烟草坏死病毒A诱导的基因沉默及其条件优化  

Development and Optimization of Tobacco necrosis virus A Induced Gene Silencing in Nicotiana benthamiana

作　　者：高阳[1] 张永亮[1] 张晓峰[1] 韩成贵[1] 于嘉林[1] 李大伟[1] 

机构地区：[1]中国农业大学农业生物技术国家重点实验室,北京100193

出　　处：《生物化学与生物物理进展》2011年第10期919-928,共10页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30325001);国家重点基础研究发展计划(973)资助项目(2006CB101903)~~

摘　　要：以烟草坏死病毒A中国分离物(Tobacco necrosis virus A Chinese isolate,TNV-AC)侵染性cDNA克隆为基础,通过基因替换、基因插入策略构建获得多种重组TNV-AC,比较了外源基因片段插入位置、插入形式及接种植物培养温度对TNV-AC诱导的基因沉默的影响.外源基因片段替换CP基因19～828 nt的重组TNV-AC丧失了在本生烟中的系统移动能力,也不能有效诱导相应基因发生明显的沉默,说明替换策略不适合于TNV-AC.向CP基因终止密码子UAG附近插入外源基因片段后,TNV-AC仍可进行复制,但最适的插入位点位于UAG之后,且容纳外源片段的长度约为120 nt.当外源片段以反向重复的形式插入UAG之后,诱导基因沉默的效率较高.接种植物的培养温度也会显著影响基因沉默的效率以及插入片段的稳定性,低温(18℃)条件下诱导NbPDS基因沉默的效率明显高于高温(24℃)条件,且沉默表型可持续110天以上.除了本生烟PDS基因,TNV-AC沉默载体还可诱导本生烟sulfur基因Su和镁离子螯合酶H亚基基因ChlH发生沉默,以上结果说明,TNV-AC具有开发为本生烟基因功能鉴定的新VIGS载体的潜力.A series of recombinant viruses were engineered by gene replacements or gene insertions into an infectious cDNA clone of Tobacco necrosis virus A Chinese isolate （TNV-AC）. TNV-AC-based virus induced gene silencing （VIGS） containing different structural arrangementg of sequences targeting specific genes and variations in the positions of exogenous fragment insertions were evaluated along with temperature effects on inoculated plants. Replacement of the coat protein （CP） gene by exogenous gene fragments could not be applied to TNV-Ac because the chimeric TNV-Ac constructs were unable to induce silencing of corresponding endogenous gene and derivatives lacking the CP could not move systematically in Nieotiana benthamiana. We found that the optimal position for insertion of exogenous gene fragments was the region immediately behind the stop codon of the CP gene, and the maximum lengths of fragments that could be accommodated within virions was around 120 nt. An NbPDS fragment inserted into TNV-Ac as an inverted repeat produced a VIGS derivative that could silence endogenous NbPDS in N. benthamiana with extremely high efficiency. We also found that temperature could significantly affect gene silencing efficiency of inoculated plants and the stability of heterologous gene fragments. Plants grown at 18 ℃ exhibited substantially higher gene silencing efficiency compared with 24℃, and the duration of silencing persisted for more than 110 days. The TNV-AC-based VIGS vectors were also able to induce efficient silencing of endogenous Su and ChlH genes in N. benthamiana. Taken together, our results show that TNV-Ac has the potential for development into a novel VIGS vector suitable for functional genomics ofN. benthamiana.

关 键 词：烟草坏死病毒A 病毒诱导的基因沉默 条件优化 

分 类 号：Q939.46[生物学—微生物学]