miR172b/c-BnMSH7.A1模块响应甘蓝型油菜中Cu2+胁迫机制

doi:10.13560/j.cnki.biotech.bull.1985.2024-0584

摘要/Abstract

摘要：

目的探究基于miRNA调控甘蓝型油菜BnMSH7.A1响应Cu²⁺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Cu²⁺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Cu²⁺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Cu²⁺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、pre-miR172c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。

关键词: Cu²⁺胁迫, BnMSH7.A1, miR172b/c, 双荧光素酶报告实验分析, 相关性分析

Abstract:

Objective To explore the mechanism of miRNA regulation in response to Cu²⁺stress in Brassica napusBnMSH7.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 Cu²⁺, 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 Cu²⁺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 Cu²⁺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 miR172b and miR172c, and miR172b/c can positively regulate the expression of BnMSH7.A1. There is no significant correlation between pre-miR172b, pre-miR172c, and miR172b/c in the leaves, but miR172b/c can negatively regulate the expression of BnMSH7.A1.

Key words: Cu²⁺ stress, BnMSH7.A1, miR172b/c, double luciferase reporter assay analysis, correlation analysis

刘芳, 杜芊芊, 何昊, 肖钢, 晏仲元, 郝小花. miR172b/c-BnMSH7.A1模块响应甘蓝型油菜中Cu²⁺胁迫机制[J]. 生物技术通报, 2025, 41(2): 139-149.

LIU Fang, DU Qian-qian, HE Hao, XIAO Gang, YAN Zhong-yuan, HAO Xiao-hua. Mechanism of miR172b/c-BnMSH7.A1 Module Responding to Cu ²⁺ Stress in Brassica napus[J]. Biotechnology Bulletin, 2025, 41(2): 139-149.

图/表 10

表1 文中所用引物

Table 1 Primers used in this article

引物名称 Primer name	正向引物 Forward primer （5′-3′）	反向引物 Reward primer （5′-3′）	用途 Purpose
DL-MSH7A1	CGTCGCAAGCTACTCAGCGT	CACATTTCGCTCCACAGAG C	RT-qPCR检测BnMSH7.A1表达量 Detect the expression level of BnMSH7.A1 by RT-qPCR
DL-MSH7C1	GAAACCGTCGCAAGCTTCAG	CATTTCGCTCCACAGAG	RT-qPCR检测BnMSH7.C1表达量 Detect the expression level of BnMSH7.C1 by RT-qPCR
DLpre-miR172b	TGGCTTTCTGAATCCTCTTCC	GATGCTGCATCTGCAACTACC	RT-qPCR检测pre-miR172b表达量 Detect the expression level of pre-miR172b by RT-qPCR
DLpre-miR172c	GCCGGTAGTTGCAGATGC	GCTGATGCAGCATCATCAAG	RT-qPCR检测miR172c前体表达量 Detect the expression level of pre-miR172c by RT-qPCR
Actin	CTGGTGATGGTGTGTCTCACAC	GTTGTCTCATGGATTCCAGGAG	内参基因 Reference gene
DLmiR172b/c	GGAATCTTGATGATGCTGCAT	随机引物Random primer	RT-qPCR检测miR172b/c表达量 Detect the expression level of miR172b/c by RT-qPCR
U6	CGATAAAATTGGAACGATACAGA	ATTTGGACCATTTCTCGATTTGT	内参基因 Reference gene
G172b	CCGCTCGAGTTGCGTATTCGTTTGGTGGT	GGAATTCTTTAGTCCTTGCTCACATCCC	克隆pre-miR172b序列并构建过表达载体 Clone the pre-miR172b sequence and construct over-expression vector
G172c	CCGCTCGAGTGACTGATTTGTCTTCTGAATCC	GGAATTCCATAAACAAAGAGAAAGAGACTTTAG	克隆pre-miR172c序列并构建过表达载体 Clone the pre-miR172c sequence and construct over-expression vector

图1 BnMSH7与AtMSH7序列比对分析结果A：蛋白序列比对结果；B：结构域分析

Fig. 1 Analysis results of sequence alignment between BnMSH7 and AtMSH7A: Protein sequence alignment results. B: Analysis of structural domains

图2 BnMSH7进化树、基序及亚细胞定位分析

Fig. 2 Phylogenetic tree, motif, and subcellular localization analysis of BnMSH7

图3 BnMSH7.A1与miR172b/c的配对及双荧光素酶验证结果A：预测到的 BnMSH7.A1与miR172b/c的配对位点；B：双荧光素酶实验验证miR172b/c与BnMSH7.A1互作关系；*P<0.05，**P<0.01。下同

Fig. 3 Pairing of BnMSH7.A1 with miR172b/c and double luciferase validation resultsA: The predicted pairing sites between BnMSH7.A1 and miR172b/c. B: Validation of the interaction between miR172b/c and BnMSH7.A1 using dual-luciferase assay. *P<0.05， **P<0.01. The same below

图4 过表达后BnMSH7.A1和miR172b/c表达情况A：过表达pre-miR172b和pre-miR172c后，油菜子叶中BnMSH7.A1的表达变化；B：过表达pre-miR172b和pre-miR172c后，油菜子叶中miR172b和miR172c的表达变化

Fig. 4 Expressions of BnMSH7.A1 and miR172b/c after over-expressionA: Expression variation of BnMSH7.A1 in rape cotyledons after the over expression of pre-miR172b or pre-miR172c. B: Expression variation of miR172b and miR172c in rape cotyledons after the over expression of pre-miR172b or pre-miR172c

图5 铜响应元件在启动子上分布情况

Fig. 5 Distribution of copper-responding elements on promoters

图6 不同浓度Cu2+处理后miR172b/c和BnMSH7.A1表达水平分析

Fig. 6 Analysis of expressions of miR172b/c and BnMSH7.A1 after treatment with different concentrations of Cu2+

表2 相关性分析

Table 2 Correlation analysis

相关性 Correlation	根 Root		叶 Leaf
相关性 Correlation	r	P	r	P
Pre-miR172b：miR172b	0.61	0.02	-0.45	0.02
Pre-miR172c：miR172c	0.73	0.03	-0.31	0.04
miR172b/c：BnMSH7.A1	0.52	0	-0.78	0.04

图7 pre-miR72b和pre-miR172c二级结构图

Fig. 7 Secondary structure diagram of pre-miR72b and pre-miR172c

图8 不同浓度Cu2+处理后pre-miR172b和pre-miR172c表达水平分析

Fig. 8 Analysis of expressions of pre-miR172b and pre-miR172c after treatment with different concentrations of Cu2+

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miR172b/c-BnMSH7.A1模块响应甘蓝型油菜中Cu²⁺胁迫机制

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

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