一株从原蚕分离的微孢子虫的生物学特性及其系统发育分析  

Biological characters and phylogenetic analysis of a microsporidian isolated from parent silkworm

作　　者：黄旭华 何强[2] 汤庆坤 蒋满贵 李田[2] 龙江琼 潘国庆[2] 郑宁 鲁成[2] 潘志新 HUANG Xu-hua;HE Qiang;TANG Qing-kun;JIANG Man-gui;LI Tian;LONG Jiang-qiong;PAN Guo-qing;ZHENG Ning;LU Cheng;PAN Zhi-xin(Guangxi Academy of Sericulture Sciences,Nanning 530007,China;State Key Laboratory of Silkworm Genome Biology,Southwest University,Chongqing 400715,China)

机构地区：[1]广西蚕业科学研究院,南宁530007 [2]西南大学家蚕基因组生物学国家重点实验室,重庆400715

出　　处：《南方农业学报》2020年第1期11-18,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31560048,31460035);家蚕基因组生物学国家重点实验室开放项目(2016)~~

摘　　要：【目的】研究从广西蚕种繁育中分离获得的微孢子虫,调查其感染性和分类地位,为蚕种生产掌握病原来源及有效控制家蚕微粒子病流行提供参考依据。【方法】采用生物试验测定从蚕种生产一线分离获得微孢子虫(GXM14)对家蚕的半数感染浓度(IC50)和胚种传染率,显微镜观察其孢子形态,采用吉姆萨染色法观察孢子的生活史;通过PCR扩增及测序获得GXM14的SSU rRNA基因序列和ITS序列,并利用MEGA 5.0和DNASTAR中的Megalign构建系统发育进化树及进行相似性和遗传距离分析。【结果】GXM14孢子呈卵圆形,大小为(1.68±0.15)μm×(3.06±0.15)μm,其孢子生活史中有双核,以二分裂方式进行增殖,符合Nosema属的特征。GXM14对家蚕的IC50为3.53×105个/mL,是家蚕微孢子虫(N.b)的5.8倍;对家蚕的胚种传染率为5.0%,只是N.b的1/9。GXM14的SSU rRNA基因序列长度1226 bp,G+C含量为34.42%;ITS序列长度503 bp,G+C含量为29.82%。GXM14的SSU rRNA基因序列与甜菜夜蛾微孢子虫(Nosema sp.SE)SSU rRNA基因序列(KT336240.1)的遗传距离为0.2,相似度达98.9%,即GXM14与甜菜夜蛾微孢子虫(KT336240.1)的亲缘关系很近。基于微孢子虫ITS序列的相似性和遗传距离分析结果表明,GXM14与12株已知Nosema属微孢子虫的相似度均低于91.0%,而遗传距离在4.0以上,证实GXM14是一种新的微孢子虫,故将其暂命名为Nosema sp.GXM14。【结论】从蚕种生产一线分离获得的家蚕病原性微孢子虫(GXM14)属于Nosema属,与N.b同属异种,是一株新的微孢子虫,可能是野外昆虫微孢子虫交叉感染家蚕的病原。因此,在蚕种繁育中加强防虫杀虫,防止昆虫微孢子虫污染桑叶交叉感染家蚕,是控制家蚕微粒子病暴发流行的重要措施。【Objective】The present study was conducted to investigate the pathogenicity and classification feature of the microsporidian that was isolated from silkworms(Bombyx mori)in egg production,that would provide reference basis for the pathogen sources and the control of pebrine in silkworm egg production.【Method】GXM14 microspofidian was isolated from the parent silkworm in silkworm egg production line.The 50%infectious concentration(IC50)and the germinative infection rate of GXM14 were investigated with biological test methods,the spore shape of GXM14 was observed with optical microscope,and its life cycle was observed with Giemsas staining.The small subunit ribosomal RNA(SSU rRNA)gene and rRNA ITS sequence of GXM14 were obtained by PCR amplification,cloning and sequencing,the phylogenetic tree based on SSU r RNA sequences was constructed with the software of MEGA5,and the genetic distance of SSU rRNA and rRNA ITS sequence were analyzed with Megalign in software of DNASTAR.【Result】The IC50 of GXM14 to silkworm was 3.53×105 ind/mL,which was 5.8 times to that of Nosema bombycis(N.b),and the germinative infection rate of GXM14 was 5.0%,which was only 1/9 of that of N.b.The spore of GXM14 was oval in shape and(1.68±0.15)μm×(3.06±0.15)μm in size.There was binuclear in the life cycle of GXM14,and propagated in binary fission,which was conformed to the taxonomic features of Nosema.The SSU r RNA gene sequence of GXM14 was 1226 bp long,the G+C content was 34.42%,the rRNA ITS sequence of GXM14 was 503 bp long,the G+C content was 29.82%.The SSU rRNA genetic distance of GXM14 and Nosema sp.SE(KT336240.1)was 0.2,and its similarity was 98.9%,showed that GXM14 had close relationship with Nosema sp.SE.Analysis on rRNA ITS of microsporidian indicated that the similarity of GXM14 and 12 known Nosema microsporidian isolates were all lower than 91.0%,and their genetic distance were more than 4.0.It showed that GXM14 was a new Nosema microsporidian,and named Nosema sp.GXM14.【Conclusion】There is a pathogenic microspo

关 键 词：家蚕 微孢子虫 蚕种繁育 感染性 Nosema属 

分 类 号：S884.21[农业科学—特种经济动物饲养]