微孢子虫感染家蚕相关基因的研究
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摘要
微孢子虫是一类广泛分布于自然界而又极其古老的专性细胞内寄生原虫,能感染从无脊椎动物到脊椎动物几乎所有的动物,是人类以及家蚕(Bombyx mori)、蜜蜂(Apis mellifera)等许多经济昆虫的重要病原。感染家蚕的微孢子虫除微粒子属(Nosema)之外,还有内网虫属(Endoreticulatus)微孢子虫等。感染家蚕的内网虫属样微孢子虫(Endoreticulatus-like Microsporidium),是蚕种生产中不时有所发现的一种小型微孢子虫,除寄生于家蚕外,还可以感染桑尺蠖(Phthonandria atrilineata)、棉铃虫(HHelicoverpa armigera)和斜纹夜蛾(Prodenia litura)等多种鳞翅目昆虫,对蚕种生产中微粒子病检验常造成一定的干扰。感染家蚕的不同种(Species)的微孢子虫,所寄生的宿主和所处的细胞内环境都不一样,可以推测不同种的微孢子虫对家蚕的感染机制不同。获得确定的不同种微孢子虫,通过比较研究是一种良好的途经。本研究直接以纯化的内网虫属微孢子虫嵊州株(Endoreticulatus sp.Shengzhou)的孢子悬浮液(109个/mL)为模板,克隆了其核糖体RNA基因,通过与其他微孢子虫的比较研究,确定了其分类定位。该株微孢子虫核糖体RNA的LSUrRNA、SSUrRNA，ITS和IGS序列长分别为1757bp、1254bp、499bp和419bp,拼接后获得核糖体基因全序列长4431bp,GC含量为43.35%,序列已登录GenBank,序列号为JN792450。内网虫属微孢子虫嵊州株核糖体RNA全基因排列顺序为LSU-ITS-SSU-IGS-5S,从核糖体RNA基因水平研究了其进化定位,进一步确定了内网虫属微孢子虫嵊州株归类为内网虫属,为蚕种生产中微粒子病的检验提供了一定理论依据。
尽管不同种的微孢子虫对家蚕的感染机制不同,但感染率的高低会受孢子发芽率的影响。诸多研究表明家蚕微孢子虫的孢壁蛋白在孢子发芽以及侵染宿主过程中发挥重要作用,目前已鉴定出了SWP25和SWP30两种内壁蛋白,以及SWP5、SWP26和SWP32三种外壁蛋白。本研究通过基因克隆、蛋白表达、SDS-PAGE分析、抗体制备、间接免疫荧光抗体实验以及免疫电镜定位分析,鉴定了家蚕微孢子虫的一种新的孢壁蛋白SWP12,预测蛋白质分子质量和等电点分别为25.56kDa和6.69,定位分析表明该蛋白是一种孢子内壁蛋白,该蛋白的多克隆抗体未能明显抑制微孢子虫对宿主细胞的粘附。该蛋白的一些分子特征有助于我们进一步理解微孢子虫与宿主的互作关系。
微孢子虫对家蚕的感染不仅与其自身的孢壁蛋白相关,也会引起宿主细胞基因的差异表达变化,微孢子虫诱导家蚕的基因差异表达往往和家蚕免疫相关基因的启动有关,但有关家蚕微孢子虫侵染的BmN细胞基因差异表达研究鲜有报道。本研究采用GeneFishing技术,通过对BmN细胞接种家蚕微孢子虫,以正常BmN细胞作对照,研究了家蚕在微孢子虫胁迫下相关基因的差异表达,从基因组水平上鉴别了10个受家蚕微孢子虫诱导或抑制表达基因。对差异表达的转录衍生片段(Transcript derived fragments,TDFs)序列进行基因克隆测序,经Blast分析发现这些差异表达基因的功能涉及如下几个方面：1)蛋白质合成;2)细胞信号转导；3)线粒体呼吸链电子传递与能量代谢；4)细胞免疫；5)未知功能蛋白。
家蚕微孢子虫感染BmN细胞的差异表达Akirin基因是新近发现于果蝇中的天然免疫系统新成员,它在果蝇免疫缺陷(Immune deficiency,Imd)通路中发挥重要作用。本研究经RT-PCR克隆测序确定其cDNA序列的ORF框含588bp,编码195个氨基酸残基,预测分子量和等电点分别为21.77kDa和8.97,属于碱性蛋白质,没有信号肽位点。Sikmap软件分析表明Akirin基因位于家蚕第22对染色体上,微点阵表达分析表明该基因在家蚕幼虫的卵巢、精巢、脂肪体和中肠等各种组织均表达,经基因克隆和原核表达获得了融合表达蛋白。这些为进一步研究Akirin基因在家蚕中的免疫作用奠定了基础。
Microsporidia, widely distributed in nature, are extremely ancient and highly specialized obligate intracellular parasites that infect a broad range of animals including vertebrates and invertebrates and are important pathogens for many economic insects such as Bombyx mori and Apis mellifera. The silkworm, Bombyx mori are also infected by Endoreticulatus besides Nosema bombycis. Endoreticulatus.sp Shengzhou is a micro microsporidia found in silkworm eggs, and this microsporidia can also infect so many kinds of other Lepidoptera insects such as Phthonandria atrilineata, Helicoverpa armigera and Prodenia litura besides Bombyx mori. There are different species of microsporidium which can bring infection to the silkworm, Bombyx mori. These microsporidium all have different hosts and internal cell environment and it is reasonable to presume that different species of microsporidium own different infection mechanism. It is a good method to make a precise identification on species of microsporidium by comparative study. In this research, we employed a sample method directly using purified spores suspension with a concentration of109spores/mL instead of genomic DNA as PCR templetes for amplifying complete rRNA genes of