【作者】 王林玲；

【导师】 陈克平； 姚勤；

【作者基本信息】 江苏大学 ， 食品科学， 2007， 博士

【摘要】 微孢子虫是一种专营细胞内寄生的单细胞生物,能感染从原生动物到哺乳动物,包括昆虫,鱼类和人类在内的几乎所有的动物。家蚕、柞蚕微孢子虫是引起家蚕和柞蚕微粒子病的病原,微粒子病对养蚕业来说是一种毁灭性的病害。我国每年因为微粒子病烧掉的蚕种价值上亿元,特别是近两年微粒子病在中国蚕业生产上又有回头的趋势。因此,对微孢子虫的研究既具有理论意义,也具有经济价值。本研究以家蚕微孢子虫和柞蚕微孢子虫为实验材料,克隆测序了柞蚕微孢子虫的核糖体基因,为柞蚕微孢子虫的分类和系统进化分析提供分子生物学的依据,也从分子水平上分析了家蚕微孢子虫和柞蚕微孢子虫的进化关系。结合二维电泳和质谱技术分离、鉴定了家蚕微孢子虫和柞蚕微孢子虫的总蛋白,在整体水平上分析了微孢子虫的蛋白质组成。用比较蛋白质组学的方法比较了家蚕微孢子虫和柞蚕微孢子虫接种五龄家蚕后不同时间段家蚕中肠的蛋白质变化,分析了家蚕中肠对微孢子虫的应答模式,从蛋白质组的水平上筛选与感染相关的特异性蛋白标志物和抗性功能相关的蛋白。一、柞蚕微孢子虫的核糖体基因用特异引物进行PCR扩增,克隆和测序了柞蚕微孢子虫的核糖体基因,得到柞蚕微孢子虫LSU rRNA基因的部分序列,SSU rRNA,5S rRNA和转录间隔区(ITS1和ITS2)的全序列(Genbank登录号为DQ073396)。柞蚕微孢子虫的核糖体基因排列顺序为LSU—ITS—SSU—ITS—5S,和家蚕微孢子虫的排列顺序相同,是一种即不同于真核生物,也不同于原核生物的排列顺序。用SSU rRNA和ITS序列进行同源比对和进化分析,柞蚕微孢子虫属于Nosema属,是和家蚕微孢子虫进化关系很近的一个种。本研究为柞蚕微孢子虫的分类提供了分子生物学的证据,将微孢子SSU rRNA基因和ITS结合起来分析微孢子虫的亲缘关系是一个很好的尝试。二、家蚕微孢子虫和柞蚕微孢子虫的蛋白质组学研究用蛋白质组学的方法研究了微孢子虫的蛋白质组。纯化的微孢子经液氮研磨和超声波破碎,用蛋白抽提液抽提微孢子总蛋白,然后二维电泳,质谱鉴定。家蚕微孢子虫鉴定到16个蛋白,其中有4种酶,2种转录调控因子,2种膜通道蛋白,1种细胞骨架结构蛋白,1种信号分子相关的蛋白,1种核糖体蛋白和5种功能未知蛋白。柞蚕微孢子虫鉴定到9个蛋白,其中有3种酶,3种转录调控因子,1种外膜糖蛋白,1种钙离子相关的膜通道蛋白和1种热激蛋白。结合生物信息学和文献检索的方法,初步分析了这些蛋白的功能。在家蚕微孢子虫和柞蚕微孢子虫鉴定的蛋白质中发现,酶、转录调控因子和膜蛋白占了较大的比例,说明酶、转录调控因子和膜蛋白确实是微孢子虫孢子时期大量表达和存在的蛋白质,这也与孢子的结构和生理吻合。三、家蚕微孢子虫,柞蚕微孢子虫和清水接种家蚕后家蚕中肠的比较蛋白质组学研究取接种家蚕微孢子虫,柞蚕微孢子虫和清水对照处理后的24h,48h,72h家蚕(品种:C108)中肠总蛋白,进行二维电泳和质谱鉴定。用比较蛋白质组的方法对接种不同微孢子虫后不同时间段的中肠蛋白质的变化和家蚕中肠对微孢子的应答机制进行了研究。总的来说,许多酶类表现出表达量的差异,在取材的三个时间段中,大部分参与碳水化合物代谢的酶在接种家蚕微孢子虫的家蚕中肠中呈现下调的趋势,如烯醇酶和羟基丙酮酸异构酶;而参与蛋白质和氨基酸降解的酶类却呈上调的趋势,如蛋白酶和延胡索酰乙酰水解酶。在接种家蚕微孢子虫的家蚕中肠的H+通道V-ATPase比接种柞蚕微孢子虫和清水的有减少的趋势。有趣的是参与免疫和应答反应的一些蛋白,接种家蚕微孢子虫和柞蚕微孢子虫24h后的家蚕中肠中的表达量都比对照高,在72h后,接种家蚕微孢子虫的表达量比接种柞蚕微孢子虫和对照的低。这个结果和PD-QUEST软件分析的二维电泳图谱的结果一致。肌动蛋白解聚合因子4(actin-depolymerizing factor 4),硫氧化/还原蛋白过氧化物酶(thiol peroxiredoxin)和抗胰糜蛋白酶前体(antichymotrypsin precursor)是差异蛋白中最值得研究和重视的蛋白。肌动蛋白解聚合因子是一种在真核生物中广泛存在的肌动蛋白结合蛋白,在调控细胞内肌动蛋白纤丝的解聚合和再聚合中起着关键作用。在接种微孢子实验中,肌动蛋白解聚合因子4表现出明显的差别,在接种了家蚕微孢子虫24h和72h时的家蚕中肠的二维电泳银染图中没有肌动蛋白解聚合因子4,而接种柞蚕微孢子虫和对照中却有该蛋白明显的染色。说明感染了家蚕微孢子虫的中肠细胞的肌动蛋白纤丝的解聚合和再聚合平衡被打破,中肠细胞的细胞骨架遭到破坏。肌动蛋白纤丝的解聚合和再聚合平衡被打破是家蚕微孢子虫在家蚕中肠细胞中生长发育的前提还是微孢子虫感染后的病理反应还需进一步的研究。肌动蛋白解聚合因子4有可能成为治疗和鉴定家蚕感染微孢子虫的目标蛋白。硫氧化/还原蛋白过氧化物酶是一种抗氧化酶系,在生物体对活性氧族的防御系统中和在宿主与寄生虫之间的相互影响中起着主要作用。硫氧化/还原蛋白过氧化物酶在取材的24h,48h和72h都被鉴定出表达量上的差异。接种家蚕微孢子虫和柞蚕微孢子虫24h时后,该蛋白在家蚕中肠中表达量都比对照高,48h时后,接种家蚕微孢子虫比接种柞蚕微孢子虫的表达量高,72h时后接种家蚕微孢子虫比接种柞蚕微孢子虫以及对照表达量低。说明硫氧化/还原蛋白过氧化物酶参与了家蚕中肠对微孢子虫的免疫应答。抗胰糜蛋白酶前体是抗胰糜蛋白酶因子的前体形式,没有活性,经加工后才能形成具有活性的抗胰糜蛋白酶因子。抗胰糜蛋白酶因子(antichymotrypsin)属于抑丝酶家族(serpins),参与蛋白质的加工、细胞凋亡、细胞外基质重构、以及保护细胞免受损伤等。人类细胞质中的抗胰糜蛋白酶因子的升高是烧死,外伤和一些癌症急性期的标志物。抗胰糜蛋白酶前体在接种家蚕微孢子虫72h后,家蚕中肠中的表达量远远高于在接种柞蚕微孢子虫以及对照的表达量,说明该时期的家蚕微孢子虫在中肠细胞中的快速发育。因此抗胰糜蛋白酶表达的增高有希望成为家蚕感染微孢子虫的标志。更多还原

【Abstract】 Microsporidia are obligate,intracellular single eukaryotic organisms that infect all major animal groups from invertebrates to vertebrates,including insects,fishes,and mammals.The microsporidian Nosema bombycis and Nosema antheraeae is a pathogen that infects bombyx mori and the Chinese oak silkworm,Antheraea pernyi,causing pebrine disease.Pebrine disease is a destructive disease,and has caused heavy losses in sericulture in china.Pebrine disease has increased in recent years,so it is the two grand requisites of theoretics and production to do research on microsporidia.In this paper,we used N.bombycis and N.antheraeae as experimental materials. The ribosomal RNA gene of N.antheraeae was cloned and sequenced.We presented here the molecular biological evidence of the taxonomy of N.antheraeae and the evolutional relation between N.antheraeae and