【作者】 罗洁；

【导师】 周泽扬；

【作者基本信息】 西南大学 ， 微生物学， 2013， 博士

【摘要】 DNA作为遗传物质在承载着生命延续的同时也为分子考古学家研究人类的起源、物种的进化史提供了新的契机。近年来,随着基因组数据库的丰富和完善,生物学家逐渐发现接近1/3的物种都具有物种特异基因,鉴于它们大多是随机产生和自然选择双重压力下被保留下来的“化石”基因,对物种的环境适应性、自然习性起着很关键的作用,有的甚至在人脑和视觉蛋白形成过程中扮演举足轻重的作用,因此逐渐受到遗传学家和分子考古学家的青睐。微孢子虫做为一种存在广泛宿主的“无线粒体”原生动物,它们兼具原核细胞和真核细胞的特征,尽管对于它们的进化地位仍然存在争议,但是近年来,越来越多的研究表明它们是进化地位较低的真核生物,并且目前已被NCBI归类为真菌类。其中家蚕微孢子虫(Nosema bombycis)作为首个被鉴定的微孢子虫,专性寄生于经济昆虫家蚕,是引发家蚕微粒子病的病原体。家蚕微孢子虫能够在外界环境刺激下,成功弹出极丝,将孢原质送入宿主体内从而成功的寄生,进而引起家蚕生理机能障碍,造成宿主细胞的病变坏死以及组织器官功能的丧失。鉴于其对人类经济生活带来的巨大危害,目前,家蚕微孢子虫已被世界各养蚕国家和地区列为唯一法定检疫对象。尽管对其研究已有近150年的历史,但是微粒子病的防控仍然是蚕业界研究的重点和难点。随着基因组时代的来临,人们获得了越来越多的微孢子虫基因组序列信息,因此,基于比较基因组学分析,探索不同种属之间的微孢子虫的差异基因的种类、数量及功能,对于研究不同种类微孢子虫生物学特征,发掘微孢子虫与其对应宿主之间的共进化关系,以及为寻找种属特异的检测靶标基因,具有十分重要的生物学意义。近年来,本实验室对家蚕微孢子虫进行了全基因组框架图的绘制,基于全基因组序列的数据,并结合已报道的其他微孢子虫基因组,我们首次鉴定到一段Nosema属特有的基因组区域,为哺乳动物寄生性微孢子虫——兔脑炎微孢子虫、肠道微孢子虫和比氏肠道微孢子虫基因组所缺失。这段Nosema属微孢子虫基因组共线性区域的演化历程是怎样的?分布于该基因组特异区域内的编码基因在家蚕微孢子虫基因组中扮演什么样的角色?鉴于物种特异基因在研究微孢子虫进化史以及不同微孢子虫间的分子鉴定两方面的重要性,同时也为了回答上述问题,本论文的主要研究内容包括：1. Nosema属微孢子虫特异基因组共线性区域的鉴定与分析对家蚕微孢子虫、兔脑炎微孢子虫和蜜蜂微孢子虫的同源基因进行比较分析,发现家蚕微孢子虫第32号Scaffold上存在一段1Nosema属特异的基因组区域,进而在已发布基因座位信息的六种微孢子虫中进行基因座位保守性分析,进一步证实该区域为脑炎属微孢子虫及比氏肠道微孢子虫基因所丢失的基因组片段。通过对其中分布的编码基因的预测及分析,发现这段特异的基因组区域存在两个编码序列,基因代号为NBO32g0034和NBO32g0035,前者编码DNA聚合酶kappa(简称NbPolκ),后者NBO32g0035的预测功能未知(简称NbOP1), RT-PCR实验证实这两个基因均能够正常转录。对该区域上游970bp序列进行启动子和调控元件预测,发现该区域存在保守的TATA框和GC框结构,并且含有SP1结合位点、CAP/CRP结合位点、bZIP (Basci-leucine zipper)转录因子蛋白、Tst-1结合位点和c-Myb等参与转录活性调节的调控基础序。该区域的GC含量明显大于N.bombycis基因组的平均GC含量,并且大量分布DNA类型的转座元件,暗示该区域是基因组不稳定区域。2. NbPolκ和NbOPl蛋白的编码基因序列特征及其染色体定位研究为了深入了解分布于该段特异基因组区域内的编码基因特征,我们首先针对NbOP1的编码基因进行预测及分析,结果显示该蛋白在家蚕微孢子虫基因组中不存在其他拷贝,其序列不具有信号肽,仅含有4个疏水簇,并且富含极性氨基酸,随后对该位置蛋白的二级结构进行预测,发现其包含20个a螺旋和11个β折叠。经过克隆测序发现该基因具有核苷酸多态性,属于典型的孤儿蛋白。随后,我们对NbPolκ蛋白的编码基因进行了生物信息分析,确定其D87至L417存在保守的DNA聚合酶kappa功能结构域,但比典型的真核生物POLK存在N-端和C-端的减缩,但是,这种两端不同程度的缺失并没有影响其氨基酸的结构特征。而用最大似然法构建的系统发育树结果显示NbPlκ与其他三种微孢子虫的POLK聚为一簇,但是这一簇基因却与真菌界的亲缘关系较远,而是落入了细菌域,并与梭状芽孢杆菌(Clostridia)亲缘关系较近。该系统发育树在一定程度上反映了NbPolκ的进化历程,推测NbPoκ可能水平转移自某一种细菌。通过对NbPolκ和NbOP1的同源信息检索,并制备探针进行Southern blot杂交,结果说明我们所分析的具有Nosema属微孢子虫基因组共线性区域位于家蚕微孢子虫的第XV染色体上。3. NbPolκ和NbOP1的在酿酒酵母中的定位首先采用亚细胞定位导肽预测软件对这两个基因进行亚细胞定位预测,结果显示NbPolκ可能分布于细胞核或者线粒体中,而NbOP1则具有细胞质定位特征。随后利用酿酒酵母表达系统,检测其各自的亚细胞定位分布。结果表明,NbOP1定位于酿酒酵母的细胞质；而NbPolK则能够靶向酿酒酵母线粒体,并且N-端导肽序列对于NbPolκ的定位信号没有任何影响。这一结果与克鲁斯锥虫的同源基因TcPolκ能够具有线粒体定位信号的特征相符合,暗示寄生生活中的NbPolK的特殊地演化地位及其与典型真核生物Po1κ的功能分化。4. NbPolκ和NbOPl在家蚕微孢子虫基因组中的功能验证为了更好的了解这两个编码基因在家蚕微孢子虫基因组的表达定位特征以及其可能存在的功能。我们构建了原核表达载体,通过重组融合蛋白的诱导表达和纯化,获得了与理论分子量一致的重组蛋白,用其免疫小鼠以制备多克隆抗体。免疫印迹结果显示所得抗血清特异性较好,能够识别与理论分子量相近的蛋白条带。分别用其与成熟孢子和处于增殖期的孢子进行间接免疫荧光,表明NbOP1为一种新鉴定到的位于家蚕微孢子虫孢壁的蛋白；而NbPolκ则是定位于家蚕微孢子虫细胞核的参与氧化损伤途径的蛋白。5. Nosema属微孢子虫基因组特异共线性区域的进化历程假说根据我们前面对两个编码基因的研究,结合微孢子虫的物种进化分析,我们通过总结实验结果并提出了该片段在微孢子虫基因组中进化历程的假说。