摘要
该研究以抗病品种中国野生毛葡萄‘商-24’和感病品种欧洲葡萄‘红地球’为材料,利用RT-PCR方法克隆TLP15基因,分别命名为VqTLP15和VvTLP15(GSVIVT01018769001),对其进行生物信息学分析、亚细胞定位、转化拟南芥,并接种不同病原菌观察分析转基因株系的抗性,采用qRT-PCR检测SA和JA/Eth信号途径以及调控气孔运动的相关基因表达。结果显示:(1)成功克隆获得VqTLP15基因的开放阅读框(ORF);氨基酸序列比对显示,VqTLP15基因与葡萄基因组网站欧洲葡萄‘黑比诺’VvTLP15和‘红地球’克隆的VvTLP15基因的同源性分别为98.99%和99.66%。(2)亚细胞定位表明,VqTLP15定位于细胞质。(3)成功获得VqTLP15转基因拟南芥株系(L1、L2、L3)。(4)接种观察发现:白粉菌处理7 d后转基因株系对白粉菌的抗性较野生型(Col-0)提高,且其叶片的白粉菌孢子浓度显著低于Col-0;灰霉菌诱导的叶片坏死性损伤在转基因株系(L1、L2和L3病斑面积>40%的比例分别为71%、62%和67%)中显著大于Col-0(43%);接种PstDC3000后转基因株系叶片的病害表型没有Col-0明显,叶片孔径减小程度大于Col-0,且细菌浓度低于Col-0。(5)组织化学染色分析表明:白粉菌处理后转基因株系叶片胼胝质沉积、细胞死亡率和O2^-·水平都显著大于Col-0;灰霉菌处理后转基因株系的细胞死亡率、H2O2和O2^-·水平均高于Col-0;PstDC3000处理后细胞死亡率和O2^-·积累水平都高于Col-0。(6)qRT-PCR检测显示:接种白粉菌后,转基因株系中PR1和ICS1的表达水平均升高,PR1表达在接种72 h时达到峰值,而ICS1在接种120 h时达到峰值,LOX3的表达水平逐渐降低,并于接种120 h时降至最低水平,但仍高于Col-0;接种灰霉菌后,转基因株系中PR1、NPR1和PDF1.2基因的表达均上调,并在接种48 h时达到峰值,LOX3基因的表达水平下降,但仍高于Col-0;接种PstDC3000后,转基因株系中PR1、PDF1.2和NHL10的表达均高于Col-0,但WRKY53的表达低于Col-0,L1中COI1、FRK1、ATPPC2、FLS2、OST1的表达水平高于Col-0;接种flg22或LPS后,L1中COI1基因的表达低于Col-0,但ATPPC2、FLS2、OST1基因的表达水平高于Col-0。研究表明,过量表达VqTLP15基因降低了对白粉菌和PstDC3000的敏感性,增加了对灰霉菌的敏感性。VqTLP15基因可能通过介导水杨酸(SA)和茉莉酸/乙烯(JA/Eth)信号转导途径以及气孔免疫反应来参与植物的抗病防御反应,为葡萄抗病分子育种提供了一个可能的候选基因。
The TLP15 was cloned from the resistant variety of Chinese wild Vitis quinquangularis‘Shang-24’and the susceptible variety of Vitis vinifera cv.‘Red Globe’by RT-PCR,named VqTLP15 and VvTLP15(GSVIVT01018769001),respectively.We studied the gene by bioinformatics analysis,subcellular localization analysis,transformed Arabidopsis thaliana to observe and analyze transgenic Arabidopsis resistance to different pathogens.We also analyzed the expression of related genes involving in the SA or JA/Eth signaling pathways and regulating stomatal movement by qRT-PCR.The results showed:(1)the open reading frame(ORF)of the VqTLP15 was obtained.VqTLP15 showed 98.99%and 99.66%sequence homology to VvTLP15 cloned from V.vinifera cv.‘PinotNoir’and VvTLP15 cloned from V.vinifera cv.‘Red Globe’by amino acid sequence alignment,respectively.(2)Subcellular localization indicates that VqTLP15 was localized in the cytoplasm.(3)VqTLP15 transgenic A.thaliana(L1,L2,L3)were successfully obtained.(4)Inoculation observation showed that the transgenic A.thaliana lines were inoculated with G.cichoracearum and found to have greater resistance than Col-0 to powdery mildew at 7 days post-inoculation,and the concentration of powdery mildew spores from infected leaves were significantly lower in the transgenic lines than that in Col-0.The leaf necrotic damage(43%)induced by Botrytis cinerea was significantly greater than that of Col-0 in transgenic lines(71%,62%and 67%of lesion area>40%in L1,L2 and L3,respectively).After PstDC3000 inoculation,the disease symptoms were less apparent in the transgenic lines than in Col-0.The reduction of leaf stomatal aperture were greater in the transgenic lines than in Col-0,and the concentration of bacteria was lower in the transgenic lines than in Col-0.(5)A histochemical staining assay showed:Callose deposition was more widely distributed,and the frequency of cell death and O2^-· levels were higher in the transgenic lines than in Col-0 after powdery mildew inoculation.The extent of cell death,and levels of H2O2 and O2^-·were higher in the transgenic lines than in Col-0 after B.cinerea inoculation.After PstDC3000 inoculation,the frequency of cell death and the degree of O2^-· accumulation were both higher in the transgenic lines than in Col-0.(6)qRT-PCR results showed:after powdery mildew inoculation,the expression levels of both PR1 and ICS1 increased in the transgenic lines,PR1 expression peaked at 72 hpi,and ICS1 peaked at 120 hpi.And the expression levels of LOX3 gradually decreased to the lowest level at 120 hpi in the transgenic lines,but remained higher than in Col-0.The expression levels of PR1,NPR1 and PDF1.2 all increased following B.cinerea inoculation,and peaked at 48 hpi,and the expression levels of LOX3 decreased,but remained higher than in Col-0.After PstDC3000 inoculation,PR1,PDF1.2 and NHL10 expression were all more highly expressed in the transgenic lines than in Col-0,but WRKY53 expression was lower in the transgenic lines than in Col-0.The expression levels of COI1,FRK1,ATPPC2,FLS2 and OST1 were higher in L1 than in Col-0 following PstDC3000 inoculation.After treatment with flg22 or LPS,the expression of COI1 was lower in L1 than in Col-0,but the expression levels of ATPPC2,FLS2,OST1 were higher in L1 than in Col-0.This study showed that overexpression of the VqTLP15 reduced the sensitivity to powdery mildew and PstDC3000 and increased the sensitivity to B.cinerea.VqTLP15 may involve in the defense response of plants via mediating salicylic acid(SA)and jasmonic acid/ethylene(JA/Eth)signaling pathways and stomatal immune response,and may present a candidate for future grape molecular breeding for disease resistance.
作者
王雅
闫筱筱
张修铭
李智
王西平
WANG Ya;YAN Xiaoxiao;ZHANG Xiuming;LI Zhi;WANG Xiping(College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China;State Key Laboratory of Crop Stress Biology in Arid Areas, Yangling, Shaanxi 712100, China;Ministry of Agriculture Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest, Yangling, Shaanxi 712100, China)
出处
《西北植物学报》
CAS
CSCD
北大核心
2020年第5期727-738,共12页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(31272136)
葡萄种质资源与育种创新团队(2013KCT-25)。