N.bombycis.Proteins of N.bombycis and N.antheraeae were separated and identificated by two-dimensional electrophoresis and mass spectrometry,and analyzed proteins in the level of all cell.We characterizated and identificated the whole proteins change in the midgut of bombyx mori of fifth instar at different time after injecting N.bombycis,N.antheraeae and water by comparative proteome analysis.Immune response pattern of silkworm to microsporidia was analyzed, infection-related biomarkers and resistance-related proteins were screened in the level of proteome.1.The ribosomal RNA gene of N.antheraeaeBy PCR amplification used specific primers,cloning and sequencing,we got partial sequence of the large subunit rRNAs(LSU rRNA) gene,the complete sequence of the small subunit rRNAs(SSU rRNA) gene,the internal transcribed spacer(ITS) and 5S rRNA gene(Genbank accession number:DQ073396).The organization of the ribosomal RNA genes of N.antheraeae is LSU-ITS1-SSU-ITS2-5S,a pattern similar to those of N.bombycis.The organization of the ribosomal RNA genes differs from that of most prokaryotes as well as eukaryotes.Based on alignment and phylogenetic analysis by using the SSU rRNA gene and ITS sequences,we can conclude that N.antheraeae is a Nosema species,closely related to N.bombycis.We present here the molecular biological evidence of the taxonomy of N.antheraeae.The phylogenetic relationships of microsporidia species by combining information from SSU rRNA and ITS sequence was elucidated,which was a new attempt.2.The Proteomics Analysis of Nosema bombycis and Noseraa antheraeaeThe proteomic approach was used to investigate the proteome of N.bombycis and N.antheraeae.Purified spore were disrupted by triturating in liquid nitrogen.Total proteins were extracted in a lysis buffer.Protein samples were then analyzed by 2D electrophoresis and analyzed through MALDI-TOF-MS.16 proteins of N.bombycis were identified,among them,four were identified as enzyme,two as transcriptional regulator,two as membrane channels protein,one as cytosdeleton protein,one as signal protein,one as ribosomal protein and 5 unknown proteins.9 proteins of N.antheraeae were identified,among them,three were identified as enzyme,three as transcriptional regulator;one as envelope glycoprotein;one as cation efflux-related outer membrane exported protein;one as heat shock protein.The function of proteins was analyzed by bioinformatics and literature search.A large proportion of proteins of N.bombycis and N.antheraeae are enzyme,transcriptional regulator and membrane protein.It demonstrated enzyme,transcriptional regulator and membrane protein are high expression proteins in spore,which met the structure and physiology of spore.3.Comparative proteomics studies in midgut of bombyx mori infected N.bombycis, N.antheraeae or waterWe characterizated and identificated the whole proteins in the midgut of bombyx mori(strain:C108) of the fifth instar after infecting N.bombycis,N.antheraeae or water 24h,48h and 72h by two-dimensional electrophoresis and mass spectrometry.The whole proteins change in the midgut of bombyx mori and immune response pattern of silkworm to microsporidia at different time after injecting were analyzed by comparative proteomics.At different time after infection,expression of many enzymes involved in carbohydrate metabolism decrease,for example enolase and hydroxypyruvate isomerase.On the other hand,expression of