甘蔗Ca^(2+)/H^+反向运转体基因的克隆与表达分析

Cloning and Expression Analysis of a Ca^(2+)/H^+ Antiporter Gene from Sugarcane

CAX(Ca^(2+)/H^+antiporter)是植物细胞膜Ca^(2+)主动运输体系的一个大类。本研究以高粱的CAX1基因(GenBank登录号为XM_002441593)为探针,利用电子克隆并结合RT-PCR技术,获得甘蔗CAX1基因的1条cDNA序列,命名为Sc CAX1(GenBank登录号为KT799799)。生物信息学分析显示,ScCAX1基因全长784 bp,包含1个645 bp的开放阅读框,编码1个214个氨基酸的蛋白质。ScCAX1蛋白被定位于叶绿体类囊体膜,为稳定的疏水性蛋白,不存在信号肽。蛋白二级结构元件多为α-螺旋,具有1个Na_Ca_ex superfamily。实时荧光定量PCR分析表明,甘蔗ScCAX1基因的表达具有组织特异性,在各组织中均表达,但在茎中表达量最低,叶中的表达量最高。在PEG、NaCl、SA、ABA和Me JA胁迫过程中,ScCAX1基因的表达均受到调控。其中ABA、SA和PEG胁迫下表达量上调,均在胁迫24 h达到最大值。SA胁迫24 h的表达量为对照的5.47倍,而ABA胁迫24 h的表达量为对照的3.5倍。NaCl胁迫6 h的表达量达最大值,为对照的2.14倍。推测ScCAX1基因能够响应逆境胁迫,其表达可能与甘蔗的抗盐、抗渗透胁迫性状有关。

CAX（Ca^2＋/H^＋ antiporter） is a major category of Ca^2＋ active transport systems in plant cell membrane. In the present study, using a CAX1 m RNA sequence from Sorghum bicolor（GenBank accession number： XM_002441593） as the probe, the full-length cDNA sequence of sugarcane CAX1 gene was cloned by in silico cloning combined with RT-PCR amplification, and named as ScCAX1（Gen Bank accession number： KT799799）. Bioinformatics analysis showed that ScCAX1 has a length of 784 bp and contains a complete open reading frame with a length of 645 bp, which encodes a 214 amino acid residues of sugarcane CAX1 protein. The ScCAX1 protein with stable acidity and hydrophobia was detected to be located in thylakoid membrane of chloroplasts with no signal peptide. It belongs to a conserved Na_Ca_ex. The mainly secondary structure element of ScCAX1 protein is alpha helix. Real time quantitative PCR（RT-q PCR） analysis revealed that the expression of Sc CAX1 was tissue-specific, with constituent expression in different tissues of sugarcane. The highest expression was observed in leaf while the lowest in stem. Besides, the expression of ScCAX1 gene could be regulated by treatments of PEG, NaCl, SA, ABA, and Me JA. The expression level of this gene was up-regulated by ABA, SA and PEG, with the highest inducible expression level in treatment of 24 hours. The expression level was 5.47 times higher than that of control under 24 hours stress of SA, and 3.5 times higher than that of control under 24 hours stress of ABA. Under 6 hours stress of NaCl, the gene had the highest inducible expression level, which was 2.14 times higher than that of control. This study suggested that ScCAX1 could response to stresses, and its expression may be associated with salt resistance and osmotic tolerance in sugarcane.