镉胁迫下岗梅内参miRNA与基因的筛选与验证  

Screening and Validation of miRNA Reference Genes and Genes in Ilex asprella under Cadmium Stress

作　　者：周新裕 仇梓杰 黄金恒 乜晓凤 杨全 廖沛然 ZHOU Xinyu;QIU Zijie;HUANG Jinheng;NIE Xiaofeng;YANG Quan;LIAO Peiran(Key Laboratory of Production&Development of Cantonese Medicinal Materials,State Administration of Traditional Chinese Medicine/Comprehensive Experimental Station of Guangzhou,Chinese Material Medica,China Agriculture Research System(CARS-21-16)/Guangdong Provincial Research Center on Good Agricultural Practice&Comprehensive Agricultural Development Engineering Technology of Cantonese Medicinal Materials,Guangzhou 510006;Yunfu Chinese Medicine Resources Germplasm Bank Management Center,Yunfu,Guangdong 527325)

机构地区：[1]国家中医药管理局岭南药材生产与开发重点研究室,国家中药材产业技术体系广州综合试验站,广东省南药规范化种植与综合开发工程技术研究中心,广东药科大学中药学院,广州510006 [2]云浮市中药资源种质库管理中心,广东云浮527325

出　　处：《中国农学通报》2025年第11期31-39,共9页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金青年科学基金项目“梅叶冬青MIRn87-5p调控囊泡靶向融合至液泡途径对镉区隔化和累积作用机制研究”(32102467);广州市科技计划项目“岗梅根MIRn77-3p调控细胞壁结构变化对镉固定和富集作用机制研究”(2024A04J7887);广东省科技厅项目“山地岗梅生态种植研究”(KTP20210111);云浮市2021年中医药(南药)产业人才项目-南药资源创新团队项目“南药资源创新团队”(YRCB(2021)2)。

摘　　要：本研究旨在从岗梅根中筛选出镉胁迫下稳定表达的内参miRNA和基因,以测定岗梅根中镉胁迫响应miRNA及其靶基因表达量。本研究提取了不同镉胁迫时间处理的岗梅根的总RNA进行逆转录后,通过RT-qPCR实验,测定了9个候选内参miRNA和8个候选内参基因Ct值,并通过geNorm、NormFinder、BestKeeper和RefFinder等工具进行了综合系统分析,筛选出最佳内参miRNA和基因。之后基于最佳内参miRNA和基因,本研究进一步测定了岗梅根中6个响应镉胁迫的miRNA及其靶基因表达量。结果显示,虽然各工具由于算法差异致使结果存在差异,但经RefFinder综合分析,本研究确定UBQ为最佳内参基因,其稳定性排序为UBQ>Actin>18S>GAPDH>UBC>EF1-α>28S>α-TUB,最佳的内参miRNA为U6,稳定性排序为U6>5S>MIR156ab>MIR396f>MIR159d>MIR390j>MIR171t>U4>MIR166ab。6个miRNA及其靶基因的表达分析结果表明,在镉胁迫下,MIR159c的表达量总体呈下降趋势,而MIR171a和MIR393a的表达量总体则呈上升趋势。此外,MIR167m-5p、MIR399f-5p和MIR529d的表达量呈先上升后下降的趋势。这些miRNA及其靶基因之间均呈显著负相关关系,这表明在镉胁迫环境下,岗梅可通过miRNA负调控其靶基因来适应镉胁迫。综上所述,本研究成功筛选出镉胁迫下岗梅根中最佳内参基因UBQ和内参miRNA U6,并检测了镉胁迫下岗梅响应miRNA及其靶基因的表达量,为深入探究镉胁迫对岗梅miRNA及其靶基因表达调控机制提供了一定的理论基础。The objective of this study was to identify stably expressed internal reference miRNAs and genes under cadmium(Cd)stress in the roots of Ilex asprella,for the purpose of determining the expression levels of Cd stress-responsive miRNAs and their target genes.In this study,total RNA was extracted from Ilex asprella roots subjected to various Cd stress durations,followed by reverse transcription.Subsequently,the Ct values of 9 candidate internal reference miRNAs and 8 candidate internal reference genes were determined through RTqPCR experiments.The optimal internal reference miRNAs and genes were subsequently identified through a comprehensive and systematic analysis using tools such as geNorm,NormFinder,BestKeeper,and RefFinder.After that,based on the best internal reference miRNAs and genes,this study further determined the expression of 6 miRNAs and their target genes in response to Cd stress in the roots of Ilex asprella.The results showed that although the results of various tools were different due to algorithm differences,in the comprehensive analysis of RefFinder,this study identified UBQ as the best internal reference gene,with the stability ranking of UBQ>Actin>18S>GAPDH>UBC>EF1-α>28S>α-TUB,the best internal reference miRNA was U6,and the stability ranking was U6>5S>MIR156ab>MIR396f>MIR159d>MIR390j>MIR171t>U4>MIR166ab.The expression analysis results of 6 miRNAs and their target genes showed that under the Cd stress,the expression of MIR159c decreased gradually,while the expression of MIR171a and MIR393a increased significantly.In addition,the expression levels of MIR167m-5p,MIR399f-5p and MIR529d increased first and then decreased.There was a significant negative correlation between these miRNAs and their target genes,which indicated that under Cd stress environment,Ilex asprella could negatively regulate its target genes through miRNAs to adapt to Cd stress.In conclusion,this study successfully screened the best internal reference gene UBQ and internal reference miRNA U6 in the roots of Ilex asprella un

关 键 词：镉胁迫 岗梅 实时荧光定量PCR 内参基因 MIRNA 

分 类 号：S567.1[农业科学—中草药栽培]