【作者】 胡军华；

【导师】 周泽扬；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2008， 博士

【摘要】 微孢子虫(Microsporidia)是一类细胞内专性寄生的单细胞真核生物,广泛寄生于无脊椎动物和脊椎动物(包括人类),是经济昆虫、鱼类、兔类、产毛动物、啮齿类及灵长类的常见病原。自19世纪发现家蚕微孢子虫以来,陆续报道发现有超过160个属1300多种微孢子虫。另外自1959年发现了微孢子虫感染免疫缺陷型病人后,相继发现8个属的14个种能感染人类,尤其是AIDS病人。由于微孢子虫与人类的健康密切相关,因此引起了科学界的广泛关注。家蚕微孢子虫是微孢子属的典型种,普遍寄生于家蚕而引发家蚕微粒子病导致毁灭性病害。但是目前国内外有关家蚕微孢子虫的分子生物学研究较薄弱,缺乏对微孢子虫的深刻认识。微孢子虫曾一度被认为是缺乏线粒体的原始的真核生物,但近年来的研究将微孢子虫归属于真菌类,而其经历了简化进化的过程才呈现出较原始的特征;同时实验证明了微孢子虫具有称之为“线体”(mitosome)的简化的线粒体细胞器。微孢子虫线体的结构、组成、功能及其在能量转运过程所扮演的角色目前已经成为微孢子领域研究的热点。目前已报道的微孢子虫线粒体相关基因共有14种,且有研究结果表明这些基因为线体的主要构成部分,其中除了铁硫簇装配系统较为完整,其它基因大部分缩减或缺失,线体在微孢子虫中所起的功能和作用仍不清楚。家蚕微孢子虫线粒体相关基因的研究还处于探索阶段,基因的序列信息及基因的功能研究均未见报道。本论文借鉴人和酵母线粒体研究成果,利用家蚕微孢子虫基因组数据和EST数据资源,对家蚕微孢子虫线粒体相关基因进行了鉴定,对一些关键基因进行了克隆,对关键的功能基因进行了共线性和系统进化分析,重点讨论了这些重要基因编码蛋白在微孢子虫中的亚细胞定位及其功能,最后对家蚕微孢子虫线体的功能进行了探讨,获得的主要研究结果如下:1家蚕微孢子虫线粒体相关基因的生物信息学分析以家蚕微孢子虫全基因组数据为基础,通过与线粒体蛋白库比对和文献分析鉴定线粒体相关基因,对这些基因进行结构预测和功能分析,推测家蚕微孢子虫线粒体相关基因可能的功能,为进一步克隆和表达家蚕微孢子虫线粒体相关基因以及蛋白的亚细胞定位研究打下基础。通过比对获得了171个线粒体相关基因。这171个基因中没有与氧化磷酸化和电子传递链相关的基因,其中FeS cluster装配途径中包含frataxin,nifu,nifs和ferredoxin;抗氧化途径含有Mn-SOD,thioredoxin reductase等,蛋白酶和蛋白质的折叠和定向等基因mtHsp70,Tim22/17和mtTom70等。结果表明,家蚕微孢子虫基因组中含有与某些昆虫类似的线粒体型的ABC transporter,这些线粒体型ABC transporter不同的昆虫中基因拷贝数并不相同。家蚕微孢子虫线粒体相关基因的数目比人和酵母少,和已报道的蝗虫微孢子虫接近、比兔脑原虫和隐孢子虫少。家蚕微孢子虫的线粒体相关基因均为细胞核编码的基因,无电子传递和产生ATP的功能。在家蚕微孢子虫基因组中,存在一套铁硫簇相关基因,比较这套线粒体型铁硫簇基因的差异,将为揭示家蚕微孢子虫线粒体相关蛋白的功能提供依据。共线性被认为是微孢子虫基因组的特征之一。在家蚕微孢子虫的线粒体相关基因中我们发现ATP/ADP carrier protein,ADP/ATP carrier protein,nifu,MnSOD,ABC transportermitochondrial type等没有共线性,而frataxin,nifs,ferredoxin(Adrenodoxin),mtHsp70,Tim22/17,mitochondrial Glycerol-3-phosphate dehydrogenase,PDH Elα,PDH Elβ具有较好的共线性。而且frataxin,ferredoxin和mitochondrial Glycerol-3-phosphate dehydrogenase一样,所在片段区域的基因之间存在较多的插入,颠倒和缺失。而在家蚕微孢子虫与酵母菌之间这些线粒体相关基因没有共线性现象,推测原因可能是家蚕微孢子虫的基因序列中具有转座子(NBO0016-0045,NBO0016-0056,NBO0084-0025)引起的。共线性分析揭示大部分的线粒体相关基因在微孢子虫的基因组里排列位置比较稳定,也在一定程度上反映出微孢子基因组的进化保守。基因或是基因组间密码子使用的差异往往反映了碱基组成的偏好性或是自然选择的强度。本研究比较了家蚕微孢子虫和兔脑原虫的核基因组以及家蚕微孢子虫线粒体相关基因在密码子偏好性及其影响因素上的差异。结果表明家蚕微孢子虫基因组受到弱的自然选择压力,其密码子偏性主要由碱基组成背景造成。相应的,兔脑原虫基因组受到的选择压力大得多,自然选择压力对密码子偏好起主要作用。对家蚕微孢子虫线粒体相关基因密码子偏好性的分析显示,线粒体相关基因与家蚕微孢子虫基因组一样,仅受到很弱的自然选择压力,其密码子偏性主要由碱基组成背景造成,而且大部分线粒体相关基因编码相同氨基酸的密码子偏好是一致的。为了研究家蚕微孢子虫的系统发生及其进化地位,将家蚕微孢子虫铁硫簇装配系统基因frataxin、nifs和nifu的氨基酸序列与GenBank中的真菌、原核生物和原生动物等物种的相对应的氨基酸序列进行聚类分析。系统分析结果显示:家蚕微孢子虫、兔脑孢虫和蝗虫微孢子虫具有相同的进化地位,聚成一类;与典型的具有线粒体的高等真核生物进化关系最为接近,与原生动物次之,而与原核生物最远。这表明微孢子的铁硫簇装配基因与高等真核生物线粒体型的相对应的基因最为相似,预示微孢子可能具有线粒体类似的细胞器及其具有线粒体的部分功能。另外,与原生动物的进化关系次之,这一结果辅证了微孢子虫是真菌的姊妹枝假说。构建家蚕微孢子虫线粒体相关同源基因的网络图,其中包括FeS簇装配系统、蛋白质的转位与折叠系统、磷酸甘油穿梭系统和抗氧化途径等。家蚕微孢子虫线粒体相关基因和兔脑原虫的线粒体相关基因虽然数目差异较大,但其主要的结构功能是相同的。2家蚕微孢子虫线粒体相关基因的克隆与表达本研究采用原核表达及RT-PCR的方法鉴定了3个家蚕微孢子虫线粒体相关基因frataxin,nifu和ADP/ATP carrier protein。frataxin基因大小为306 bp,在frataxin-like domain区存在3个α螺旋和1个β折叠,3个α螺旋的位置分别位于蛋白质的N-端,中间及C端,在二级结构上高度保守,且β折叠的位置也非常保守,推测这些结构在行使蛋白的功能上是必须的。