Endoreticulatus sp. Shengzhou, and studied its phyletic evolution of the isolate by comparing it with that of other microsporidia. The rRNA gene sequences of LSUrRNA, SSUrRNA, ITS and IGS are1757bp,1254bp,499bp and419bp, respectively. The complete rRNA gene sequences of the isolate is4,43lbp (GenBank number. JN792450) in length with43.35%G+C and the arrangement of the rRNA genes was reversed as LSU-ITS-SSU-IGS-5S. Morphological character and phylogenetic analysis based on the rRNA gene sequences indicated that Endoreticulatus sp. Shengzhou is closely related to Endoreticulatus genus, which provides some theoretical basis for inspection of the pebrine in silkworm eggs production.
Though there exist different infection mechanism about different species of microsporidium, infection percentage on the silkworm, Bombyx mori is always effected by germination rate of microsporidia. It is reported that the spore wall proteins of Nosema bombycis play an important role in the process of spores germination and infection. Five kinds of spore wall proteins have been identified which contain two kinds of endosporal proteins (SWP25and SWP30) and three exosporal proteins (SWP5, SWP26, and SWP32) up todate. In the current study, we identified another endosporal protein SWP12with calculated molecular mass of25.56kDa and an isoelectric point (pI) of6.69through gene cloning, SDS-PAGE analysis, protein expression, polyclonal antibody production, indirect immunofluorescence antibody test, and immunoelectron microscopy analysis, the results indicated that this protein is localized to the endospore and the special polyclonal antibody has no obvious inhibition on adherence to host cells. The characterization of this novel spore wall protein from Nosema bombycis may facilitate our further investigation on the relationship between Nosema bombycis and its hosts.
Infection of microsporidia on the silkworm, Bombyx mori is not only related to spore wall proteins, but also induce differential genes expression on host cells. At the same time, it is switch on the related immune genes that results in differential genes expression. However, there are few reports on differential expression genes of Bombyx mori after infection by Nosema bombycis. Differential expression genes of BmN cells after infection by N.bombycis were investigated, and ten differential expression genes induced or suppressed by N. bombycis in BmN cells had been identified. Based on blast analysis on the sequences of transcript derived fragments, these differentially expressed genes were divided into five categories such as protein synthesis, signal transduction on cells, transporting ATP synthase and NADPH participate in electron transfer through mitochondria respiratory chain, cell energy metabolism, cellular immunity; and unknown function protein. Identification of differential expression genes in BmN cells is helpful for revealing infect mechanism of Nosema bombycis on BmN cells and also helpful for making an illumination on mechanism of host-pathogen interactions.
