冬虫夏草菌染色体数量的初步研究  

A preliminary study of chromosome number in Ophiocordyceps sinensis

作　　者：孟国良 杜卓 张晓兰[3] 杨瑞恒[4] 王永会[1,2] 陆焦焦 余丹 董彩虹 姚一建[1] 李熠[5] MENG Guo-Liang;DUZhuo;ZHANG Xiao-Lan;YANG Rui-Heng;WANG Yong-Hui;LU Jiao-Jiao;YU Dan;DONG Cai-Hong;YAOYi-Jian;LI Yi(State Key Laboratory of Mycology,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;University of Chinese Academy of Sciences,Beijing 100049,China;Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences,Shanghai 201403,China;College of Food Science and Engineering,Yangzhou University,Yangzhou 225127,China)

机构地区：[1]中国科学院微生物研究所真菌学国家重点实验室,北京100101 [2]中国科学院大学,北京100049 [3]中国科学院微生物研究所,北京100101 [4]上海市农业科学院食用菌研究所,上海201403 [5]扬州大学食品科学与工程学院,扬州225127

出　　处：《应用昆虫学报》2021年第6期1330-1338,共9页Chinese Journal of Applied Entomology

基　　金：国家自然科学基金(32170001)。

摘　　要：【目的】冬虫夏草Ophiocordyceps sinensis是一种重要的食药用真菌,仅分布在青藏高原及其周边高海拔地区,具有独特的生存环境、生物学特性以及显著扩张的基因组。本研究旨在确定冬虫夏草菌的染色体数量,为该物种的基因组测序、进化、遗传和分子生物学研究奠定基础,也为其他虫草菌染色体计数研究提供参考。【方法】二、三代测序辅以Hi-C组装,结合显微观察研究冬虫夏草的染色体数量。选择已组装获得基因组完成图的冬虫夏草1229菌株,首先利用昆虫细胞培养液培养获得芽生孢子,芽生孢子经秋水仙素和溶壁酶处理,获得细胞有丝分裂中期分散的染色体,DAPI荧光染色后使用激光共聚焦显微镜观察计数,结合基因组组装和显微观察的结果确定冬虫夏草染色体数量。【结果】二、三代测序辅以Hi-C组装,得到6条包含两端端粒的完整染色体,染色体之间显示出较强的端粒互作;显微观察结果也支持冬虫夏草1229菌株的染色体数量为6条。【结论】冬虫夏草菌的基因组包含6条染色体,芽生孢子是观察冬虫夏草菌染色体的理想材料。[Objectives] To investigate the chromosome number of Ophiocordyceps sinensis, an important edible and medicinal fungi endemic to Tibetan Plateau and surrounding areas of high elevation, and thereby lay a foundation for genome sequencing and other research on the evolution, genetics and molecular biology of this species, as well as help determine the chromosome number of other entomogenous fungi. [Methods] A genome assembly using second and third generation sequencing technology supplemented with Hi-C data, in addition to microscopic observation, was used to determine the chromosome number of O. sinensis. Strain 1229, the genome of which has been sequenced and assembled to a complete level,was cultured in insect cell culture medium to acquire blastospores. Blastospores were co-incubated with colchicines, then treated with lywallzyme to obtain dispersed metaphase chromosomes. Those chromosomes were stained with fluorescent DAPI,then observed and counted under a laser confocal microscope. [Results] Based on the genome assembly obtained from second and third generation sequencing and Hi-C data, strain 1229 of O. sinensis was found to possess six chromosomes with a strong telomere interaction between chromosomes. This number was supported by microscopic observations. [Conclusion] The genome of O. sinensis possesses six chromosomes. Blastospores are suitable for counting the chromosomes of O. sinensis.

关 键 词：真菌 基因组组装 芽生孢子 染色体计数 Hi-C 

分 类 号：S567.35[农业科学—中草药栽培]