岷江百合病程相关蛋白4基因及其启动子的克隆与分析  

Isolation and analysis of pathogenesis-related protein 4 gene and its promoter from Lilium regale

作　　者：王自娥 李有玉 邓婕 王瀚林 孙皓 陈晓华 葛锋[1] 刘迪秋[1] WANG Zie;LI Youyu;DENG Jie;WANG Hanlin;SUN Hao;CHEN Xiaohua;GE Feng;LIU Diqiu(Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China)

机构地区：[1]昆明理工大学,生命科学与技术学院,昆明650500

出　　处：《植物生理学报》2022年第9期1747-1756,共10页Plant Physiology Journal

基　　金：国家自然科学基金项目(31760586)。

摘　　要：为了揭示岷江百合(Lilium regale)抗枯萎病菌防卫反应的分子机制,从岷江百合转录组数据中挖掘了一个响应尖孢镰刀菌(Fusarium oxysporum)侵染的病程相关蛋白(PR)4基因。通过RT-PCR和染色体步移扩增得到LrPR4的DNA序列及其启动子片段,并初步分析其功能。LrPR4包含一个长432 bp的ORF,编码143个氨基酸残基,具有保守的Barwin结构域,属于II类PR4。qPCR结果显示LrPR4在岷江百合的根、花和鳞片中均有表达,外源信号分子处理和尖孢镰刀菌侵染均诱导LrPR4的表达。LrPR4启动子片段长501 bp,具有MYB、MYC等转录因子结合位点、响应低温、干旱、光、生长素、水杨酸的顺式作用元件。将LrPR4启动子驱动的GUS基因转入烟草(Nicotiana tabacum)中表达,转基因烟草的GUS活性受植物激素茉莉酸甲酯和水杨酸以及病原真菌尖孢镰刀菌、交链格孢、厚垣镰刀菌的诱导。在大肠杆菌中表达并纯化的LrPR4重组蛋白对厚垣镰刀菌、尖孢镰刀菌的菌丝生长具有抑制作用。本研究为后续进一步研究岷江百合PR4蛋白的功能奠定了基础。To unravel the regulatory mechanisms of defense responses against Fusarium wilt in Lilium regale,a pathogenesis-related protein(PR)4 gene in response to Fusarium oxysporum infection was identified from L.regale transcriptome data.The DNA sequence of Lr PR4 and its promoter fragment were cloned through RT-PCR and genome-walking,respectively,and its function was analyzed.LrPR4 contains an ORF of 432 bp,encoding 143 amino acids with a conserved Barwin domain,the deduced LrPR4 belongs to class II PR4.The qPCR results showed that LrPR4 was expressed in the roots,flowers and scales of L.regale,and its expression level was induced by four signaling molecules treatment and F.oxysporum infection.The LrPR4 promoter fragment was 501 bp in length,which has a lot of cis-acting elements,such as the transcription factor binding sites including the MYB and MYC,the low temperature,drought,light,auxin,and SA responding elements.The GUS driven by LrPR4 promoter was transferred into tobacco(Nicotiana tabacum)for expression.The GUS activity of transgenic tobacco was evidently induced by MeJA and SA as well as pathogenic fungi infections of F.oxysporum,Alternaria alternata,and F.chlamydosporum.The purified LrPR4 recombinant protein from Escherichia coli inhibited the mycelial growth of F.chlamydosporum and F.oxysporum.This study lays the foundation for further studies on the function of PR4 protein in L.regale.

关 键 词：岷江百合 病程相关蛋白 尖孢镰刀菌 原核表达 启动子 

分 类 号：S436.8[农业科学—农业昆虫与害虫防治]