Molecular Identification of Pathogenicity Associated Genes in Honeybee Fungal Pathogen, Ascosphaera apis, by Restricted Enzyme-Mediated Integration (REMI) Constructed Mutants

文献类型： 外文期刊

第一作者： Getachew, Awraris

作者： Getachew, Awraris;Wubie, Abebe J.;Wu, Jiangli;Xu, Jin;Wu, Pengjie;Abejew, Tessema Aynalem;Tu, Yangyang;Zhou, Ting;Xu, Shu-Fa;Wubie, Abebe J.;Abejew, Tessema Aynalem

作者机构：

关键词： Mutant; hph gene; Ascosphaera apis; Southern blot; Pathogenicity; qRTPCR

期刊名称：INTERNATIONAL JOURNAL OF AGRICULTURE AND BIOLOGY （ 影响因子：0.822； 五年影响因子：0.906 ）

ISSN： 1560-8530

年卷期： 2018 年 20 卷 12 期

页码：

收录情况： SCI

摘要： Chalkbrood disease in honeybees, caused by Ascosphaera apis (Onygenales: Ascosphaeraceae), exclusively affects brood. Restricted Enzyme-Mediated Integration (REMI) was used to construct stable A. apis transformants to investigate the infectivity of transformed pathogens to honeybee larvae. PCR, Clustal W sequence alignment, and Southern blot analyses confirmed insertion of hph gene and successful integration into host chromosomes in 184 stable A. apis transformants, of these 12 were selected for further study to identify pathogenicity associated genes. In vitro bioassay confirmed that 10 of the selected mutants had notable differences in pathogenicity among themselves and with the wild-type strain. More specifically, transformed A. apis mutants were relatively less pathogenic to in vitro reared honeybee larvae than that of the wild-type with mutant-7 having the lowest pathogenicity. Following the in vitro bioassay result, the wild-type and non-pathogenic mutant-7 were selected to identify virulence related genes in A. apis. Real-time PCR validated a total of eleven virulence related candidate genes and tested for their expression levels. Six genes (OmtA, Ski3, TOXD, Hts1, Nor-1 and melanin) were highly expressed in wild-type A. apis strain samples compared to its corresponding non-pathogenic mutant-7, which had lost pathogenicity on honeybee larvae and had relatively the lowest expression levels of selected candidate genes compared to the wild-type. Furthermore, the expression of these virulence related genes suggests that they might have an important role as virulence factors in the pathogenicity of A. apis through their involvement in the secondary metabolism processes that support pathogenicity functions. (C) 2018 Friends Science Publishers

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