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

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

Abstract

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

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.

Figures/Tables 10

References 33

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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

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

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

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

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

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