小桐子早期光诱导蛋白ELIP基因的克隆及其与靶向miRNA的互作与低温下共表达分析
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1.云南师范大学生命科学学院;2.云南师范大学 生命科学学院;3.云南师范大学

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国家自然科学(31860062,31460059)


Cloning of the Early Light-Induced Protein ELIP Gene and Its Interaction and Co-expression Analysis with Targeting miRNAs in Jatropha curcas under Low Temperature
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    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基因并对其进行了系统的生物信息学分析,采用RT-qPCR分析了JcELIP基因在根、茎、叶中及12 ℃低温锻炼过程中的表达变化,鉴定了与JcELIP互作的miRNAs,进行了12 ℃低温锻炼过程中的共表达分析。【结果】结果显示,该基因完整的开放阅读框(ORF)为585 bp,编码194个氨基酸,蛋白大小为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。RT-qPCR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显著的负调控。

    Abstract:

    【Objective】As a kind of thermophilic and chilling-sensitive plants, low temperatures severely affect the growth, development, geographical distribution, and yield of Jatropha curcas L. Previous studies have found that chill-hardening at 12°C can significantly enhance the chilling resistance of J. curcas. The Early Light-Inducible Protein (ELIP) gene in J. curcas is a highly responsive gene to low temperatures. To explore the role of JcELIP in response to low temperatures in J. curcas, to comprehensively understand the structure, regulatory mechanisms, evolutionary relationships of JcELIP, and its interaction with miRNAs, and to provide an important candidate gene resource for subsequent molecular breeding of cold resistance in J. curcas. 【Methods】 This study cloned the JcELIP gene from J. curcas by RT-PCR and conducted a comprehensive bioinformatics analysis. The expression changes of the JcELIP gene in the roots, stems, and leaves, as well as during the chill-hardening at 12°C, were analyzed by RT-qPCR. The miRNAs interacting with JcELIP were identified, and a co-expression analysis was conducted during the chill-hardening at 12°C. 【Results】 The results showed that the complete open reading frame (ORF) of the JcELIP is 585 bp, encoding 194 amino acids. The size of the protein is 2.04 kD with a theoretical isoelectric point of 9.59. It is a stable hydrophobic alkaline protein, with 3 hydrophobic transmembrane helices. The tertiary structure mainly consists of α-helices and irregular coils and possesses chlorophyll a/b binding sites. Cis-acting element prediction shows that JcELIP has hormone response elements such as abscisic acid. Evolutionary analysis showed that the JcELIP from J. curcas has the highest homology with MeELIP from Manihot esculenta. RT-qPCR analysis revealed that under normal growth conditions, there is no significant difference in the expression of JcELIP in the roots, stems, and leaves of J. curcas; During the chill-hardening at 12°C, the expression of JcELIP in the leaves quickly up-regulated, reaching 64.8 times of the control at 48 h, indicating that JcELIP is involved in the response and adaptation of J. curcas to cold stress. Based on the degradome data of J. curcas, eight miRNAs, including miR390-x, miR6476-x, and novel-m0090-3p, were identified as having regulatory effects on the expression of JcELIP. Co-expression analysis showed that the expression of JcELIP was significantly negatively regulated by miR390-x and novel-m0090-3p during the chill-hardening at 12°C.

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  • 收稿日期:2023-07-16
  • 最后修改日期:2023-09-19
  • 录用日期:2023-10-12
  • 在线发布日期: 2024-01-19
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