摘要
【目的】番茄(Solanum lycopersicum)作为重要的蔬菜作物,其生长受到包括害虫、真菌、细菌和病毒等各种生物因素的危害。明确番茄抗性基因SlN-like的抗病毒功能与机制,为番茄的抗病毒育种与抗病毒药剂的靶向开发提供理论依据。【方法】从茄科植物基因组数据库Solanaceae Genomics Network中获得SlN-like的全长,并将其分为4段,利用融合聚合酶链式反应(fusion PCR)扩增获得SlN-like的序列全长;通过生物信息学分析SlN-like的进化关系、蛋白特征、保守结构域、亚细胞定位以及互作关系;通过实时荧光定量PCR分析SlN-like在番茄根、茎、叶、花和果实中的表达情况及其在烟草花叶病毒(tobacco mosaic virus,TMV)侵染后的叶片表达量;借助烟草脆裂病毒(tobacco rattle virus,TRV)介导的基因沉默技术(virus induced gene silencing,VIGS)沉默番茄内源SlN-like,摩擦接种TMV-GFP于沉默植株,明确SlN-like对病毒侵染的影响。实时荧光定量PCR分析沉默植株中脱落酸(abscisic acid)、茉莉酸(jasmonic acid)和乙烯(ethylene)激素相关基因的表达量及SlN-like在外施乙烯利(ethephon,ETH)3、6、12、24 h后的表达情况,最终明确SlN-like调控激素途径响应病毒侵染的机制。【结果】通过分子克隆与融合PCR技术,从番茄品种Micro-Tom中克隆获得全长3444 bp的SlN-like,上传至NCBI获得序列号MW792493。通过生物信息学分析发现SlN-like含有TIR、NB-ARC和NACHT结构域,并与马铃薯(Solanum tuberosum)N-like(AAP44394.1)亲缘关系最近。SlN-like在番茄各组织中均有表达,在茎中的表达量最高,其次是根、花,叶和果实中的表达量最低。TMV-GFP侵染番茄后第5、7天SlN-like的表达显著高于PBS处理,分别是PBS处理的1.6和2.2倍,并且TMV-GFP侵染会使SlN-like的表达持续升高。TRV载体介导沉默番茄的SlN-like,发现沉默78.3%的SlN-like不会影响番茄生长表型,但可促进TMV-GFP侵染;实时荧光定量PCR分析发现�
【Objective】As an important vegetable crop,tomato(Solanum lycopersicum)is endangered by various biological factors including pests,fungi,bacteria and viruses.The objective of this study is to clarify the antiviral function and mechanism of S.lycopersicum resistance gene SlN-like,and to provide a theoretical basis for the genetic breeding of antiviral S.lycopersicum and the targeted development of the antiviral agents.【Method】The full length of SlN-like was obtained from the Solanaceae Genomics Network database and was divided into four segments,fusion PCR was used to amplify the full length of sequence.Bioinformatics was used to analyze the evolutionary relationship,protein characteristics,conserved domains,subcellular location and interaction relationship of SlN-like.Real-time fluorescent quantitative PCR was used to analyze the SlN-like expression in S.lycopersicum roots,stems,leaves,flowers and fruits and its response after tobacco mosaic virus(TMV)infection.S.lycopersicum endogenous SlN-like was silenced using tobacco rattle virus(TRV)-mediated gene silencing technology,and the silent plants were inoculated with TMV-GFP to clarify the influence of SlN-like on virus infection.The expressions of abscisic acid(ABA),jasmonic acid(JA)and ethylene(ET)hormone-related genes in silenced plants,and the expression of SlN-like after application of ethephon(ETH)for 3,6,12 and 24 h were analyzed by real-time fluorescence quantitative PCR to investigate the mechanism of SlN-like regulatory hormone pathway in response to virus infection.【Result】Through molecular cloning and fusion PCR technology,a 3444 bp SlN-like was cloned from S.lycopersicum variety Micro-Tom,and uploaded to NCBI to obtain the sequence number MW792493.Through bioinformatics analysis,it was found that SlN-like contains TIR,NB-ARC and NACHT domains,and is closely related to Solanum tuberosum N-like(AAP44394.1).SlN-like expressed in all tissues of S.lycopersicum,with the highest expression in stems,followed by roots,flowers,leaves and fruits.After
作者
刘昌云
李欣羽
田绍锐
王靖
裴悦宏
马小舟
樊光进
汪代斌
孙现超
LIU ChangYun;LI XinYu;TIAN ShaoRui;WANG Jing;PEI YueHong;MA XiaoZhou;FAN GuangJin;WANG DaiBin;SUN XianChao(College of Plant Protection,Southwest University,Chongqing 400715;Key Laboratory of Horticulture Science for Southern Mountainous Regions of Ministry of Education,College of Horticulture and Landscape Architecture,Southwest University,Chongqing 400715;Chongqing Tobacco Science Research Institute,Chongqing 400715)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第20期4348-4357,共10页
Scientia Agricultura Sinica
基金
国家自然科学基金(31870147,31670148)
西南大学大学生创新创业训练计划(X202010635495)
中国烟草总公司重庆公司科技项目(A20201NY02-1306,B20211-NY1315,B20202NY1338)。
关键词
番茄
SlN-like
烟草花叶病毒
基因表达
乙烯
Solanum lycopersicum
SlN-like
tobacco mosaic virus(TMV)
gene expression
ethylene