Akirin gene, one of the differential expression genes, is reported to be a new nuclear factor which was discovered in the innate immune response of Drosophila melanogaster and plays a critical role in the immune deficiency (Imd) pathway. In the present study, Akirin gene of Bombyx mori was identified by screening differential expression genes in BmN cells and was cloned with the method of RT-PCR. Analysis on sequence shows that its cDNA was564bp and encoded a hypothetical proein of195amino acids with a predicted molecular mass of21.77kDa and an isoelectric point (pI) of8.97. The hypothetical protein belongs to alkaline proteins without any signal peptide site. Sikmap software analysis indicated that Akirin gene is located on chromosome22of Bombvx mori, and microarray expression analysis demonstrated that the gene is expressed in a variety of organizations such as ovary, spermary, fat body and midgut. The recombinant protein was obtained by gene cloning and prokaryotic expression, and these assays made a foundation work for the further research on the immunity of Akirin gene in Bombyx mori.
尽管不同种的微孢子虫对家蚕的感染机制不同,但感染率的高低会受孢子发芽率的影响。诸多研究表明家蚕微孢子虫的孢壁蛋白在孢子发芽以及侵染宿主过程中发挥重要作用,目前已鉴定出了SWP25和SWP30两种内壁蛋白,以及SWP5、SWP26和SWP32三种外壁蛋白。本研究通过基因克隆、蛋白表达、SDS-PAGE分析、抗体制备、间接免疫荧光抗体实验以及免疫电镜定位分析,鉴定了家蚕微孢子虫的一种新的孢壁蛋白SWP12,预测蛋白质分子质量和等电点分别为25.56kDa和6.69,定位分析表明该蛋白是一种孢子内壁蛋白,该蛋白的多克隆抗体未能明显抑制微孢子虫对宿主细胞的粘附。该蛋白的一些分子特征有助于我们进一步理解微孢子虫与宿主的互作关系。
微孢子虫对家蚕的感染不仅与其自身的孢壁蛋白相关,也会引起宿主细胞基因的差异表达变化,微孢子虫诱导家蚕的基因差异表达往往和家蚕免疫相关基因的启动有关,但有关家蚕微孢子虫侵染的BmN细胞基因差异表达研究鲜有报道。本研究采用GeneFishing技术,通过对BmN细胞接种家蚕微孢子虫,以正常BmN细胞作对照,研究了家蚕在微孢子虫胁迫下相关基因的差异表达,从基因组水平上鉴别了10个受家蚕微孢子虫诱导或抑制表达基因。对差异表达的转录衍生片段(Transcript derived fragments,TDFs)序列进行基因克隆测序,经Blast分析发现这些差异表达基因的功能涉及如下几个方面：1)蛋白质合成;2)细胞信号转导；3)线粒体呼吸链电子传递与能量代谢；4)细胞免疫；5)未知功能蛋白。
家蚕微孢子虫感染BmN细胞的差异表达Akirin基因是新近发现于果蝇中的天然免疫系统新成员,它在果蝇免疫缺陷(Immune deficiency,Imd)通路中发挥重要作用。本研究经RT-PCR克隆测序确定其cDNA序列的ORF框含588bp,编码195个氨基酸残基,预测分子量和等电点分别为21.77kDa和8.97,属于碱性蛋白质,没有信号肽位点。Sikmap软件分析表明Akirin基因位于家蚕第22对染色体上,微点阵表达分析表明该基因在家蚕幼虫的卵巢、精巢、脂肪体和中肠等各种组织均表达,经基因克隆和原核表达获得了融合表达蛋白。这些为进一步研究Akirin基因在家蚕中的免疫作用奠定了基础。
Microsporidia, widely distributed in nature, are extremely ancient and highly specialized obligate intracellular parasites that infect a broad range of animals including vertebrates and invertebrates and are important pathogens for many economic insects such as Bombyx mori and Apis mellifera. The silkworm, Bombyx mori are also infected by Endoreticulatus besides Nosema bombycis. Endoreticulatus.sp Shengzhou is a micro microsporidia found in silkworm eggs, and this microsporidia can also infect so many kinds of other Lepidoptera insects such as Phthonandria atrilineata, Helicoverpa armigera and Prodenia litura besides Bombyx mori. There are different species of microsporidium which can bring infection to the silkworm, Bombyx mori. These microsporidium all have different hosts and internal cell environment and it is reasonable to presume that different species of microsporidium own different infection mechanism. It is a good method to make a precise identification on species of microsporidium by comparative study. In this research, we employed a sample method directly using purified spores suspension with a concentration of109spores/mL instead of genomic DNA as PCR templetes for amplifying complete rRNA genes of Endoreticulatus sp. Shengzhou, and studied its phyletic evolution of the isolate by comparing it with that of other microsporidia. The rRNA gene sequences of LSUrRNA, SSUrRNA, ITS and IGS are1757bp,1254bp,499bp and419bp, respectively. The complete rRNA gene sequences of the isolate is4,43lbp (GenBank number. JN792450) in length with43.35%G+C and the arrangement of the rRNA genes was reversed as LSU-ITS-SSU-IGS-5S. Morphological character and phylogenetic analysis based on the rRNA gene sequences indicated that Endoreticulatus sp. Shengzhou is closely related to Endoreticulatus genus, which provides some theoretical basis for inspection of the pebrine in silkworm eggs production.
