宁夏枸杞液泡膜H+-ATPase亚基E like基因克隆及表达分析  

Cloning and Expression Analysis of Vacuolar H+-ATPase Subunit E like Gene in Lycium barbarum L.

作　　者：毛桂莲[1,2] 姚晓翠 杨迎月 张虹 郑蕊[1,2] 郑国琦 Mao Guilian;Yao Xiaocui;Yang Yingyue;Zhang Hong;Zheng Rui;Zheng Guoqi(School of Life Science,Ningxia University,Yinchuan,750021;Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia,School of Agriculture,Ningxia University,Yinchuan,750021;School of Agriculture,Ningxia University,Yinchuan,750021)

机构地区：[1]宁夏大学生命科学学院,银川750021 [2]宁夏大学农学院,宁夏优势特色作物现代分子育种重点实验室,银川750021 [3]宁夏大学农学院,银川750021

出　　处：《分子植物育种》2022年第3期826-831,共6页Molecular Plant Breeding

基　　金：本研究由宁夏自然科学基金项目(2018AAC02001;2020AAC03097;2020AAC03106);宁夏枸杞产业发展中心宁夏枸杞核心种质资源差异性分析及功能基因挖掘项目(HSZB-2020ZC242) 国家自然科学基金项目(31360168;81170682)共同资助。

摘　　要：为了了解液泡膜H+-ATPase亚基的分子基础,揭示其在枸杞耐盐碱机制中的作用,本研究以宁夏枸杞为材料,采用TA克隆获得枸杞液泡膜H+-ATPase亚基E like (LbVHA-E like)的基因序列,采用实时荧光定量PCR的方法检测其在不同程度盐胁迫下的表达变化。研究结果显示:该基因全长1 116 bp,开放阅读框为696 bp,编码231个氨基酸,预测分子量约为26.42 kD,等电点7.23;LbVHA-E like进化关系上与茸毛烟草和番茄的亲缘关系最近。LbVHA-E like蛋白质二级结构由19.05%的随机卷曲、71.43%的α-螺旋和6.93%的延伸主链构成,不存在信号肽、跨膜区,属于一种亲水的不稳定性蛋白,亚细胞定位预测其主要定位于细胞质。实时荧光定量PCR分析结果显示,在盐胁迫初期(1 d),该基因在根和叶中的表达量均高于对照,在低浓度盐胁迫时(100 mmol/L)根中的基因表达量均高于对照,由此表明在盐胁迫初期,LbVHA-E like基因可能参与了盐胁迫的Na+离子的排出和区隔化。本研究结果将为LbVHA-E like基因参与宁夏枸杞耐盐分子调控机理的揭示提供理论依据。To learn about the role of the vacuolar H+-ATPase subunits in salt stress, the vacuolar H+-ATPase subunit E like gene in Lycium barbarum L. was cloned in this study, and the expression pattern of LbVHA-E like gene under salt stress was also investigated using TA cloning and RT-qPCR technology. The results showed that the full length of LbVHA-E like was 1 116 bp, containing a 696 bp the open reading frame, encoding a protein of 231 amino acids. The predicted molecular weight of LbVHA-E like protein was 26.42 kD and the isoelectric point was7.23. Evolutionary analysis showed that LbVHA-E like protein was closely related to Solanum lycopersicum and Nicotiana tomentosiformis in evolutionary relationship. Its protein secondary structure consists of 19% random coil,71.43% alpha-helix and 6.93% extended backbone. The LbVHA-E like protein was a hydrophilic unstable protein and excluded signal peptide or transmembrane domains. Subcellular localization prediction results showed that LbVHA-E like protein mainly accumulated in the cytoplasm. The quantitative real-time PCR analysis revealed that the relative expression level of LbVHA-E like gene in Lycium barbarum L. roots and leaves were higher than that of control at 1 d stress. Meanwhile, the relative expression level of LbVHA-E like gene of roots was higher than that of control under low NaCl(100 mmol/L) concentration. It indicated that LbVHA-E like gene could excrete Na+from the cytoplasm and comparted it into vacuolar cells at the early stage of treatment. The results of this study will provide a theoretical basis for revealing the molecular regulation mechanism of LbVHA-E like gene in salt tolerance of Lycium barbarum in Ningxia.

关 键 词：宁夏枸杞(Lycium barbarum) 液泡膜H+-ATPase亚基E like 基因克隆 RT-QPCR 

分 类 号：S567.19[农业科学—中草药栽培]