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[目的]研究玉米Na~+/H~+逆向转运蛋白基因ZmSOS1的克隆与鉴定,为研究该基因在玉米逆境信号转导与生长发育中的作用提供参考。[方法]采用电子克隆、RT-PCR、生物信息学方法,从玉米基因组中克隆1个质膜型Na~+/H~+逆向转运蛋白基因,并鉴定该基因编码产物的跨膜结构预测以及在盐胁迫下的表达模式等信息。电子克隆具体步骤:将水稻质膜型Na~+/H~+逆向转运蛋白OsSOS1的氨基酸序列作为种子序列输入GenBank,对玉米基因组数据库和EST数据库进行tBLASTN分析,结果检索到含有候选基因的基因组序列AC186524和1批高度相似的玉米EST。为确定候选基因的编码区,将上述EST序列进行拼接,并将拼接所得的contig通过BLASTN分析定位到基因组序列AC186524中。为获得完整的编码区序列,利用与contig间隔区相对应的水稻OsSOS1蛋白的氨基酸序列,进一步对玉米基因组序列进行tBLASTN分析;同时,结合GENSCAN软件的基因预测结果,最终确定了候选基因的编码区序列。最后,通过比较编码区序列和基因组序列,确定ZmSOS1基因的外显子和内含子结构。[结果]利用电子克隆方法,从玉米中克隆出1个质膜型Na~+/H~+逆向转运蛋白基因ZmSOS1(编码序列反向互补于AC186524中的78 964～96 731 bp处);ZmSOS1基因的开放阅读框长3 411 bp,编码1 136个氨基酸的蛋白。ZmSOS1蛋白的理论等电点pI=6.46,分子质量为126.2kDa,理论半衰期大于10 h,不稳定参数为41.74,属于不稳定蛋白。通过在线软件TMHMM2.0和TMPRED分析,发现ZmSOS1蛋白含有12个跨膜结构区,且有一个长的高度亲水性的羧基端尾巴,暗示质膜型Na~+/H~+逆向转运蛋白在进化上是较为保守的。ZmSOS1基因至少含有21个内含子,如果将ZmSOS1基因的结构分别与水稻、拟南芥和苔藓植物(Phycomitrella patens)的同源基因OsSOS1、AtSOS1、PpSOS1相比较,可以发现质膜型Na~+/H~+逆向转运蛋白基因编码区的结构是比较保守的且研究发现质膜型Na~+/H~+逆向转运蛋白基因在进化过程中很可能发生过内含子丢失事件;序列分析表明,ZmSOS1蛋白与拟南芥和水稻同源物AtSOS1、OsSOS1的氨基酸序列一致性分别为61%和82%,鉴于ZmSOS1的氨基酸序列与同源序列的多重比对,猜想质膜型Na~+/H~+逆向转运蛋白在进化过程中,N-端序列可能承受着较为严谨的净化选择压作用,而C-端序列所受净化选择压则相对松弛;RT-PCR分析表明,ZmSOS1基因的表达可以被盐胁迫诱导增强,表明其可能在玉米耐盐性上发挥功能。[结论]ZmSOS1基因可能参与玉米的渗透胁迫反应。
[Objective] The research aimed to study the cloning and identification of ZmSOS1, a maize Na + / H + antiporter gene, and provide references for studying the role of ZmSOS1 in maize stress signal transduction and growth. [Method] A plasma membrane type Na ~ + / H ~ + antiporter gene was cloned from maize genome by electronic cloning, RT-PCR and bioinformatics methods. The transmembrane structure of the gene was identified and Under salt stress expression patterns and other information. Specific steps of electronic cloning: The amino acid sequence of rice plasma membrane type Na ~ + / H ~ + antiporter OsSOS1 was input into GenBank as a seed sequence, and tBLASTN was performed on the maize genomic database and EST database. As a result, the genomic sequence containing the candidate gene AC186524 and 1 batch of highly similar corn EST. To determine the coding region of the candidate gene, the above EST sequences were spliced and the spliced contig was mapped to the genomic sequence AC186524 by BLASTN analysis. In order to obtain the complete coding region sequence, the amino acid sequence of rice OsSOS1 corresponding to the contig spacer region was further used for tBLASTN analysis of the maize genomic sequence. At the same time, with the gene prediction result of GENSCAN software, the coding region of the candidate gene was finally determined sequence. Finally, the exon and intron structures of ZmSOS1 gene were determined by comparing coding region sequences and genomic sequences. [Result] A plasmids Nam + / H + antiporter gene ZmSOS1 was cloned from maize by using the method of electronic cloning (the coding sequence was reverse complementary to 78 964-96 731 bp in AC186524). ZmSOS1 The open reading frame of the gene is 3 411 bp, encoding a protein of 1 136 amino acids. The theoretical isoelectric point of ZmSOS1 protein is pI = 6.46, the molecular mass is 126.2 kDa, the theoretical half-life is more than 10 h, and the instability parameter is 41.74, which belongs to the unstable protein. The online software TMHMM2.0 and TMPRED analysis showed that the ZmSOS1 protein contained 12 transmembrane domains and had a long, highly hydrophilic carboxy-terminal tail, suggesting that the plasma membrane Na ~ + / H ~ + antiporter Evolutionary is more conservative. ZmSOS1 gene contains at least 21 introns. If the structure of ZmSOS1 gene is compared with the homologous genes OsSOS1, AtSOS1 and PpSOS1 of rice, Arabidopsis thaliana and bryophyte (Phycomitrella patens) The structure of the coding region of / H ~ + antiporter gene is relatively conservative and it is found that the intron loss event may occur during the evolution of the plasma membrane type Na ~ + / H ~ + antiporter gene. Sequence analysis showed that The amino acid sequence identities of ZmSOS1 protein and Arabidopsis thaliana and rice homologues AtSOS1 and OsSOS1 were 61% and 82%, respectively. Given the multiple alignments of ZmSOS1 amino acid sequences with homologous sequences, In the process of evolution, the N-terminal sequence of H ~ + antiporter may undergo a more stringent purifying selection pressure, while the C-terminal sequence is subjected to a relatively relaxed purifying selection pressure. RT-PCR analysis showed that the expression of ZmSOS1 gene It can be induced by salt stress, indicating that it may play a role in salt tolerance of maize. [Conclusion] The ZmSOS1 gene may be involved in osmotic stress response in maize.