本氏烟半胱氨酸蛋白酶基因家族特征及其在TMV侵染中的功能  

Characteristics of Cysteine Proteinase Gene Family in Nicotiana benthamiana and Its Function During TMV Infection

作　　者：裴悦宏 李凤巍 刘维娜 温玉霞 朱鑫 田绍锐 樊光进 马小舟 孙现超[1] PEI YueHong;LI FengWei;LIU WeiNa;WEN YuXia;ZHU Xin;TIAN ShaoRui;FAN GuangJin;MA XiaoZhou;SUN XianChao(Chongqing Key Laboratory of Plant Disease Biology,College of Plant Protection,Southwest University,Chongqing 400715)

机构地区：[1]西南大学植物保护学院植物病害生物学重庆市重点实验室,重庆400715

出　　处：《中国农业科学》2022年第21期4196-4210,共15页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31870147);中国烟草总公司重庆公司科技项目(A20201NY02-1306,B20202NY1338,B20211-NY1315);广西中烟工业有限责任公司项目(2021450000340029);国家大学生创新创业训练项目(202210635030)。

摘　　要：【目的】明确抗病毒化合物氯吲哚酰肼(chloroinconazide,CHI)诱导的本氏烟(Nicotiana benthamiana)半胱氨酸蛋白酶(cysteine proteinase,CP)在烟草花叶病毒(tobacco mosaic virus,TMV)侵染过程中的作用及其基因家族特征,为理解茄科作物抗病毒分子机制及化学调控提供理论依据。【方法】利用全基因组策略,从茄科作物基因组数据库Sol Genomics Network中检索获得本氏烟NbCP的全家族基因序列,利用生物信息学分析NbCP基因家族的进化关系、Motif基序及启动子顺式作用元件。根据生物信息分析结果,利用实时荧光定量PCR技术分析TMV侵染后NbCP基因的表达,以此筛选出潜在抗病蛋白。使用烟草脆裂病毒(tobacco rattle virus,TRV)介导的基因沉默技术和马铃薯X病毒(potato virus X,PVX)介导的基因过表达技术验证该关键蛋白对TMV-GFP侵染的影响,结合实时荧光定量PCR手段探索该关键蛋白的抗病毒机制。【结果】NbCP家族共有24个成员,根据其染色体定位命名为NbCP1—NbCP24。进化关系分析表明NbCP分成5个亚家族,其中Group V含有7个NbCP,而Group I仅含2个NbCP;Motif分析表明不同分支NbCP具有相似的motif分布;启动子顺式作用元件分析表明NbCP家族基因受光调控,并且多个NbCP家族基因启动子含有茉莉酸甲酯、水杨酸甲酯、脱落酸、赤霉素和生长素等激素响应元件。结合生物信息学分析,从上述各个亚家族中分别筛选1个关键潜在抗病相关NbCP基因(NbCP8、NbCP12、NbCP13、NbCP18、NbCP22),并利用实时荧光定量PCR技术分析上述基因在TMV侵染第5天的表达,发现NbCP8表达差异性最大,上升了2.6倍,NbCP8在叶中的表达量最高,其次是茎和根,在花中的表达量最低。TRV介导的NbCP8沉默能显著增加TMV-GFP的侵染,而PVX介导的NbCP8过表达能显著抑制TMV-GFP侵染,表明NbCP8作为植物正调控因子抑制病毒侵染。沉默NbCP8显著抑制了水杨酸信号途径相关基因PR1以及茉【Objective】The objective of this study is to identify the effects of Nicotiana benthamiana cysteine proteinase(CP)induced by the antiviral compound chloroinconazide(CHI)on the infection of tobacco mosaic virus(TMV)and its gene family characteristics,and to provide a theoretical basis for understanding the antiviral molecular mechanism and chemical regulation of Solanaceae crops.【Method】Genome-wide analysis of NbCP genes was identified from Sol Genomics Network and the evolutionary relationships,motifs and promoter cis-acting elements of NbCP gene family were analyzed by bioinformatics.According to the results of bioinformatics analysis,the expression of NbCP genes after TMV infection was analyzed by real-time fluorescence quantitative PCR(qRT-PCR),so as to screen out potential disease-resistant protein.The effect of this key protein on TMV-GFP infection was verified by gene silencing mediated by tobacco rattle virus(TRV)and gene overexpression mediated by potato virus X(PVX).qRT-PCR and biochemical methods were combined to determine the antiviral mechanism of this key protein.【Result】There were 24 NbCP genes in N.benthamiana,which were named NbCP1-NbCP24 according to their chromosome location.The phylogenetic analysis showed that the NbCPs were divided into five subfamilies,of which Group V contained seven NbCPs,while Group I contained only two NbCPs.Motif analyses showed that NbCPs of different branches had similar motif distribution.Promoter cis-acting elements analysis showed that NbCP family genes were all regulated by light,and several NbCP family gene promoters contained hormone response elements such as methyl jasmonate(MeJA),methyl salicylate(MeSA),abscisic acid(ABA),gibberellin(GA)and auxin(IAA).Combined with bioinformatics analysis,five key potential disease-resistant related NbCP genes(NbCP8,NbCP12,NbCP13,NbCP18,and NbCP22)were screened from the above subfamilies,and the expression of these NbCP genes under TMV infection at the 5th day was analyzed by qRT-PCR,and it was found that the expre

关 键 词：本氏烟 全基因组 半胱氨酸蛋白酶 NbCP8 烟草花叶病毒 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]