与原核生物相比,微孢子虫的frataxin蛋白缺乏52-65位置的α螺旋;与酵母相比较,缺乏40-45和61-66位置的α螺旋,推测61-66位置的α螺旋与该蛋白在细胞质中的定位有关,而40-45位置的α螺旋与该蛋白在线粒体中的定位有关。没有发现与线粒体定位相关的区段。nifu序列编码140个氨基酸残基,比兔脑原虫和典型的真核生物和哺乳动物的nifu要短。nifu基因编码的蛋白在其结构域非常保守,但在N端和C端变异很大,与E.cuniculi的蛋白同源性高度相似,有78%的一致性(identity)。nifu和兔脑孢虫的一样,均无任何跨膜结构及信号肽,仅有一个保守结构域Nifu N存在。没有发现与线粒体定位相关的区段。ADP/ATP carrier protein基因只有674bp,224aa。分子量为20kDa,等电点为9.15,根据SMART软件发现其有3个跨膜区,具有典型的膜蛋白特征。与其它生物的同源基因比较,此基因不完整,缺乏大部分序列信息,与兔脑原虫的ADP/ATP carrier proteinl同源性最高。推测ADP/ATP carrier protein存在于线粒体膜上,扮演着将线粒体合成的ATP运输到细胞质中,将细胞质中ADP运回线粒体的功能。RT-PCR试验结果表明frataxin,nifu和ADP/ATP carrier protein 3个基因都具有转录活性,但是表达活性都比较弱。western blot结果显示,微孢子总蛋白中存在frataxin蛋白,且仅存在frataxin蛋白的一种形式,不存在蛋白的前体。在家蚕微孢子的成熟孢子中能够表达frataxin蛋白,但其表达丰度较低。3家蚕微孢子虫线粒体相关基因编码蛋白的定位研究应用IFA和免疫电镜技术对线粒体相关基因编码蛋白的表达特异性和亚细胞定位特点进行研究。MitoTracke Red CMXRos染色后的家蚕细胞可以看见明显的细胞核区域及圆球状的线粒体,家蚕微孢子虫的孢子大部分出现红色荧光,分区明显,而且DAPI染色的细胞核区域和MitoTracke Red CMXRos染色区域不相同。比较抗体标记、MitoTracker Red CMXRos染色和DAPI染色的位置发现,在家蚕微孢子虫孢子中前两种标记的位置是重叠的,和DAPI标记的细胞核的位置不相同,说明家蚕微孢子虫的孢子中存在线粒体类似细胞器及存在核基因编码的线粒体蛋白,此蛋白的存在预示着微孢子的线粒体类似细胞器是有功能的,但其具体功能尚不清楚。8种线粒体相关基因的抗体均显示与家蚕微孢子虫孢子结合,与DNA染料DAPI染色的位置不重叠,且与家蚕微孢子虫的宿主(家蚕)细胞无交叉反应。其中frataxin,ferredoxin,tim22/17和ATP/ADP carrier protein抗体标记的孢子荧光信号位于孢子的孢原质中,以孢子壁为边界,出现球状的荧光信号,每个孢子中至少有6个球状信号,推测这些荧光信号可能代表着线粒体的残留部分一线体。Nifs和nifu的荧光信号较弱,随机分布在孢原质中,推测这2个蛋白是分布在孢原质中的蛋白。ADP/ATP carrier protein和mtTom70则以孢子壁为边界,出现连续的线状荧光信号,推测这2个蛋白可能位于孢子的原生质膜上。同时,采用免疫胶体金标记的方法对线粒体相关基因Frataxin,nifs,ferredoxin和mtTom70 4个蛋白进行定位研究。电镜下可以清楚地看到家蚕微孢子的内部结构,包括极管,后极泡,细胞核,细胞壁等。胶体金颗粒大多分布在孢原质中,位于孢原质的中间和靠近细胞壁的边缘,在细胞核,细胞壁上没有胶体金颗粒。在家蚕微孢子虫中标记线粒体相关蛋白Frataxin和ferredoxin的胶体金颗粒常常2—4个聚在一起,可能代表着线体的位置。标记nifs的胶体金颗粒分散分布在孢原质中,而mtTom70则分布在孢原质中靠近孢壁的位置,与IFA定位的情况基本一致。4家蚕微孢子虫线粒体相关蛋白初步分析首先,采用percoll密度梯度离心和蔗糖密度梯度离心方法进行了微孢子虫细胞器的分离探索,结果表明:2种方法都不能有效地富集和纯化家蚕微孢子虫的细胞器。利用免疫共沉淀-质谱方法从家蚕微孢子虫孢质蛋白中分离、富集和鉴定线粒体相关基因多克隆抗体的抗原靶蛋白。发现与frataxin作用的蛋白中鉴定出11个蛋白,其中2个为未知蛋白;与支架蛋白nifu相联系的蛋白有1个未知蛋白,还有一个腺苷酸环化酶;与TIM22/17相联系8个蛋白中有4个未知蛋白和3个假设蛋白;与ATP/ADP carrier protein作用的蛋白为一个假设蛋白;与nifs作用的4个蛋白中1个蛋白为未知蛋白,2个蛋白为假设蛋白;有2个蛋白与ferredoxin相联系;3个蛋白与tom70相联系;1个蛋白与adp/atp carrierprotein相联系。部分蛋白具有信号肽或跨膜区。通过本章的研究,我们获得了线粒体相关基因的关联蛋白,为微孢子线体的进一步研究打下了基础。此外,免疫沉淀-质谱这一技术路线简单易行,为线粒体相关蛋白的分离筛选提供了新思路和新方法。更多还原

【Abstract】 Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites.They can infect a wide variety of animals ranging from invertebrates to vertebrates,and they are the common fateful pathogens for sericulture,fishery and shrimp farms.Since the first microsporidia,Nosema bombycis,has been discovered in the middle of the nineteenth century,thus far,more than 1 300 microsporidia species belonging to 160 genera have been reported,from which 14 species belonging to 8 genera can lead to several syndromes in immunocompetent hosts of human and cause opportunistic infections in acquired immunodeficiency syndrome(AIDS) patients. Microsporidia draws great interest to scientists because they are closely related to human health. Microsporidia