基于过表达策略验证烟草PVY抗性候选基因eIF4E的功能

【作者】 李杰；

【导师】 任如意；

【作者基本信息】 牡丹江师范学院 ， 遗传学， 2015， 硕士

【摘要】 马铃薯Y病毒(Potato virus Y,PVY)是马铃薯Y病毒组的主要成员之一。马铃薯Y病毒侵染寄主的机制是与位于植物体内的一个翻译起始因子e IF4E有关,e IF4E与病毒RNA5’端的类似于帽子结构的连接蛋白VPg互作,加快了病毒RNA的翻译。在辣椒和番茄中均已得到证实,因此尝试在烟草中找到与PVY抗性相关的e IF4E家族中的对应成员。本研究首先参照豌豆、辣椒、番茄、莴苣及普通烟草中e IF4E和/或e IF(iso)4E m RNA序列,在烟草基因组数据库(红花大金元)中比对,获得11个烟草中的e IF4E序列。再通过同源性分析以及RT-PCR与测序,得到2个PVY抗性候选基因-e IF4E-6、e IF4E-10。分别设计e IF4E-6、e IF4E-10的特异性引物,从感病材料LJ925中克隆,得到大小相同的750 bp的e IF4E-6、e IF4E-10基因,构建e IF4E-6基因和e IF4E-10基因的植物表达载体,用农杆菌介导法将e IF4E-6基因和e IF4E-10基因转入抗性烟草品种VAM中,经过在预培养2-3 d、农杆菌浸染6min、共培养3 d、100 mg/L的卡那霉素进行抗性筛选、炼苗和移栽等一系列步骤,得到了e IF4E-6的抗性苗300株和e IF4E-10的抗性苗280株,从得到的转化苗中各选出120株进行PCR扩增检测,e IF4E-6转入烟草后的阳性转化率为88.3%,e IF4E-10基因转入烟草的阳性转化率为84.2%。e IF4E-6、e IF4E-10各选出60株转化株和10株对照植株,采用摩擦接种的方式对植株进行病毒接种,统计结果e IF4E-6转化苗的发病率达到81.7%,e IF4E-10转化苗的发病率仅有5%,可能原因是出现了抗性突破。结果表明,e IF4E-6与PVY抗性相关,e IF4E-10与PVY抗性不相关。更多还原

【Abstract】 Potato virus Y(Potato virus Y, PVY) is one of the main members of the potato virus Y group.Mechanism of potato Y virus infected host is associated with a translation initiation factor e IF4 E in plants, e IF4 E interacted with viral RNA5’ end similar to the cap structure connection protein VPg, to speed up the translation of viral RNA.In pepper and tomato have been confirmed, in order to verify in tobacco and PVY resistance related details of which members of the e IF4 E family, a series of experiment design.This study first referred to the pea, chili, tomato, lettuce and tobacco e IF4 E and/or e IF(iso)4E m RNA sequence in the genome database(tobacco,Honghuadajinyuan) in comparison,obtained 11 e IF4 E sequences in tobacco. Through the analysis of homology, 2 PVY candidate resistance genes-e IF4E-6, e IF4E-10.Specific primers were designed and e IF4E-6, e IF4E-10, cloned from susceptible material LJ925, e IF4E-6, e IF4E-10 gene 750 bp of the same size, construction of expression vector of e IF4E-6 gene and e IF4E-10 gene in plants, the e IF4E-6 gene and e IF4E-10 gene into tobacco resistance cultivar VAM by Agrobacterium mediated method, through the training of 2-3 d,agrobacterium 6min, co culture 3 d, 100 mg/L kanamycin resistant screening, hardening and transplanting a series of steps, obtained the e IF4E-6 300 transformation seedlings and e IF4E-10 280 transformation seedlings,PCR method for identifying positive plants obtained from each selected 120 transformation seedlings strain was amplified by PCR detection, e IF4E-6 into the positive tobacco transformation rate was 88.3 % after transformation into tobacco e IF4E-10 gene, positive rate was 84.2 %. EIF4E-6 and e IF4E-10 were elected for each of 60 transgenic strains and 10 control plants using friction inoculation method of virus inoculation on plant, the statistical results of the onset of e IF4E-6 transformed seedling rate reached 81.7 %, the incidence of e IF4E-10 transformed seedling rate of only 5 %, individual transformation seedlings resistance breakthrough, preliminary results indicated that e IF4E-6 and PVY resistance is strong correlation. e IF4E-10 and PVY resistance is not related.更多还原