小桐子早期光诱导蛋白基因ELIP克隆及其与靶向miRNA的互作与低温下共表达分析  

Cloning of early light-induced protein gene ELIP and its interaction and co-expression analysis with targeting miRNA in

作　　者：陈建南 朱晓琴 吕宇婧 龚明 CHEN Jiannan;ZHU Xiaoqin;LÜYujing;GONG Ming(School of Life Sciences,Key Laboratory of Biomass Energy and Environment of Yunnan Province,Engineering Research Center of Sustainable Development and Utilization of Biomass Energy of Ministry of Education,Yunnan Normal University,Kunming 650500,China)

机构地区：[1]云南师范大学生命科学学院,生物能源持续开发利用教育部工程研究中心,云南省生物质能与环境生物技术重点实验室,昆明650500

出　　处：《西北植物学报》2024年第3期430-442,共13页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金项目(31860062,31460059)。

摘　　要：【目的】作为一种喜温冷敏植物,低温严重影响小桐子的生长发育、地域分布与产量。前期工作发现12℃低温锻炼可显著提高小桐子的抗冷性,小桐子早期光诱导蛋白(ELIP)基因是低温高响应基因。为探究JcELIP在小桐子低温响应中的作用,全面了解JcELIP的结构、调控机制、进化关系及JcELIP与miRNAs的互作关系,也为后续小桐子抗冷分子育种提供一个重要的候选基因资源。【方法】通过RT-PCR克隆了小桐子JcELIP基因并对其进行系统的生物信息学分析,采用RT-qPCR分析JcELIP基因在根、茎、叶中及12℃低温锻炼过程中的表达变化,鉴定与JcELIP互作的miRNAs,进行了12℃低温锻炼过程中的共表达分析。【结果】该基因完整的开放阅读框(ORF)为585 bp,编码194个氨基酸,蛋白的分子质量为2.04 kD,理论等电点为9.59,属于稳定的疏水碱性蛋白,具有3个疏水跨膜螺旋;三级结构主要由ɑ-螺旋和无规则卷曲组成,具有叶绿素a/b结合位点。顺式作用元件预测显示JcELIP具有脱落酸等激素响应元件。进化分析显示:小桐子JcELIP与木薯MeELIP同源性最高。RTqPCR分析显示:正常生长条件下小桐子JcELIP在根、茎、叶中的表达量无显著差异;12℃冷锻炼条件下JcELIP在叶中表达快速上调,48 h时其表达量达到对照组的64.8倍,表明JcELIP参与了小桐子对冷胁迫的响应与适应。基于小桐子降解组数据,鉴定到miR390-x、miR6476-x和novel-m0090-3p等8个miRNAs对JcELIP的表达具有调控作用;共表达分析结果表明在12℃低温锻炼过程中JcELIP的表达受miR390-x和novel-m0090-3p显著负调控。【结论】JcELIP高响应低温胁迫并与miRNAs互作,表明其在小桐子响应低温胁迫过程中发挥重要作用。[Objective]As a kind of thermophilic and chilling-sensitive plants,low temperature severely af-fected the growth,development,geographical distribution,and yield of Jatropha curcas L..Previous studies showed that chill-hardening at 12℃significantly enhanced the chilling resistance of J.curcas.The early light-inducible protein(ELIP)gene in J.curcas was a highly responsive gene to low tempera-ture.The study aimed to explore the role of JcELIP in response to low temperature in J.curcas,to com-prehensively understand the structure,regulatory mechanisms,evolutionary relationships of JcELIP and its interaction with miRNAs,and to provide an important candidate gene resource for subsequent molecu-lar breeding of cold resistance J.curcas.[Methods]This study cloned the JcELIP gene from J.curcas by RT-PCR and conducted a comprehensive bioinformatics analysis.The expression of JcELIP gene in the roots,stems,and leaves,as well as during the chill-hardening at 12℃,were analyzed by RT-qPCR.The miRNAs interacting with JcELIP was identified,and a co-expression analysis was conducted during the chill-hardening at 12℃.[Results]The results showed that the complete open reading frame(ORF)of JcELIP was 585 bp,encoding 194 amino acids.The size of the protein was 2.04 kD with a theoretical iso-electric point of 9.59.It was a stable hydrophobic alkaline protein,with 3 hydrophobic transmembrane helices.The tertiary structure mainly consisted ofα-helices and irregular coils and possessed chlorophyll a/b binding sites.Cis-acting element prediction showed that JcELIP had hormone response elements such as abscisic acid.Evolutionary analysis showed that the JcELIP from J.curcas had the highest homology with MeELIP from Manihot esculenta.RT-qPCR analysis revealed that under normal growth conditions,there was no significant difference in the expression of JcELIP in the roots,stems,and leaves of J.cur-cas.During chill-hardening at 12℃,the expression of JcELIP in the leaves was quickly up-regulated,reaching 64.8 times of the control at 48

关 键 词：小桐子 早期光诱导蛋白(ELIP)基因 冷锻炼 表达分析 miRNA互作 

分 类 号：Q943.2[生物学—植物学] Q945.78[农业科学—作物学] S565.9