激发子诱导的过敏性细胞死亡调控基因的筛选

Screening of genes regulating hypersensitive cell death induced by elicitors through VIGS in Nicotiana benthamiana

采用病毒诱导基因沉默技术从本氏烟(Nicotiana benthamiana)cDNA文库中筛选受3种不同激发子(Nep1、harpin和INF1)诱导的过敏性细胞死亡的调控基因。以农杆菌Gv3101为宿主、利用PVX病毒表达载体构建了含有7 000个单克隆的本氏烟cDNA文库,采用牙签刺伤法和注射接种法,从6 000个克隆中筛选到了34个能使激发子诱发的过敏性细胞死亡表型发生改变的克隆。对上述阳性克隆进行序列测定和分析,发现其中13个与已知的基因具有较高的同源性,其中克隆Nb14-4编码1个特异的ALY蛋白;克隆Nb3-9和Nb4-26分别编码1个苹果酸脱氢酶和果糖二磷酸醛缩酶;Nb4-28编码普通烟草中的14-3-3 d-2蛋白。还有21个克隆在公共数据库中没有同源基因。上述结果表明:利用病毒表达载体建立cDNA文库,并采用病毒诱导基因沉默技术可从烟草全基因组内筛选受不同激发子诱导的过敏性细胞死亡的调控基因,为揭示植物过敏性细胞死亡分子机制提供试验依据。

Virus-induced gene silencing（VIGS）was used to isolate cDNAs from tobacco（Nicotiana benthamiana）.Here,we used VIGS to screen genes regulating hypersensitive cell death（HCD）triggered by three various elicitors including Nep1,harpin and INF1.The cDNA library was constructed into a binary potato virus X（PVX）-based expression vector and Agrobacterium tumefeciens（Gv3101）.6 000 clones were individually toothpick-inoculated or injection-inoculated into leaves of N.benthamiana to induce targeted gene silencing.These tobacco leaves were infiltrated by different elicitor 3-4 weeks after inoculation with A.tumefeciens,and 34 clones were identified to suppress or attenuate HCD.13 different genes were identified by sequencing,in which Nb14-4,Nb3-9,Nb4-26 and Nb4-28 encode a specific ALY protein,malate dehydrogenase,fructose-bisphosphate aldolase and 14-3-3 d-2 protein,respectively.These genes may be involved in elicitor-trigger HCD.This work will help to elucidate molecular base of elicitor-trigger HCD.And the result indicated that this functional screening method via VIGS was a feasible strategy to identify cDNAs of N.benthamiana that regulate HCD.