烟草中一个LTR类反转录转座子基因的克隆与功能分析  

Isolation and function analysis of a LTR-retrotransposons from tobacco( Nicotiana tabacum)

作　　者：郭玉双 陈静[2] 刘筱嘉 余世洲 余婧 林世锋 邹颉 付强 张孝廉 赵杰宏 任学良 

机构地区：[1]烟草行业分子遗传重点实验室/贵州省烟草科学研究院,贵州贵阳550081 [2]遵义医学院生化与分子生物学教研室,贵州遵义563009 [3]番禺出入境检验检疫局,广东番禺510006

出　　处：《南京农业大学学报》2015年第6期936-942,共7页Journal of Nanjing Agricultural University

基　　金：中国烟草总公司重大专项项目(110201301004)

摘　　要：[目的]分析烟草中LTR类反转录转座子对植物生长发育的影响。[方法]利用同源克隆及RACE的方法,从烟草栽培品种‘贵烟1号’c DNA中克隆得到了1个烟草反转录转座子的全长c DNA序列,命名为Ntrt1(Gen Bank登录号:KC899077)。利用实时定量RT-PCR研究了该基因在烟草不同组织中的表达水平;构建了基于双生病毒卫星诱导的Ntrt1的基因沉默载体,以中国番茄黄化曲叶病毒(tomato yellow leaf curl China virus,TYLCCNV)为辅助病毒在烟草中沉默了该基因,并调查了基因沉默后的表型。[结果]序列分析表明:该基因全长5 046 bp,编码3个ORF,分别为Gag/pol多聚蛋白、反转录转座子的中心蛋白以及一个整合酶。该基因能在烟草的根、茎、叶中表达,在叶片中的表达量最高。Ntrt1基因沉默烟草与对照相比,烟草植株明显变矮。[结论]Ntrt1在烟草的生长发育中具有重要作用。[Objectives]The aim of the paper is to understand the function of long terminal repeat（LTR）-retrotransposons in plant decelopment. [Methods]A novel（LTR）-retrotransposon gene named Ntrt1 was isolated from Nicotiana tabacum using homology-based and RACE（rapid amplification of cDNA ends）technology. Ntrt1 gene silencing vector was constructed based on the geminivirus satellite and then introduced into Agrobacterium tumefaciens strain EHA105. Agrobecterium harboring recombinant silencing vector and（tomato yellow leaf curl China virus,TYLCCNV）infectious clone were co-agroinoculated into tobacco plants. Meanwhile,tobacco plants co-agroinoculated with TYLCCNV and empty silencing vector was used as the control. [Results]The full-length of Ntrt1 was 5 046 bp and was predicted to encode three important proteins that were Gag/pol polyprotein,central protein of retrotransposon and retrotransposon integrase protein. Real-time quantitative PCR analysis revealed that Ntrt1 was expressed ubiquitously in tobacco including leaf,root and stem and the expression level was highest in leaf. Real-time quantitative PCR analysis revealed that the expression level of Ntrt1 was reduced to 15% compared with control. Virus-induced gene silencing mediated silencing of Ntrt1 led to dwarfing in tobacco. [Conclusions]These results suggested that Ntrt1 was required for plant development.

关 键 词：反转录转座子 烟草植株 基因沉默 同源克隆 R类 全长CDNA序列 实时定量RT-PCR 中国番茄黄化曲叶病毒 

分 类 号：Q943.2[生物学—植物学]