苹果炭疽叶枯病菌GcAP1复合体β亚基基因的克隆及功能分析

发布时间：2018-05-21 06:53

本文选题：衔接蛋白 + 果胶酶　；参考：《中国农业科学》2017年08期

【摘要】：【目的】明确衔接蛋白(adaptor protein)GcAP1复合体β亚基在苹果炭疽叶枯病菌(Glomerella cingulata)生长发育和致病过程中的功能,检测GcAP1β在该菌中的时空表达模式,并揭示其是否调控多聚半乳糖醛酸内切酶(endopolygalacturonase)基因CgPG1和CgPG2、果胶裂解酶(pectin lyase)基因pnl-1和pnl-2以及果胶酸酯裂解酶(pectate lyase)基因pelA和pelB的表达,为深入开展苹果炭疽叶枯病菌衔接蛋白在致病信号传导途径中的分子机制研究打下基础。【方法】通过构建GcAP1β基因敲除载体和GcAP1β-gfp融合表达载体,利用农杆菌介导的遗传转化技术(ATMT)获得Δgcap1β突变体和GcAP1β恢复菌株Δgcap1β-GcAP1β,并由RT-PCR和Southern杂交分析进行鉴定。以野生型菌株W16为对照,对Δgcap1β突变体和GcAP1β恢复菌株Δgcap1β-GcAP1β的生长速度、产孢能力、分生孢子萌发率及附着胞形成率和致病性进行测定。利用生物信息学软件Prot Comp 9.0和TMHMM对GcAP1β蛋白进行结构分析,并结合GcAP1β-GFP信号观测,进行GcAP1β的亚细胞定位。利用qRT-PCR技术,检测GcAP1β在菌丝、分生孢子、芽管、附着胞和侵染阶段的表达量,并检测CgPG1、CgPG2、pnl-1、pnl-2、pelA和pelB在野生型菌株和Δgcap1β突变体中的表达量。【结果】GcAP1β基因全长2 321bp,含有3个内含子,编码720个氨基酸。与野生型菌株W16相比,Δgcap1β突变体菌落成褶皱状,菌丝生长速度明显减慢,而分生孢子产量、分生孢子萌发率、附着胞形成率无显著差异。Δgcap1β致病力明显降低,仅在苹果叶片上引起极小的点状斑。GcAP1β基因恢复菌株Δgcap1β-GcAP1β完全修复了因GcAP1β基因缺失造成的表型缺陷。荧光检测显示,融合蛋白GcAP1β-GFP分布于细胞质中。qRT-PCR检测结果表明,GcAP1β在苹果炭疽叶枯病菌各个发育阶段都有表达,且在侵染后表达量相对最高。GcAP1β的缺失导致CgPG1表达量降低至20.3%,CgPG2表达量降低至16.5%,pnl-1表达量降低至8.2%,pnl-2表达量降低至14.4%,pelA表达量降低至4.4%,pelB表达量降至0.8%。【结论】衔接蛋白GcAP1复合体分布于细胞质中,是苹果炭疽叶枯病菌生长发育所需要的;GcAP1调控CgPG1、CgPG2、pnl-1、pnl-2、pelA和pelB的表达,是苹果炭疽叶枯病菌一个重要的毒力因子。
[Abstract]:[objective] to investigate the function of 尾 subunit of adaptor protein)GcAP1 complex in the growth, development and pathogenicity of Glomerella cingulata, and to detect the temporal and spatial expression pattern of GcAP1 尾 in this bacterium. The expression of CgPG1 and CgPG2, pnl-1 and pnl-2 of pectin lyase, and pelA and pelB of pectate lyase were investigated. To lay a foundation for the further study of the molecular mechanism of the binding protein of anthracis in the pathogenicity signal transduction pathway. [methods] the GcAP1 尾 gene knockout vector and the GcAP1 尾 -gfp fusion expression vector were constructed. The 螖 gcap1 尾 mutants and GcAP1 尾 restoring strain 螖 gcap1 尾 -GcAP1 尾 were obtained by Agrobacterium tumefaciens-mediated genetic transformation and identified by RT-PCR and Southern hybridization. The growth rate, sporulation ability, conidia germination rate, attachment formation rate and pathogenicity of 螖 gcap1 尾 mutant and GcAP1 尾 restoring strain 螖 gcap1 尾 -GcAP1 尾 were determined by using wild-type strain W16 as control. The structure of GcAP1 尾 protein was analyzed by bioinformatics software Prot Comp 9.0 and TMHMM, and the subcellular localization of GcAP1 尾 was carried out with the observation of GcAP1 尾 -GFP signal. QRT-PCR technique was used to detect the expression of GcAP1 尾 in hyphae, conidia, bud tube, appressorium and infection stage, and to detect the expression of CgPG1CgPG2pnl-1pnl-2pnl-2pnl-2pelA and pelB in wild type strains and 螖 gcap1 尾 mutants. [results] the whole length of GcAP1 尾 gene was 2 321bpp, containing three introns. Encoding 720 amino acids. Compared with wild-type strain W16, 螖 gcap1 尾 mutant colony became fold shape, hyphal growth rate slowed obviously, but conidial yield, conidia germination rate and appressant formation rate were not significantly different. 螖 gcap1 尾 pathogenicity was significantly decreased. The phenotypic defect caused by the deletion of GcAP1 尾 gene was completely repaired by 螖 gcap1 尾 -GcAP1 尾 gene restoring strain 螖 gcap1 尾 in apple leaves. Fluorescence detection showed that the fusion protein GcAP1 尾 -GFP was distributed in the cytoplasm. QRT-PCR results showed that GcAP1 尾 was expressed in all developmental stages of anthracis. After infection, the relative highest expression of .GcAP1 尾 resulted in the decrease of CgPG1 expression to 20.3kg. the expression of CgPG2 decreased to 16.5pnl-1. The expression of pnl-1 decreased to 8.2pnl-2, and the expression of pnl-2 decreased to 14.4pelA to 4.4pelB. [conclusion] the expression of the cohesion protein GcAP1 is reduced to 0.80.The expression of pnl-1 is reduced to 8.2pnl-2. [conclusion] the expression of the cohesion protein GcAP1 is reduced to 0.84pelB. [conclusion] The zygote is distributed in the cytoplasm, GcAP1 is an important virulence factor for the growth and development of anthrax leaf blight in apple, which regulates the expression of CgPG1, CgPG1, pnl-1, pnl-2pnl-2pnl-2pnl-2, and is an important virulence factor of anthrax leaf blight in apple.
【作者单位】：中国农业科学院果树研究所;
【基金】：国家自然科学基金青年科学基金(31501596) 中央级公益性科研院所基本科研业务费专项(1610182016002) 中国农业科学院科技创新工程
【分类号】：S436.611.12

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