many enzymes involved in protein and amino acid metabolism increases,for example,proteasome and fumarylacetoacetate hydrolase.The vacuolar ATP synthase(V-ATPase) is a membrane-related protein which can pump H+ outside of the cells or into the lumens of some vacuolar organelles.Expression of V-ATPase in midgut of bombyx mori infected with N.bombycis is lower than in midgut of bombyx mori infected with N.antheraeae or water.At 24h,proteins involved in immune response of silkworm to microsporidia express increase in midgut of bombyx mori infected N.bombycis and N.antheraeae than the control,while at 72h,proteins decrease in midgut of bombyx mori infected with N.bombycis than the treatments infected with N.antheraeae or the control.The result is consistent with the analysis of two-dimensional electrophoresis gel by PD-OUEST.We should pay whole attention to research expression change of actin-depolymerizing factor 4,thiol peroxiredoxin and antichymotrypsin precursor.Actin-depolymerizing factor 4 is an actin-binding protein expressed in all eukaryotic cells,which has important functions in equilibrium between monomeric and filamentous actin.In our experiment,expression change of actin-depolymerizing factor 4 is obvious.At 24h and 72h,there is little expression of actin-depolymerizing factor 4 in midgut of bombyx mori infected with N.bombycis,however there are high expression in midgut infected with N.antheraeae or water.This phenomenon explains the broken balance between monomeric and filamentous actin.Whether the balance between monomeric and filamentous actin is a precondition to microsporidian glow or a pathological process still needs to study.The actin-depolymerizing factor 4 is expected to become the target protein that identifies the silkworm infected with N.bombycis.Thiol peroxiredoxin is an antioxidant enzyme that catalyzes the reduction of peroxides,and may be involved in functions such as protection ROS released by immune effector cells and interaction between parasite and host.There are expression changes after infecting with N.bombycis,N.antheraeae or water at 24h,48h and 72h. At 24h,expression of thiol peroxiredoxin in midgut of bombyx mori infected with N. bombycis and N.antheraeae is higher than control;at 48h,expression in midgut of bombyx mori infected N.bombycis is higher than infected N.antheraeae;at 72h, expression in midgut of bombyx mori infected with N.bombycis is lower than the treatments infected with N.antheraeae or the control.The phenomenon explains thiol peroxiredoxin participate in immune response of mudgut of silkworm to microsporidia.Antichymotrypsin precursor is a precursor of antichymotrypsin,has no function, and must be processed to be active antichymotrypsin.Antichymotrypsin is a member of the serine protease inhibitor family known as serpins.Antichymotrypsin has been demonstrated a functional role in protein processing,apoptosis,restructuration of extracellular matrix and protection of cells from damage.Plasma level of antichymotrypsin has been implicated in acute-phase responses to burn injuries,surgery, and certain cancers.Expression of antichymotrypsin in midgut of bombyx mori infected with N.bombycis at 72h is far higher than the treatments infected with N.antheraeae or the control,which implicated that the expression of the Antichymotrypsin is expected to become the infection-related biomarkers of the silkworm infected with N.bombycis更多还原