were even thought to be "primitive" eukaryotes without microspordial but are now widely recognized as being fungi that have undergone reductive evolution as so to appear to be primitive organisms.And now,a highly reduced microspordial organelle has been identified, so-called "mitosome".In general,the structure,composition and function in energy transfer of microspordia may play an important role in the process of microsporidia infecting.Thus microspordia has been a focus of research in microsporidian pathology.14 microspordial relative genes were reported in microsporidia which have also been demonstrated to involve in the metabolism of mitosome.However,most of those genes greatly reduced and the function and action mechanism of them in the microsporidia were unknown yet.N. bombycis is known as a pathogen of silkworm pebrine,which usually prevails in sericulture.The prevention and treatment of pebrine is still an important and difficult topic for sericulture.Now,the complete genomic sequence of N.bombycis has been finished in our labs.By comparative with genome datas and EST datas between N.bombycis and other species,such as human and yeast,we identified the candidate of mitochondrial-related genes.Among candidate genes,we have cloned some important genes,analyzed and discussed their location and function in the Nosema bombycis.Some major research results were obtained as following:1 Bioinformatics analysis of mitochondrial-related gene of microsporidia N.bombycisAfter comparison of total genome datas of N.bombycis and mitochondrial protein library,we obtained mitochondrial-related genes from N.bombycis,the potential function of mitochondrial-related genes of N.bombycis was predicted by analysis of function and structure.We further finished the cloning,expressing and location of mitochondrial-related genes of N. bombycis.We obtained 171 mitochondrial-related proteins include frataxin,nifu,nifs and ferredoxin FeS cluster assembly system,Mn-SOD,thioredoxin reductase for antioxidation way, mtHsp70,Tim22/17and mtTom70 for the folding of protease and proteins except the related protein for oxidative phosphorylation and electron transport chain.N.bombycis have mitochondrial type ABC Transporter same as other insect,nevertheless,the number of copy is distinctly less to Encephalitozoon cuniculi,Cryptosporidium parvum,human and yeast,but closer to the Antonospora locustae.Even if N.bombycis exsisted partily mitochondrial related proteins coded by genome,it have no the function of electron transport and production of ATP.We have found a series genes involved in Fe-S cluser,that might will provide us clues to the function of mitochondrial related gene about N.bombycis by the comparation of Fe-S cluser in other microspordia species.Collinearity was been known as one of the major characteristic of microspordia