Though there exist different infection mechanism about different species of microsporidium, infection percentage on the silkworm, Bombyx mori is always effected by germination rate of microsporidia. It is reported that the spore wall proteins of Nosema bombycis play an important role in the process of spores germination and infection. Five kinds of spore wall proteins have been identified which contain two kinds of endosporal proteins (SWP25and SWP30) and three exosporal proteins (SWP5, SWP26, and SWP32) up todate. In the current study, we identified another endosporal protein SWP12with calculated molecular mass of25.56kDa and an isoelectric point (pI) of6.69through gene cloning, SDS-PAGE analysis, protein expression, polyclonal antibody production, indirect immunofluorescence antibody test, and immunoelectron microscopy analysis, the results indicated that this protein is localized to the endospore and the special polyclonal antibody has no obvious inhibition on adherence to host cells. The characterization of this novel spore wall protein from Nosema bombycis may facilitate our further investigation on the relationship between Nosema bombycis and its hosts.
Infection of microsporidia on the silkworm, Bombyx mori is not only related to spore wall proteins, but also induce differential genes expression on host cells. At the same time, it is switch on the related immune genes that results in differential genes expression. However, there are few reports on differential expression genes of Bombyx mori after infection by Nosema bombycis. Differential expression genes of BmN cells after infection by N.bombycis were investigated, and ten differential expression genes induced or suppressed by N. bombycis in BmN cells had been identified. Based on blast analysis on the sequences of transcript derived fragments, these differentially expressed genes were divided into five categories such as protein synthesis, signal transduction on cells, transporting ATP synthase and NADPH participate in electron transfer through mitochondria respiratory chain, cell energy metabolism, cellular immunity; and unknown function protein. Identification of differential expression genes in BmN cells is helpful for revealing infect mechanism of Nosema bombycis on BmN cells and also helpful for making an illumination on mechanism of host-pathogen interactions.
Akirin gene, one of the differential expression genes, is reported to be a new nuclear factor which was discovered in the innate immune response of Drosophila melanogaster and plays a critical role in the immune deficiency (Imd) pathway. In the present study, Akirin gene of Bombyx mori was identified by screening differential expression genes in BmN cells and was cloned with the method of RT-PCR. Analysis on sequence shows that its cDNA was564bp and encoded a hypothetical proein of195amino acids with a predicted molecular mass of21.77kDa and an isoelectric point (pI) of8.97. The hypothetical protein belongs to alkaline proteins without any signal peptide site. Sikmap software analysis indicated that Akirin gene is located on chromosome22of Bombvx mori, and microarray expression analysis demonstrated that the gene is expressed in a variety of organizations such as ovary, spermary, fat body and midgut. The recombinant protein was obtained by gene cloning and prokaryotic expression, and these assays made a foundation work for the further research on the immunity of Akirin gene in Bombyx mori.
引文
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