假说核心为,微孢子虫的最近共同祖先(Last Common Ancester, LCA)通过水平基因转移或垂直遗传获得Nosema属微孢子虫基因组特异共线性区域,之后,该区域在微粒子属与脑炎属微孢子虫在物种分化之后各自独立进化。,NbPolκ勺保留可能是为了应对家蚕微孢子虫和蜜蜂微孢子虫来自昆虫宿主的剧烈地氧化应激(酚氧化物酶通路)的攻击所造成的DNA损伤,但是可能由于功能退化或者功能分化进而NbPolκ仅保留在四种感染昆虫的微孢子虫的基因组中中然而,家蚕微孢子虫物种特异基因NbOP1的保留是由于家蚕微孢子虫宿主专性适应性需要而衍生出的新的功能基因。当然,这些推论亟待进一步的实验验证。综上所述,本研究首次对家蚕微孢子虫物种特异基因进行细致的研究,并同其他微孢子虫的基因座位保守性进行了比较研究,证实这段Nosema属微孢子虫基因组特异共线性区域中的两个功能基因均能够编码家蚕微孢子虫生理代谢途径不可或缺的重要蛋白。最后,结合前期的分析数据我们对该区域的成因进行了初步探讨,并提出微孢子虫中差异基因序列的形成机制的假说。总之,本研究初步探索了家蚕微孢子虫专性细胞内寄生生活所形成的寄生虫与宿主协同进化历程,同时,也为筛选家蚕微粒子病的分子检测靶标基因奠定分子生物学基础。更多还原

【Abstract】 DNA genetic information is not only contributed to life inheritance, but also offered clues to trace back to the origins and evolution of human beings for molecular archaeologists studies. In recent years, with the abundant and enrichment of genome database, biologists has figured out that nearly1/3species-specific genes existed in almost all of the species, which were randomly generated but be preserved under natural selection, and even plays a pivotal role in environmental adaption and natural habitats.Microsporidia is a kind of amitochondrion protozoa with a wide range of host. Although most of its genome exhibits both prokaryotes and eukaryotes characteristics, their evolutionary status remains controversial. Microsporea is placed within fungi by NCBI nowadays, as platitudinous published results demonstrated they are closer related to eukaryotes or sister-group of fungi with a common ancestor. Nosema bombycis, the first microsporidia to be identified, is an obligate intracellular parasite that causes pebrine. They can severely infected silkworm as eject sporoplasm into host cells/tissues through polar tube, which in turn resulting in host insects metabolic disturbance, cells necrosis and organs dysfunction. Pebrine can lead to the great loss of silk industry, thus it was pronounced as the only legal quarantine disease amongst worldwide sericulture countries. Despite the study on N. bombycis has experienced nearly150years, the prevention and control of pebrine are still suspended at present. Recent year witness the booming era of microspora genome, as well the indefatigable exploring of the distinction characteristics among different genus for species-specific genes selection. Basing on the draft genome of N. bombyis carried out by our lab, we predicted and analyzed a Nosema-specific genome region, which contains two CDS genes named as NbPolκ and NbOPl in N. bombycis, but absent in the genome of Encephalitozoon cuniculi, Encephalitozoon intestinalis and Enterocytozoon bieneusi. NbPolκ has homologue genes in Nosema ceranae, whereas its neighbor ones were only kept in No.32Scaffold in N. bombycis. The objective of this work is to study where does it comes from and and