西伯利亚白刺NsCBL2基因克隆及表达分析

Cloning and expression analysis of NsCBL2 gene from Nitraria sibirica

CBL家族在许多植物应对非生物胁迫的调控中扮演了重要角色,西伯利亚白刺作为典型稀盐型盐生植物,在恶劣生境有极强的适应能力。对西伯利亚白刺CBL家族成员进行研究,进一步丰富植物耐盐分子机制,发掘耐盐抗逆等基因资源。以西伯利亚白刺为试材,克隆得到NsCBL2基因CDS序列,通过生物信息学分析,结合亚细胞定位及模拟盐、碱及干旱胁迫下的实时荧光定量PCR(qRT-PCR)对NsCBL2功能进行初步研究。生物信息学分析结果显示:西伯利亚白刺Ns CBL2基因全长681 bp,编码226个氨基酸;氨基酸序列相对分子量为26.10 kDa,分子式为CHNOS,是不稳定亲水的酸性蛋白;系统发育进化树上与其距离最近的是AtCBL2。亚细胞试验鉴定结果显示NsCBL2蛋白定位于细胞膜及液泡膜上。对西伯利亚白刺进行模拟盐、碱及干旱胁迫处理,基因表达量均发生变化,尤以盐及碱胁迫下变化较为显著且表达趋势相似。拟南芥AtCBL2在Na~+存储至液泡过程中发挥重要的调控作用,参与了离子调控机制,推测进化关系上与其相近的NsCBL2可能在调控机制上也有共通之处。

The CBL family plays an important role in the regulation of abiotic stress in many plants.Nitraria sibirica,as a typical halophyte,has strong adaptability in harsh habitats.The CBL family members of N.sibirica were studied to further enrich the molecular mechanism of salt tolerance and explore the genetic resources of salt tolerance and resistance.In this study,the CDS sequence of NsCBL2 gene was cloned from N.sibirica,further analyzed through bioinformatics,real-time fluorescence quantitative PCR(qRT-PCR) under salt,alkali and drought stress combined with subcellular localization,and the function of NsCBL2 was preliminarily understood.The results by bioinformatics showed that:the total length of the NsCBL2 gene was 681 bp,encoding 226 amino acids;the relative molecular weight of the amino acid sequence was 26.10 kDa,and the molecular formula was CHNOS,which was an unstable hydrophile acid protein;AtCBL2 was closest to it on the phylogenetic tree.Subcellular identification showed that NsCBL2 protein was localized on the cell membrane and vacuole membrane.The expression levels of this gene were changed under simulated salt,alkali and drought stress,especially under salt and alkali stress.AtCBL2 in Arabidopsis thaliana plays an important role in the regulation of Nastorage into vacuoles and participated in the ion regulation mechanism.It was speculated that AtCBL2 and NsCBL2 have a similar evolutionary relationship in the regulation mechanism.