysisdemonstrated that the protein of MoSOS1 contained a conserved Na＋/H＋ exchanger superfamily,a c NMP（Cyclic nucleotide-monophosphate）and a CAP_ED（Catabolite gene activator protein-effector domain）superfamily. The bioinformatics analysis showed that MoSOS1 proteinbelonged to unstable acidic protein with mainly secondary structure of α spiral and random coil,and in which there was no signal peptide.Quantitative real-time RT-PCR analysis showed that expression levels of MoSOS1 presented an increase trend in both shoots and roots with theincrease of external Na Cl concentrations,and its levels in roots were higher than those in shoots,indicating that the expression of MoSOS1 wasinduced and regulated by salt.