本生烟脂质转移蛋白基因克隆及VIGS沉默载体和过量表达载体构建  

Cloning of LTP1 in Nicotiana benthamiana and Construction of VIGS Silencing and Over Expression Vectors

作　　者：梁子健 朱鹏翔 纪兆林[1] 陈孝仁[1] 朱峰[1] Liang Zjian;Zhu Pengxiang;Ji Zhaolin;Chen Xiaoren;Zhu Feng(College of Horticulture and Plant Protection,Yangzhou University,Yangzhou,225009)

机构地区：[1]扬州大学园艺与植物保护学院,扬州225009

出　　处：《分子植物育种》2020年第3期924-930,共7页Molecular Plant Breeding

基　　金：国家自然科学基金资助项目(31500209);2018年度扬州园林科学与工程研究院课题研究项目(YZYL2018-0015);现代农业产业技术体系建设专项资金(CARS-30-3-02)共同资助。

摘　　要：非特异性脂质转移蛋白(non-specific lipid transfer proteins,nsLTPs)是植物中大量存在的小分子脂类结合蛋白,具有多种生物学功能。为研究nsLTPs在调控植物应答生物与非生物胁迫的反应中起的作用,本研究以本生烟(Nicotiana benthamiana)为材料,成功克隆非特异脂质转移蛋白NbLTP1全长基因,其开放阅读框序列为360 bp,编码119个氨基酸,分子重量为12.44 kD,为疏水性蛋白,等电点为7.45。对NbLTP1蛋白质三级结构进行了预测和分析,并进一步构建了烟草脆裂病毒(Tobacco rattle virus,TRV)介导的病毒诱导的基因沉默载体TRV:NbLTP1。荧光定量PCR结果表明通过VIGS可以显著抑制NbLTP1基因的表达。此外还通过In-Fusion克隆技术快速成功构建了pCAMBIA1305-NbLTP1-3*HA过量表达载体。本研究为下一步遗传转化、后续研究NbLTP1调控植物防御生物与非生物胁迫的分子机理、培育新种质提供理论依据。Non-specific lipid transfer proteins(nsLTPs)are small molecular lipid-binding proteins abundant in plants and have various biological functions.In order to study the role of nsLTPs in the regulation of plant response to biotic and abiotic stresses,this study used Nicotiana benthamiana as a material to successfully clone the full-length non-specific lipid transfer protein NbLTP1 gene.Its open reading frame sequence.Is 360 bp and encodes 119 amino acids with a molecular weight of 12.44 kD.It is a hydrophobic protein with an isoelectric point of 7.45.The three-dimensional(3-D)modeling of NbLTP1 was predicted and analysed.We have further successfully constructed the virus-induced gene silencing(VIGS)vector TRV:NbLTP1 mediated by Tobacco rattle virus(TRV).Real-time quantitative PCR showed that the expression of NbLTP1 gene was significantly inhibited by VIGS.In addition,an overexpression vector of pCAMBIA1305-NbLTP1-3*HA was also constructed by In-Fusion cloning technique.This study provides a theoretical basis for genetic transformation,the follow-up study on the molecular mechanism of NbLTP1 regulating plant defense against biotic and abiotic stress,and cultivation of new germplasm.

关 键 词：本生烟(Nicotiana benthamiana) 非特异性脂质转移蛋白 病毒诱导的基因沉默 基因克隆 过量表达 

分 类 号：Q943.2[生物学—植物学] S572[农业科学—烟草工业]