摘要
[目的]本文旨在研究E3泛素连接酶基因的沉默对植物基础免疫及线虫寄生的影响,为揭示植物寄生线虫侵染寄主的分子机制提供理论依据。[方法]利用烟草脆裂病毒(TRV)介导的基因沉默技术(VIGS)沉默本氏烟E3泛素连接酶基因NbE 3R14,用flg22处理基因沉默烟草叶片,RT-qPCR检测PTI(病原相关分子模式触发免疫,PAMPs-triggered immunity)相关基因的表达水平;接种辣椒疫霉于基因沉默烟草叶片,观察叶片病斑症状;采用室内人工接种法测定靶基因的沉默对南方根结线虫寄生的影响。[结果]RT-qPCR检测显示:农杆菌转化的烟草植株中NbE 3R14基因已被成功沉默,flg22处理基因沉默烟草叶片激发的PTI相关基因GRAS 2、WRKY7及WRKY8表达量分别下降41.3%、63.9%及61.8%,与野生型对照(WT)相比均有显著性差异(P<0.05);接种辣椒疫霉36 h后,TRV∷NbE 3R14处理烟草叶片的病斑比WT增大28.5%;南方根结线虫在TRV∷NbE 3R14处理植株上产生的根结数、雌虫数及卵块数与WT相比分别显著增加22.1%、27.4%和17.2%(P<0.05),单卵块卵量则没有差异;而在TRV∷GFP处理植株上产生的根结数、雌虫数、卵块数以及单卵块卵量与WT均无明显差异。[结论]沉默NbE 3R14基因能干扰植物基础免疫,从而导致烟草对辣椒疫霉及南方根结线虫的侵染更为感病。
[Objectives]The E3 ubiquitin ligase plays an important role in manipulating the response of plant to biotic and abiotic stresses.Understanding the effects of E3 gene silencing on plant basal immunity and nematode parasitism will provide a theoretical basis for revealing the molecular mechanism for the infection of plant-parasitic nematodes to plants.[Methods]The silencing of E3 ubiquitin ligase gene NbE 3R14 in Nicotiana benthamiana was carried out using tobacco rattle virus-mediated gene silencing technology(VIGS).After silencing NbE 3R14,the tobacco leaves were treated with synthetic peptide flg22 and the expression levels of PAMP-triggered immunity(PTI)-related genes were detected by RT-qPCR.Meanwhile,the tobacco leaves were inoculated with Phytophthora capsici and the symptoms on leaves were observed.The effects of silencing target gene on the parasitism of Meloidogyne incognita were tested by artificial inoculation in the greenhouse.[Results]The detection of RT-qPCR showed that the NbE 3R14 gene in tobacco plants agroinfiltrated with TRV∷NbE 3R14 was successfully silenced,and the expression levels of genes related with PTI triggered by flg22,including GRAS 2,WRKY 7 and WRKY 8 gene,decreased 41.3%,63.9%and 61.8%,respectively,which were significantly different from those in the wild type(WT)tobacco(P<0.05).The average diameter of lesion on the leaves of TRV∷NbE 3R14 tobacco plants after 36 h of inoculation with P.capsici increased 28.5%when compared with those on the WT tobacco.The number of root knots,females and egg masses produced by M.incognita on TRV∷NbE 3R14 tobacco plants increased 22.1%,27.4%and 17.2%,respectively,when compared with those on the WT tobacco,which showed the significant differences(P<0.05),while the number of eggs per egg mass didn’t show significant difference.However,the numbers of root knots,females,egg masses and eggs per egg mass produced on TRV∷GFP were significantly different from those on WT tobacco.[Conclusions]The silencing of NbE 3R14 in N.benthamiana reduced the level of plant basal immunity,which resulted in the higher sensitivity to the infection either with P.capsici or with M.incognita.
作者
刘琳硕
贺祥
李红梅
元青
王暄
LIU Linshuo;HE Xiang;LI Hongmei;YUAN Qing;WANG Xuan(College of Plant Protection/Key Laboratory of Integrated Management of Crop Diseases and Pests,Ministry of Education,Nanjing Agricultural University,Nanjing 210095,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2020年第1期65-71,共7页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(31872923)
