小桐子cystatin家族基因和相应miRNAs的鉴定及其在低温响应中可能的作用 

吴丹丹,陈永坤,杨宇,孔春艳,龚明*
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

通信作者:龚明;E-mail: gongming6307@163.com

摘 要:

半胱氨酸蛋白酶抑制剂(cystatin)是一组抑制植物中木瓜类半胱氨酸蛋白酶C1A和豆类蛋白酶C13活性的小分子蛋白。小桐子(Jatropha curcas)是一种具有开发潜力的能源植物及冷敏植物, cystatin基因家族及作用目前尚不清楚。本研究在全基因组水平上对小桐子cystatin基因家族及靶向该家族成员的miRNAs进行了鉴定, 并对cystatin家族系统进化、保守结构域、染色体定位、表达特性及成员基因与miRNAs互作参与调控小桐子对低温的响应进行了解析。结果表明, 小桐子基因组中共鉴定到5cystatin基因(命名为JcCYS1~5), 定位到6810号染色体, 可分为3个亚家族(ABC), 编码106~242个氨基酸,理论等电点4.97~9.17, 序列比对发现cystatin的结构域基本保守且位于N端。表达分析显示, JcCYS1在种子、花蕾、叶、茎端表达量较高, JcCYS2在授粉期间表达量高。基于miRNA组和降解组测序结果, 发现有10miRNAs可能靶向调控cystatin基因家族的3个成员JcCYS1JcCYS2JcCYS5RT-qPCR分析表明, 小桐子cystatin家族的5个基因的表达水平在12°C冷锻炼期间总体呈现出显著响应, 尤其是在冷锻炼早期(12~24 h)。此外, 对靶向JcCYS2JcCYS5miRNAs在冷锻炼过程中的表达分析表明, miR11036-ymiR396-zJcCYS2以及miR8154-xJcCYS5的表达在冷锻炼期间呈明显负相关, 暗示着这些miRNAs的确参与了cystatin基因的调控, 且这种调控应该与冷锻炼诱导的小桐子耐冷性有关。这些结果有助于更好了解小桐子cystatin家族功能及其与相应miRNAs的互作、以及这种互作如何调控小桐子对低温胁迫的响应。

关键词:小桐子; cystatin; 基因家族; miRNAs; 低温响应

收稿:2020-11-19   修定:2020-12-17

资助:国家自然科学基金(31860062, 31460059)

Identifcation of cystatin family genes and their corresponding miRNAs in Jatropha curcas and possible roles in response to low temperature

WU Dandan, CHEN Yongkun, YANG Yu, KONG Chunyan, GONG Ming*
Key Laboratory of Biomass Energy and Environmental Biotechnology of Yunnan Province, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming 650500, China

Corresponding author: GONG Ming; E-mail: gongming6307@163.com

Abstract:

Cystatin is a group of small proteins to inhibit the activities of cysteine proteases of papain-like
C1A and leguminosase C13 family in plants.
Jatropha curcas is a promising energy plant and chilling-sensitive plant, and the cystatin gene family and their roles are still unclear in J. curcas. In the present study, cystatin gene family and miRNAs targeted the genes of the family were identifed at the genome-wide level. The phylogenetics, conserved domains, chromosomal localization, expression characteristics of cystatin family genes were analyzed, and interactions among cystatin genes and their corresponding miRNAs involving in regulation of low temperature response of J. curcas were also evaluated. The results indicated that fve cystatin genes were identifed in J. curcas genome (named as JcCYS15), which located to chromosome 6, 8 and 10, and cystatin family could be divided into three subfamilies (A, B, C), encoding 106 to 242 amino acids, with theoretical isoelectric points of 4.97 to 9.17. Sequence alignment showed that the structure domain of cystatin was basically conservative and located at the N-terminal. Expression analysis demonstrated that JcCYS1 was highly expressed in seeds, flower buds, leaves and stems, while JcCYS2 was highly expressed during pollination. Based on the sequencing results of miRNAome and degradome, 10 miRNAs were identifed to participate in targeted regulation of JcCYS1, JcCYS2 and JcCYS5. RT-qPCR analysis showed that the expression level of 5 genes in cystatin family totally responded signifcantly to the chill-hardening at 12°C in J. curcas, especially at early phase of the hardening (12–24 h). In addition, expression analysis of the miRNAs targeted to JcCYS2 and JcCYS5 revealed signifcantly negative correlation between miR11036-y/miR396-z and JcCYS2, as well as miR8154-x and JcCYS5, implying that those miRNAs were indeed involved in regulating the cystatin genes, which may be correlated with the chill-hardening induced chilling tolerance. These results will be helpful for better understanding functions of the cystatin family and its interaction with the corresponding miRNAs, and how this interaction regulates responses of J. curcas to low temperature stress.

Key words: Jatropha curcas; cystatin; gene family; miRNAs; low temperature response

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