what’s the functional role of this divergent genome region in N. bombycis.To answer these questions, as well as take into account of the significant role of species-specific genes involved in evolution analysis, we performed this study in following aspects:1. The identification and analysis of Nosema-specific genome collinearity regionBy comparing with the homologue genes among N. bombycis, N. cernaea, and E. cuniculi genome data, we analyzed two encoding genes NbPolκ and NbOP1distributed within a Nosema-specific genome region at No.32scaffold in N. bombycis. RT-PCR results indicated that both genes are transcribed in midguts of silkworm infected by N. bombycis.The characteristics analysis on their upstream sequence of this region exhibit both core promoter and regulatory element motifs that could regulated the transcriptional levels. In addition, the GC content of the specific region was higher than the average GC%of (the) whole N. bombycis genome. Transposable elements were widely distributed at this region, which implies the flexible of this region.2. The sequences characteristics and chromosomal localization of NbPolκ and NbOPl genesTo further analyze the characteristics of encoding genes that distributed in this region, we first analyzed the composition of amino acids of NbOP1, and the results showed that NbOPl is a kind of typical orphan gene that contains4hydrophobic clusters and with abundant of polar amino acids. The prediction of NbOP1secondary structure showed that it is a folded protein with20a-helices and11β-sheet. Moreover, this single copy gene also showed nucleotide polymorphism. We thereafter analyzed the NbPolκ gene, and confirmed it has DNA polymerase kappa functional domain from D87to L417. But the reduction occurred at both N-and C-termini ends, comparing with POLK from other eukaryotes. It is notably that this termini reduction has no effect on the protein folding. Finally, the phylogenic analysis demonstrated this gene is closer related to prokaryotes, which implied that the unique evolution process of this gene in N. bombycis. Moreover, southern hybridization results showed that this Nosema-specific genomic region is located on the chromosome XV of N. bombycis.3. The localization of NbPolκ and NbOP1protein in Saccharomyces cerevisiaeThe subcellular localization signals were primarily predicted, and the results indicate NbPolκ contains N-terminal transite peptide that target to nuclear or mitochondrion, however the NbOP1is equipped with cytoplasm target peptides. In order to verify the subcellular localization of these two proteins, we constructed recombinant vectors of NbPolκ-pUG35,NbPolκ-pUG36, and NbOP1-pUG35, respectively. After that, positive expression recombinant yeasts