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

doi:10.3864/j.issn.0578-1752.2017.08.007

摘要/Abstract

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ProtComp 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2 321 bp，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。

关键词: 衔接蛋白, 果胶酶, 苹果, 炭疽菌, 致病力

Abstract: 【Objective】 The objectives of this study are to determine the function of β subunit of adaptor protein GcAP1 complex in growth and pathogenicity of Glomerella leaf spot of apple pathogen Glomerella cingulata, investigate expression patterns of the GcAP1β in the fungal growth and pathogenicity, decipher whether or not GcAP1β regulate the expression of endopolygalacturonase genes CgPG1 and CgPG2, pectin lyase genes pnl-1 and pnl-2, pectate lyase genes pelA and pelB, and to lay a foundation for further studies of adaptor protein in pathogenic signal transduction pathways of G. cingulata. 【Method】 Based on the GcAP1β deletion vector and GcAP1β-gfp fused expression vector, the Δgcap1β mutant and the GcAP1β complementation strain Δgcap1β-GcAP1β were structured using ATMT, respectively, verified by RT-PCR and Southern blot analysis. Colony growth rate, sporulation, germination rate, appressorial formation rate and pathogenicity of the Δgcap1β mutant and the GcAP1β complementation strain Δgcap1β-GcAP1β were assayed, compared with the wild-type strain W16. GcAP1β subcellular localization was carried out with the bioinformatics softwares ProtComp 9.0 and TMHMM, along with signal observation of GcAP1β-GFP. The GcAP1β expression levels in hyphae, conidia, appressoria and pathogenicity stage were identified by qRT-PCR. Moreover, the expression levels of CgPG1, CgPG2, pnl-1, pnl-2, pelA and pelB in the wild-type strain W16 and the Δgcap1β mutant were detected, respectively. 【Result】 GcAP1β is 2 321 bp in length, including 3 introns, which encodes a 720 amino acids. Compared with the wild-type strain W16, the Δgcap1β mutant showed a rill-like fold colony and decreased growth, while sporulation, germination rate and appressorial formation rate were unaffected. Virulence of the Δgcap1β mutant reduced significantly, which induced tiny spots on the leaves. Moreover, the GcAP1β complementation strain Δgcap1β-GcAP1β fully restored the phenotype flaws by reintroducing GcAP1β to the Δgcap1β mutant. Fluorescent signal showed that the fused protein GcAP1β-GFP was distributed to the cytoplasm. qRT-PCR analysis showed that GcAP1β expresses through the lifecycle of G. cingulata, and the highest expression level of GcAP1β occurred at the post-invasion to leaves. Compared with WT, the Δcgap1β mutant showed a drastic reduction of CgPG1 transcripts (20.3%), CgPG2 transcripts (16.5%), pnl-1 transcripts (8.2%), pnl-2 transcripts (14.4%), pelA transcripts (4.4%) and pelB transcripts (0.8%). 【Conclusion】 The adaptor protein GcAP1 complex is distributed to the cytoplasm and is necessary for growth and development of G. cingulata; GcAP1 regulates the expression of CgPG1, CgPG2, pnl-1, pnl-2, pelA and pelB and is a vital virulence factor of G. cingulata.

Key words: adaptor protein, pectinase, apple, Colletotrichum, virulence

张俊祥，冀志蕊，王娜，徐成楠，迟福梅，周宗山. 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[J]. 中国农业科学, 2017, 50(8): 1430-1439.

ZHANG JunXiang, JI ZhiRui, WANG Na, XU ChengNan, CHI FuMei, ZHOU ZongShan. Gene Cloning and Functional Analysis of GcAP1 Complex Beta Subunit in Glomerella cingulata[J]. Scientia Agricultura Sinica, 2017, 50(8): 1430-1439.

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