genome.We have found that some mitochondrial-related genes are not have collinearity,such as ATP/ADP carrier protein,ADP/ATP carrier protein,nifu,MnSOD,ABC transporter microspordial type,while some have better relation of coilinearity such as frataxin,nifs,Adrenodoxin(ferredoxin),mtHsp70, Tim22/17,microspordiall Glycerol-3-phosphate dehydrogenase,PDH E1α,PDH E1β.Several event of insert,invert and loss of frataxin gene have occurred among different related species,for instance adrenodoxin and mitochondrial glycerol-3-phosphate dehydrogenase found neither in yeast nor in microspordia,which maybe result from the transposon(NBO0016-0045,NBO0016-0056, NBO0084-0025) near the loci.This suggested that most of mitochondrial-related genes sequences in microspordia were steady and conservative.The differences of codon bias between mitochondrial-related genes or genome reveals that the strength of natural select depend on nucleotide composition.Comparative of mitochondrial-related genes,genome of N.bombycis and E.cuniculi,the result had shown that the selection strength of genome of N.bombycis appears to be very poor,whose baise code mainly result from nucleotide composition.Analysis of codon bias of mitochondrial-related genes of N.bombycis suggested that mitochondrial-related genes of N.bombycis appear to very poor,only been influced by natural selection strength,whose codon bias is mainly about compositon of nucleotide the same as that the mitochondrial-related gene nuclear coding.In order to study of the phylogenetic evolution of N.bombycis,multiple alignment were done by using amino acid sequences of frataxin,nifs and nifu from fungal,prokaryote,protozoa,together with the gene of N.bombycis.The result indicated that mitochondrial-related genes of N.bombycis, Encephalitozoon cuniculi and Antonospora locusta fall in one clade,close to typical evolution relation of high eukaryote,junior to protozoa,and is far from prokaryote.It can be supposed that the mitochondrial type frataxin protein of high eukaryote and microspordia is most similar,indicating microspordia may have a mitochondrial-like organelle and posses a part of mitochondrial function. Besides,the phylogenetic tree has shown that N.bombycis is closer to protozoa which also proved that microspordia is the sister group of fungi.We have constructed the pathway of 171 mitochondrial-related homologous genes of N. bombycis include Fe-S cluster assembly system,protein transportation and folding system, glycerophosphate shuttle system,antioxidation way and so on.Aithrough the copies numbers of relative gene between N.bombycis and Encephalitozoon cuniculi microspordiai varies,the major structure and function is same.2 Cloning,expressing and sequences analysis of mitochondrial-related protein ofN.bombycisWe focused 3 mitochondrial-related proteins:frataxin,nifu,ADP/ATP