strain for NbPolκ and NbOP1were induced in SC-Ura-Met for expression. The result indicated orphan protein has no specific targeting signals, but NbPolK could target on yeast mitochondrion regardless of N-terminal transit peptide, it is notable that the later result is consistent with the characteristics of TcPolκ, indicating the un-perspective evolutionary process and functional divergent of Polκ.4. The existence meanings of NbPolK and NbOP1in N. bombycisTo better understand the function of these two proteins, we reconstructed recombinant vectors of NbPolκ and NbOP1, and then transformed them into E. coli BL21, and the fusion protein purified by Ni-NTA affinity columns, was immunized with mice for polyclonal antibody preparation, repectively. Western blot results showed that both NbPolK and NbOP1were expressed in N. bombycis. Immunolocalization through indirect fluorescent assay displayed that orphan protein is localized on the surface of spores, the result of which implied it could be a spore wall protein (SWP) or a protein interact with SWP. Whereas, NbPolκ can recognize DNA but involving in oxidative damage pathway.5. The formation of this Nosema-specific collinearity region in N. bombycisAccording to the analysis of two coding sequences and microsporidia speciation, a hypothesis on the evolution of this genomic fragment was proposed. The core of this hypothesis is that after the Last Common Ancestor (LCA) of microsporidia obtained this fragment from HGT or Vertical inheritance, but diverged during the independent evolution of different microsporidia. In Nosema genus, NbOP1was only reserved in N. bombycis to meet the needs of neofunctionalisation. But, to the Encephalitozoon genus, the genomes of which faced more serious reduction evolution, both NbPolκ and NbOP1were lost at last because of degeneration or subfunctionlisation. While NbPolκ reservation may contribute to both N. bombycis and N. ceranae bypass the DNA damage caused by the attack of host phenol Oxidase Activation Pathway, but NbOP1 was only kept in N. bombycis because of the needy of specific host recognization or interaction. However, our hypothesis is still needs further verification in future.In summary, this study aimed to study the encoding genes within Nosema-specific genome collinearity region, and finally confirmed that both of these two genes encode important proteins indispensably involving in physiological metabolisms of N. bombycis. We finally imagined the evolutionary process of this region in microsporidia according to the bioinformatic analysis and experimental results. This study not only enriches the functional genome data of N. bombycis, but also lays the basis of the molecular biological targets of pebrine.更多还原