miR172b/c-BnMSH7.A1模块响应甘蓝型油菜中Cu^(2+)胁迫机制  

Mechanism of miR172b/c-BnMSH7.A1 Module Responding to Cu^(2+) Stress in Brassica napus

作　　者：刘芳 杜芊芊 何昊 肖钢[3] 晏仲元 郝小花 LIU Fang;DU Qian-qian;HE Hao;XIAO Gang;YAN Zhong-yuan;HAO Xiao-hua(College of Life and Environmental Science,Hunan University of Arts and Science,Changde 415000;Changde Research Centre Agricultural Biological Macromolecule,Changde 415000;College of Agriculture,Hunan Agriculture Universtity,Changsha 410000)

机构地区：[1]湖南文理学院生命与环境科学学院,常德415000 [2]常德市农业生物大分子研究中心,常德415000 [3]湖南农业大学农学院,长沙410000

出　　处：《生物技术通报》2025年第2期139-149,共11页Biotechnology Bulletin

基　　金：国家重点研发计划项目(2022YFD2300103);湖南省教育厅重点项目(22A0167);国家自然科学基金项目(32071930);湖南文理学院校级基金项目(20BSQD12);湖南省自然科学基金项目(2022JJ40288)。

摘　　要：【目的】探究基于miRNA调控甘蓝型油菜BnMSH7.A1响应Cu^(2+)胁迫的作用机理,为解析重金属胁迫响应机制中miRNA的调控功能提供新的研究思路。【方法】通过克隆甘蓝型油菜BnMSH7基因,利用生物信息学方法进行序列特征及潜在功能分析,预测筛选出调控甘蓝型油菜BnMSH7基因的候选miRNA,并使用烟草双荧光素酶报告系统对其进行体外验证,过表达pre-miR172b和pre-miR172c侵染油菜子叶,进行体内验证。经不同浓度Cu^(2+)处理油菜幼苗,RT-qPCR检测miR172b、miR172c和BnMSH7.A1表达情况,进行相关分析,推断Cu^(2+)胁迫下,miR172b和miR172c对BnMSH7.A1的调控情况。【结果】野生型萤火虫荧光值与海肾荧光值比值比空白对照显著下降,说明BnMSH7.A1受miR172b和miR172c调控。同时体内验证发现miR172b和miR172c与BnMSH7.A1的表达规律相反,表现为抑制调控,进一步说明miR172b和miR172c可以调控BnMSH7.A1。经生物信息学预测发现,miR172b、miR172c和BnMSH7.A1启动子上均存在铜响应元件;且Cu^(2+)处理后,根中pre-miR172b和pre-miR172c分别可以促进miR172b和miR172c表达,进一步促进BnMSH7.A1表达。叶中miR172b和miR172c负向调控BnMSH7.A1的表达,pre-miR172b、premiR172c与miR172b、miR172c呈负相关关系,但相关性不显著。【结论】在不同组织中miR172b/c-BnMSH7.A1模块的调控方式存在差异,在根中pre-miR172b、pre-miR172c与miR172b、miR172c均为正相关关系,miR172b/c可正向调控BnMSH7.A1表达。在叶中pre-miR172b、pre-miR172c与miR172b/c相关性不显著,但miR172b/c可负向调控BnMSH7.A1的表达。【Objective】To explore the mechanism of miRNA regulation in response to Cu^(2+)stress in Brassica napus BnMSH7.A1,and to provide new research ideas for exploring the regulatory function of miRNA in response to heavy metal stress.【Method】The BnMSH7 gene was cloned from B.napus,bioinformatics methods were used to analyze its sequence characteristics and potential functions,and to predict and screen the candidate miRNAs regulating the BnMSH7 gene in B.napus,which was verified by the tobacco dual luciferase reporter system in vitro.Pre-miR172b and pre-miR172c were transfected into rape cotyledons for in vivo validation.Rape seedlings were treated with different concentrations of Cu^(2+),and qRT-PCR was used to detect the expressions of miR172b,miR172c,and BnMSH7.A1.Correlation analysis was conducted to infer the regulation of BnMSH7.A1 by miR172b and miR172c under Cu^(2+)stress.【Result】It was found that the ratio of fluorescence values of wild-type fireflies(Watasenia scintillans)to sea kidney(Renilla reniformis)fluorescence values significantly decreased compared to the blank control,indicating that BnMSH7.A1 was regulated by miR172b and miR172c.Meanwhile,in vivo validation revealed that the expression patterns of miR172b and miR172c were opposite to those of BnMSH7.A1,exhibiting inhibitory regulation,further indicating that miR172b and miR172c regulated BnMSH7.A1.According to bioinformatics predictions,copper responsive elements were found on the promoters of miR172b,miR172c,and BnMSH7.A1.After Cu^(2+)treatment,pre-miR172b and pre-miR172c in roots positively regulated miR172b and miR172c,respectively,further promoting the expression of BnMSH7.A1.MiR172b and miR172c in the leaves negatively regulated the expressions of BnMSH7.A1,and pre-miR172b and pre-miR172c showed negative regulation with miR172b and miR172c,but not significantly.【Conclusion】The regulatory mode of miR172b/c-BnMSH7.A1 module varies in different organizations.In the root,pre-miR172b and pre-miR172c are positively correlated with miR1

关 键 词：Cu^(2+)胁迫 BnMSH7.A1 miR172b/c 双荧光素酶报告实验分析 相关性分析 

分 类 号：R73[医药卫生—肿瘤]