地黄内生真菌GG22侵染下miR160家族成员及靶基因的鉴定分析  

Identification of miR160 family and their target genes in Rehmannia glutinosa in response to endophytic fungus GG22 infection

作　　者：许姣 赵乐[1,2] 董诚明 朱畇昊[1,2] XU Jiao;ZHAO Le;DONG Cheng-ming;ZHU Yun-hao(School of Pharmacy,Henan University of Chinese Medicine,Zhengzhou 450046,China;Respiratory Disease Diagnosis and Treatment and New Drug of Henan Collaborative Innovation Center,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学药学院,河南郑州450046 [2]呼吸疾病诊疗与新药研发河南省协同创新中心,河南郑州450046

出　　处：《中国中药杂志》2022年第12期3198-3207,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81603232);国家重点研发计划项目(2017YFC1702800);河南省重大科技专项(171100310500);河南省科技攻关项目(172102310539)。

摘　　要：为明确地黄miR160家族成员在响应内生真菌尖孢镰刀菌Fusarium oxysporum GG22侵染中的作用,该研究从高通量测序技术获得的小RNA数据库中筛选出地黄miR160家族成员,利用RNAfold分析其前体结构,DNAMAN和MEGA对前体序列和成熟序列进行保守性和进化性分析;对地黄miR160家族成员的靶基因进行预测功能注释以及互作关系分析;结合降解组数据对靶基因进行鉴定分析。结果发现miR160前体具有完整的茎环结构;序列比对显示在前体、成熟序列上都具有较高的保守性,成熟序列中5′端第3~16位碱基高度保守;系统进化树说明了地黄与拟南芥、水稻、丹参及芝麻之间的亲缘关系;预测出22个miR160的靶基因中生长素响应因子(ARF)居多;GO功能分析主要富集到生物学过程、细胞组分以及分子功能;降解组测序获得miR160的4个靶基因注释到ARF18和ARF22;表达特性分析表明,地黄经过内生真菌侵染后,通过改变miR160和靶基因的表达来调控植物生长;对差异表达的rgl-miR160a和rgl-miR160a-3p进行qRT-PCR验证,结果显示与测序结果一致。该研究结果进一步明确了地黄在响应GG22胁迫的分子机制,为地黄今后育种改良以及相关研究提供理论依据。This study aims to reveal the possible role of miR160 family in Rehmannia glutinosa in response to the infection of endophytic fungus Fusarium oxysporum GG22.Specifically,miR160 precursors and mature miR160 were retrieved from the small RNA database yielded by high-throughput sequencing.RNAfold was used to analyze the precursor structure,and DNAMAN and MEGA to analyze conservation and evolution of miR160 precursors and mature miR160.The target genes of miR160 were predicted and annotated,and the interaction was analyzed.Based on degradome sequencing,the target genes were further identified.The results showed that miR160 precursors had intact stem-loop structures.The precursor and mature sequences were conserved,particularly the 3 rd-16 th bases of the 5′-terminal.According to the phylogenetic tree,R.glutinosa had close evolutionary relationship with Arabidopsis thaliana,Oryza sativa,Salvia miltiorrhiza,and Sesamum indicum.A total of 22 target genes of miR160 were predicted and most of them were auxin response factor(ARF)genes.The target genes were involved in the Gene Ontology(GO)terms of biological processes,cellular components,and molecular functions.According to the degradome sequencing results,four target genes of miR160 were ARF(ARF18,ARF22)genes.R.glutinosa regulated its growth in response to the infection of endophytic fungus by changing the expression of miR160 and the target genes.qRT-PCR result of the differentially expressed rgl-miR160a and rgl-miR160a-3p was consistent with the sequencing result.This study clarifies the molecular mechanism of R.glutinosa in response to GG22 stress,laying a theoretical basis for the improvement and future research of R.glutinosa.

关 键 词：地黄 miR160 靶基因 GG22 

分 类 号：S567.234[农业科学—中草药栽培]