摘要
【目的】蜜蜂球囊菌Ascosphaera apis是一种专性侵染蜜蜂幼虫而导致白垩病的致死性真菌病原。基于前期已获得的高质量纳米孔(Nanopore)长读段测序数据,对蜜蜂球囊菌菌丝(AaM)和孢子(AaS)中的毒力因子相关全长转录本进行鉴定和分析,为毒力因子相关剪接异构体的功能研究提供参考信息和基础。【方法】利用BLAST工具,将AaM和AaS中的所有全长转录本比对Nr数据库,以鉴定蜜蜂球囊菌的毒力因子(几丁质酶、脂肪酶、水解酶和蛋白酶)相关的全长转录本。使用minimap2软件,将AaM和AaS中的全长转录本序列与蜜蜂球囊菌参考基因组注释的已知转录本序列进行比对,将比对到参考基因组的全长转录本进行归一化处理,再通过每百万计数法(Counts per million,CPM)计算毒力因子相关全长转录本的表达量。利用百迈克云平台的相关工具绘制转录本的表达量聚类热图。通过IGV浏览器对部分毒力因子相关全长转录本结构进行可视化。【结果】在AaM鉴定到毒力因子相关的367个基因及407个全长转录本,包括12条几丁质酶相关全长转录本,48条脂肪酶相关全长转录本,289条水解酶相关全长转录本,58条蛋白酶相关全长转录本。在AaS鉴定到毒力因子相关367个基因及400个全长转录本,包括14条几丁质酶相关全长转录本,63条脂肪酶相关全长转录本,267条水解酶相关全长转录本,56条蛋白酶相关全长转录本。另外,AaM和AaS特有的毒力因子(几丁质酶、脂肪酶)相关全长转录本分别有0条和17条,共有的毒力因子相关全长转录本有60条。进一步分析发现,蜜蜂球囊菌的部分毒力因子基因可通过可变剪接形成多条剪接异构体。【结论】共鉴定到蜜蜂球囊菌毒力因子(几丁质酶、脂肪酶、水解酶和蛋白酶)相关的367个基因和486条全长转录本;相比于蜜蜂球囊菌参考基因组注释的转录本,绝大多数毒力因子基因对应的全长转录本数量更多且结构更为复杂。研究结果丰富了蜜蜂球囊菌毒力因子相关基因和转录本的注释信息,为毒力因子相关剪接异构体的功能研究提供了基础,也为白垩病防控提供了潜在靶点。
[Objectives]To identify and analyze virulence factor-related full-length transcripts of Ascosphaera apis,a lethal fungal pathogen that exclusively infects honeybee larvae causing chalkbrood disease,using previously gained high-quality long-read sequencing data,thereby providing reference information and a foundation for the functional study of virulence factor-associated isoforms of this pathogen.[Methods]All full-length transcripts of A.apis mycelium(AaM)and spores(AaS)were aligned to the Nr database using BLAST tool to identify full-length transcripts relative to virulence factors,such as chitinase,lipase,hydrolase and protease.Sequences of full-length transcripts in AaM and AaS were aligned to those of known transcripts annotated in the A.apis reference genome,followed by normalization of the numbers of full-length transcripts mapped to the reference genome.The expression levels of each virulence factor-related,full-length transcript were then calculated.A heatmap showing the spatial pattern of transcript expression was drawn using the appropriate tool in BMKCloud and the structures of partial virulence factor-related full-length transcripts were visualized in IGV browser.[Results]367 genes and 407 full-length transcripts associated with virulence factors were identified in AaM,of which 12 were chitinase-related,48 lipase-related,289 hydrolase-related and 58 protease-related.367 genes and 400 full-length transcripts associated with virulence factors were identified in Aas,of which 14 were chitinase-related,63 lipase-related,267 hydrolase-related and 56 protease-related.In addition,there were 0 and 17 unique virulence factor-related,full-length transcripts(lipase-related and chitinase-related transcripts)in AaM and Aas,respectively,and 60 that were common to mycelia and spores.Further analysis indicated that part of the virulence factor-related genes can generate several isoforms via alternative splicing.[Conclusion]A total of 367 genes and 486 full-length transcripts associated with virulence factors,such as chitinase,lipase,hydrolase and protease were identified.Most full-length transcripts corresponding to virulence factor-related genes were relatively abundant and had more complex structures.Our findings enrich the annotation of genes and transcripts relevant to Ascosphaera apis virulence factors,provide a foundation for the functional study of virulence factor-associated isoforms and offer potential targets for the prevention and control of chalkbrood in honey-bees.
作者
隆琦
余岢骏
吴鹰
孙明会
冯睿蓉
赵萧
胡颖
徐细建
付中民
陈大福
郭睿
LONG Qi;YU Ke-Jun;WU Ying;SUN Ming-Hui;FENG Rui-Rong;ZHAO Xiao;HU Ying;XU Xi-Jian;FU Zhong-Min;CHEN Da-Fu;GUO Rui(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Apitherapy Research Institute,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Apicultural Research Institute of Jiangxi Province,Nanchang 330000,China)
出处
《应用昆虫学报》
CAS
CSCD
北大核心
2021年第5期1073-1082,共10页
Chinese Journal of Applied Entomology
基金
国家自然科学基金(31702190)
国家现代农业产业技术体系建设专项(CARS-44-KXJ7)
福建农林大学硕士生导师团队项目
福建省病原真菌与真菌毒素重点实验室开放课题
江西省应用研究培育计划(20181BBF68003)
福建省大学生创新创业训练计划项目(202010389016,202010389162)。
关键词
蜜蜂球囊菌
菌丝
孢子
毒力因子
全长转录本
第三代测序
Ascosphaera apis
mycelium
spore
virulence factor
full-length transcript
third-generation sequencing