carrier protein by eukaryote express and RT-PCR.Frataxin gene is 306 bp.There are 3α-spiral which is loaded in N-terminal,center and C-terminal of protein,highly conservative in secondary structure and 1β-fold whose folding loaction also very conservative.In that,we have supposed that these structure is essential in the function of protein.Frataxin of microspordia is lack ofα-spiral with the location of 52-65 by compared of eukaryote,is lack ofα-spiral with the location of 40-45,61-66 by compared of yeast,can be guessed thatα-spiral with the location of 61-66 is related to protein location in cytoplasm,α-spiral with the location of 40-45 is related to protein location in microspordia.This conclusion also proved that the protein from microspordia is different from the typical eukaryote,the former lacks of loaction information maybe interpret reduction of loaction information or very different in nature.There are no mitochondrial targeting sequence by prediction analysis of Nbfra using PRORT program or MITOPROTII.Nbfra have a Arg in the location of 100 is same to CyaY analogues of plant,yeast and bacterial.Also,human frataxin is been found a His residues binding with iron in this Ioaction.Nbnifu gene which coded 140 amino acid residues is shorter to the nifu of typical eukaryote and Encephalitozoon cuniculi.The nifu gene is relative conservative in its structure,but have differences between N-terminal and C-terminal,which is highly similarity to the protein of E.cuniculi(78% identity at amino acid level).Nifu is lack of any transmembrane structure or signal peptide,and only has one conservative domain Nifu-N.The microspordia mitochondrial protein might be different from of typical eukaryote mitochondrial proteins,or the former is lack of location sigal,which caused highly reduction of location sigai or very different.There are no mitochondriai location sequence by prediction analysis of Nbfra using PRORT program or MITOPROTII.Nbadp/atp is 674bp which coded 224 amino acid,and its pI value is 9.15.In this study,we have found 3 transmembrane area by SMART software as typical membrane proteins.Compared with other homologue gene,Nbadp/atp is incomplete,lacking of most sequence information,and have high homology with ADP/ATP carrier proteinl of Encephalitozoon cuniculi.We predicated that ADP /ATP carrier protein located in mitochondrial membrane,transfers the ATP synthesised from mitochondrion to cytoplasm,transfers ADP back to mitochondrion.Frataxin,nifu,ADP/ATP carrier protein also have the activity of tanscription but poorer express activity.Frataxin exists in the total protein of N.bombycis and without a proprotein by western blotting analysis.3 Study on the location of mitochondrial-related gene encoding protein of N.bombycis To verify the expressing specificity and subcellular location characteristics