作物学报2025，Vol.51Issue(5)：1156-1165,10.DOI:10.3724/SP.J.1006.2025.44161
黄麻U6启动子克隆及其转录活性分析
Cloning and transcriptional activity analysis of U6 promoter in jute(Corchorus capsularis)
摘要
Abstract
The U6 promoter is a critical element for driving the transcription of single guide RNA(sgRNA)in the CRISPR/Cas9 system,with endogenous U6 promoters often exhibiting higher efficiency than exogenous ones.However,no studies to date have focused on endogenous U6 promoters in jute(Corchorus L.).In this study,two candidate U6 promoters,CcU6.1 and CcU6.3,were cloned from the genome of the jute cultivar"Meifeng 4"using conserved sequences from the Arabidopsis thaliana U6-26 sgRNA promoter(AtU6-26).Fusion expression vectors carrying GUS reporter genes driven by the CcU6.1 and CcU6.3 promoters were constructed,and the transcriptional activities of these promoters were evaluated through Agrobacterium-mediated transfor-mation of tobacco(Nicotiana benthamiana)leaves and jute hairy roots.Promoter activity was determined based on GUS histo-chemical staining.Homology analysis revealed that both CcU6.1 and CcU6.3 promoters contained two essential elements for U6 promoter activity:the USE and TATA boxes.GUS staining demonstrated that both jute U6 promoters exhibited transcriptional activity,although the CcU6.1 promoter showed weaker activity compared to the CcU6.3 promoter in both Nicotiana benthamiana leaves and jute hairy roots.Quantitative PCR further confirmed these findings.Since excessively long U6 promoters may reduce transcriptional efficiency,a comparative cis-regulatory element analysis of the CcU6.3 promoter and the AtU6-26 promoter were conducted.This analysis suggested that a truncated version of the CcU6.3 promoter,spanning from the transcriptional start site to the-550 bp region,could enhance transcriptional activity.This study is the first to identify and characterize the CcU6.3 promoter,which exhibits relatively high transcriptional activity in jute.The CcU6.3 promoter holds significant potential as a strong and efficient promoter for constructing CRISPR/Cas9 gene-editing systems in Corchorus species.关键词
黄麻/U6启动子/CRISPR/Cas9/本氏烟草/毛状根/转录活性Key words
jute/U6 promoter/CRISPR/Cas9/Nicotiana benthamiana/hairy roots/transcriptional activity引用本文复制引用
黄梦欣,庄灵玲,程佩佩,李秦,徐建堂,陶爱芬,方平平,祁建民,张立武..黄麻U6启动子克隆及其转录活性分析[J].作物学报,2025,51(5):1156-1165,10.基金项目
本研究由国家自然科学基金项目(32472219),福建省自然科学基金项目(2023J01443),财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-16)和福建农林大学科技创新专项基金(KFB23001,KFB24080)资助. This study was supported by the National Natural Science Foundation of China(32472219),the Fujian Provincial Natural Science Foundation(2023J01443),the China Agriculture Research System of MOF and MARA(CARS-16),and the Science and Technology Innovation Project of Fujian Agriculture and Foresty University(KFB23001,KFB24080). （32472219）
