甘蔗Na^+/H^+逆转运蛋白基因的克隆与表达分析

Isolation and Characterization of a Na^+/H^+ Antiporter Gene from Sugarcane

Na^+/H^+逆向转运蛋白基因SOS1(salt overly sensitive 1)是植物耐盐性的必需基因之一,在植物抵御盐胁迫过程中发挥十分重要的作用。本研究以小麦EST序列KJ563230为探针,利用电子克隆技术结合RT-PCR,获得一条甘蔗SOS1基因的cDNA序列,命名为ScSOS1(GenBank登录号为KT003285)。序列分析结果表明,该基因全长1403 bp,包含一个1272 bp的开放阅读框,编码423个氨基酸的蛋白质。ScSOS1蛋白的相对分子质量为47.6 kD,理论等电点(pI)为9.12。氨基酸序列分析表明,ScSOS1蛋白具有一个CAP-ED superfamily结构域。生物信息学预测显示,ScSOS1的编码蛋白为亲水性蛋白,不存在信号肽,二级结构元件多为无规则卷曲,主要参与中间代谢。实时荧光定量PCR分析表明,ScSOS1基因的表达具有组织特异性,在甘蔗叶鞘、蔗皮、蔗髓、侧芽和根中均有表达,其中在叶鞘中的表达量最高,根中的表达量最低。此外在NaCl、PEG、ABA、SA和MeJA的胁迫过程中,该基因表达均受到调控,其中受NaCl和PEG诱导后上调表达,均在24 h表达量达最高,分别约为对照组的1.5倍和4.0倍。推测该基因的表达与甘蔗耐盐性和抗渗透胁迫有关。

Salt overly sensitive 1（SOS1） gene, encoding a Na^＋/H^＋ antiport protein, plays an important role in biological processes of plants against salt stress. Using a SOS1 m RNA sequence from Triticum aestivum（KJ563230） as the probe, the homologous ESTs of sugarcane were obtained from NCBI database. A sugarcane c DNA sequence of SOS1 gene was cloned by in silico cloning combined with RT-PCR, and named as Sc SOS1（Gen Bank accession number： KT003285）. The bioinformatics analysis showed that Sc SOS1 has a length of 1403 bp with a complete open reading frame（ORF, 107 to 1423 bp）, encoding a 423 amino acid residues of sugarcane SOS1 protein with an estimated molecular weight of 47.6 k D and a calculated isoelectric point（p I） of 9.12. The protein of Sc SOS1 belongs to a conserved CAP-ED superfamily. Yet the Sc SOS1 protein has no signal peptide and belongs to hydrophilic protein with the main function for intermediary metabolism. The mainly secondary structure element of Sc SOS1 protein is random coil. Real-time quantitative PCR（RT-q PCR） analysis revealed that Sc SOS1 was tissue-specifically expressed in leaf sheath, bark, pulp, bud and root of sugarcane, with the highest expression in leaf sheath and the lowest in root. Besides, the expression of Sc SOS1 gene could be regulated by the treatments of Na Cl, PEG, ABA, SA, and Me JA, and up-regulated by the stresses of Na Cl and PEG, with the highest inducible expression levels of 1.5 times and 4.0 times as high as those of control at 24 hours, respectively. This paper suggested that Sc SOS1 involves in sugarcane tolerance salt and osmotic stresses. It can set up a basis for the elucidation of sugarcane salt resistance mechanism.