features,IFA and immune electron microscopy were been used.It is obvious clear of nuclear field and ball-shaped mitochondria in the cell by MitoTracke Red CMXRos dying.Most of spores possessed red flurescence and differentiated clearly,nuclear field by DAPI dying and MitoTracke Red CMXRos is different.The cell location by fluorescent antibody and MitoTracker Red CMXRos dying is overlapped partialy,which can explained both mitochondria-like organelle and mitochondrial proteins coding by genome are exsist in N.bombycis.However,the function of these protein remains to be further researched.The results were shown that 8 mitochondrial-related proteins binding to spore of N.bombycis, is not overlapping with DAPI dying,and have no cross reaction with host cell of N.bombycis.The antibody signals of frataxin,ferredoxin,tim22/17,ATP/ADP carrier protein exsist in cytoplasm near the spore wall,have ball-shaped fluorscence sigai which might be michondrial relict organelle-mitosome,with 6 point-shaped signals in a spore.Nifs and nifu were just locating in the cytoplasm.MtTom70 and ADP/ATP carrier protein that possess continuous line-shaped flurescence sigal,can be guessd in the spore membrane.Location analysis of mitochondriai-related genes of frataxin,nifs,ferredoxin and mtTom70 proteins were done by immuno-gold labeling.It is clear to see that inner structure of spores of N. bombycis,such as polar tube,posterior vacuole,nucleus,spore wall and so on.Two to four Colloidal particle gold particles often cluster together in frataxin and ferredoxin which may be a mitosome. Nifs diffused in spore cytoplasm freedomly while mtTom70 along with spore wall.4 A preliminary analysis of mitochondrial-related proteins of microsporidia N.bombycisFirstly,organelles of N.bombycis were seperated by using percoll density gradient centrifugation and sucrose density gradient centrifugation methods.The results showed that two methods could not enrich and purify organelle.Then co-immunoprecipitation were used to obtain more mitochondrial-related proteins by polyclonal antibody,negative blood as a control.Cytoplasm protein was prepared from spores of N. bombycis by frost thawing method.Eleven proteins effected on frataxin,two were unkown proteins. One unknown protein associated with scaffolding protein nifu was found by analysis of MALDI-TOF-MS cutting gel method.The conjecture was that the eight proteins associated with TIM22/17 were four unknown proteins and three hypothesis proteins.One hypothesis protein effected on ATP/ADP carrier protein.Four proteins associated with nifs,in which one was unkown protein and two were hypothetical proteins.Two proteins were connected with ferredoxin and three proteins with tom70.Only one protein was related with adp/atp carrier protein.Some proteins had a signal peptide or transmembrane domain.更多还原