2021年10月17日 星期日
芹菜AgLIMS基因的克隆和生物信息学 分析及其表达特性
Cloning and bioinformatic analysis on AgLIMS gene from Apium graveolens and its expression characteristics
2020年 第29卷 第6期 页码[23-31]    下载全文[12.6MB]  
摘要

 以芹菜(Apium graveolens Linn.)品种‘六合黄心芹’(‘Liuhe Huangxinqin’)和‘津南实芹’(‘Jinnan Shiqin’)为研究对象,分别克隆获得芹菜柠檬烯合酶基因,命名为AgLIMS,其开放阅读框长1 800 bp,编码599个氨基酸。结果显示:2个芹菜品种的AgLIMS基因核苷酸序列存在2个位点的差异,其编码的氨基酸序列也存在2个位点的差异;AgLIMS基因编码的氨基酸序列在第50至第586位存在植物萜类合酶(Terpene cyclase plant C1)保守区域,具有典型的类异戊二烯合成酶(Isoprenoid Biosyn C1)超家族结构域。2个芹菜品种的AgLIMS蛋白的理论相对分子质量分别为69 120和69 190,理论等电点均为pI 5.52。AgLIMS蛋白的氨基酸序列中,亲水性氨基酸比例高于疏水性氨基酸;其二级结构主要由α螺旋和无规则卷曲构成,但α螺旋、延伸链和β转角的比例在2个芹菜品种间略有差异;其三级结构均含有1个β折叠,但α螺旋数量在2个芹菜品种间略有差异。AgLIMS基因与多种植物LIMS基因编码的氨基酸序列一致性为61.33%;在系统树上,2个芹菜品种与同科种类的进化关系较近,而与其他科木本种类的进化关系较远。qRT-PCR结果显示:AgLIMS基因在2个芹菜品种的叶片、叶柄和根中均能表达,其相对表达量在叶片中最高、在根中最低,且在不同组织间差异显著;该基因在‘六合黄心芹’不同组织中的相对表达量总体上显著高于‘津南实芹’。在高温(38 ℃)、低温(4 ℃)、干旱(质量体积分数20%PEG6000)和高盐(0.2 mol·L-1NaCl)条件下,2个芹菜品种AgLIMS基因的相对表达量呈现不同的变化趋势;总体上看,‘津南实芹’AgLIMS基因的相对表达量均不同程度升高,而品种‘六合黄心芹’AgLIMS基因的相对表达量则在高温和干旱条件下显著升高。综合分析结果表明:2个芹菜品种的AgLIMS基因核苷酸序列及其编码的氨基酸序列差异很小;AgLIMS蛋白为中性、亲水性蛋白,其进化具有一定的保守性;AgLIMS基因在芹菜中的表达具有组织特异性,且在受到非生物胁迫时其表达特性发生变化,但这种变化效应存在品种间差异。

Abstract

Taking cultivar ‘Liuhe Huangxinqin’ and ‘Jinnan Shiqin’ of Apium graveolens Linn. as research objects, limonene synthase gene was cloned and named as AgLIMS, and the length of its open reading frame. is 1 800 bp, encoding 599 amino acids. The results show that there are two sites of difference in the nucleotide sequences of AgLIMS gene from two A. graveolens cultivars, and there are also two sites of difference in their encoding amino acid sequences; there is a conserved region of plant terpene synthase (Terpene cyclase plant C1) from 50th to 586th in amino acid sequence encoded by AgLIMS gene, which possesses typical isoprenoid synthase (Isoprenoid Biosyn C1) superfamily domain. The theoretical relative molecular masses of AgLIMS protein of two A. graveolens cultivars are 69 120 and 69 190, respectively, and their theoretical isoelectric points are both pI 5.52. Among the amino acid sequence of AgLIMS protein, the percentage of hydrophilic amino acid is higher than that of hydrophobic amino acid; their secondary structures are mainly composed of αhelix and random coil, but the percentages of α-helix, extended strand and β-turn are slightly different between two A. graveolens cultivars; their tertiary structures all contain one β-sheet, but the number of α-helix is slightly different between two A. graveolens cultivars. The identity of amino acid sequences encoded by AgLIMS gene and LIMS gene from multiple plants is 61.33%; in the phylogenetic tree, the evolutionary relationships of two A. graveolens cultivars are relatively close with species in the same family, but are relatively far with woody species in other families. The qRT-PCR result shows that AgLIMS gene can express in leaf, petiole and root of two A. graveolens cultivars, its relative expression is the highest in leaf and the lowest in root, and the difference in different tissues is significant; the relative expression of this gene from different tissues of ‘Liuhe Huangxinqin’ is significantly higher than that from ‘Jinnan Shiqin’ in general. Under high temperature (38 ℃), low temperature (4 ℃), drought (mass volume fraction of 20% PEG6000) and high salt (0.2 mol·L-1NaCl) conditions, the relative expression of AgLIMS gene from two A. graveolens cultivars shows different change tendencies; in general, the relative expression of AgLIMS gene from ‘Jinnan Shiqin’ all increases at different degrees; while that of ‘Liuhe Huangxinqin’ significantly increases under high temperature and drought conditions. The comprehensive analysis result shows that the differences in nucleotide sequences of AgLIMS gene and its encoding amino acid sequences of two A. graveolens cultivars are very small; AgLIMS protein is neutral and hydrophilic protein, and its evolution is conserved with some degree; the expression of AgLIMS gene from A. graveolens is tissue-specific, and its expression characteristics vary under abiotic stress, but the variation effect is different among cultivars.

关键词芹菜; 柠檬烯合酶基因(LIMS基因); 蛋白质理化特性; 非生物胁迫; 基因表达特性
Key wordsApium graveolens Linn.; limonene synthase gene (LIMS gene); physicochemical property of protein; abiotic stress; gene expression characteristics
作者贾丽丽a,b,c, 尹莲a,b,c, 刘洁霞a,b,c, 王昊a,b,c, 沈迪a,b,c, 冯凯a,b,c, 熊爱生a,b,c
所在单位南京农业大学: a. 园艺学院, b. 作物遗传与种质创新国家重点实验室,
c. 农业农村部华东地区园艺作物生物学与种质创制重点实验室, 江苏 南京 210095
点击量7344
下载次数135
基金项目江苏省农业自主创新资金项目〔CX(18)2007〕; 江苏高校优势学科建设项目(PAPD)