线粒体型蛋白frataxin在家蚕微孢子虫中的鉴定与分析

[目的]Frataxin是铁硫簇相关蛋白,在线粒体代谢中起着重要作用.分析家蚕微孢子中该蛋白的结构特征,系统进化关系以及在孢子体内的转录翻译活性.[方法]基于家蚕微孢子全基因组序列,同源序列搜索获得该基因序列.进行蛋白二级结构比较,近缘物种共线性特征分析及系统进化树构建.此外构建pGEX-4T-1-Nbfra原核重组表达载体,转化E coli BL21(DE3)进行目的蛋白表达纯化,以此为抗原免疫小鼠,制备抗体,并与家蚕微孢子总蛋白进行Western免疫杂交.[结果]Nbfra蛋白缺乏进入线粒体的信号序列,功能区缺乏部分α-螺旋;frataxin基因在不同微孢子基因组中的分布具有共线性特征,表明其在基因组进化中非常保守.系统进化分析显示微孢子形成独立进化支,并且与高等真核生物近缘,说明微孢子在进化地位上比其它原虫更加高等,支持了微孢子虫是真菌的姊妹枝的进化地位假说.[结论]免疫杂交结果表明Nbfra基因家蚕微孢子虫中能正常表达与翻译.本研究为微孢子的分类地位及线体假说提供了重要的补充依据.     

SWP25, A Novel Protein Associated with the Nosema bombycis Endospore1

ABSTRACT. Microsporidia are eukaryotic, obligate intracellular, spore-forming parasites. The resistant spores, which harbor a rigid cell wall, are critical for their host-to-host transmission and persistence in the environment. The spore wall comprises two major layers: the exospore and the endospore. In Nosema bombycis, two spore wall proteins have been characterized—an endosporal protein, SWP30, and an exosporal protein, SWP32. Here, we report the identification of the third spore wall protein of N. bombycis, SWP25, the gene of which has no known homologue. SWP25 is predicted to posses a signal peptide and a heparin-binding motif. Immunoelectron microscopy analysis showed that this protein is localized to the endospore. This characterization of a new spore wall protein of N. bombycis may facilitate our investigation of the relationship between N. bombycis and its host, Bombyx mori.     

家蚕微粒子病原虫(Nosema bombycis)小亚基核糖体RNA全基因的克隆及其二级结构的构建

在用PCR技术扩增、克隆、测序了家蚕微粒子病原虫Nosema bombycis (镇江株)小亚基核糖体RNA基因核心序列(5'-端起1 200 bp)的基础上,用SSP-PCR技术克隆了核心序列3'-端下游序列,从而获得了家蚕微粒子病原虫小亚基核糖体RNA基因的全序列共1 233 bp。 用RnaViz 、Forcon、DCSE等生物软件构建了家蚕微粒子病原虫小亚基核糖体RNA的二级结构,与其它微孢子虫及真核生物小亚基核糖体RNA的二级结构相比,该二级结构缺乏螺旋10、E10-1、11、18、E23-n和43。     

家蚕微孢子虫中小RNAs的全基因组鉴定与分析

【目的】家蚕Bombyx mori微粒子病是蚕业生产上的毁灭性病害,家蚕微孢子虫Nosema bombycis是该病的病原,可经卵垂直传播和经口水平传播。为了探索家蚕微孢子虫中对重复元件的抵御以及对基因转录调控的潜在方式,本研究拟在基因组水平上对该物种的小RNAs进行全面系统的分析,鉴定与转座子相关的小RNAs和潜在的miRNAs。【方法】从感染家蚕微孢子虫的家蚕中肠中提取总RNA,分离小片段RNA并反转录后,进行Solexa高通量测序。通过生物信息学方法对小RNAs进行分类及功能注释,鉴定起源于家蚕微孢子虫不同类型转座子的小RNAs,并对潜在的miRNA进行预测分析。【结果】家蚕微孢子虫小RNAs的长度主要是24和25 nt,其中大部分序列表现出5′末端的尿嘧啶偏好性。家蚕微孢子虫中存在丰富的与转座子相关联的小RNAs,并且与转座子标准序列匹配的反义小RNAs明显多于正义小RNAs。同时,鉴定获得了31个候选miRNAs,部分为Nosema属的其他孢子虫中所共有,暗示其在微孢子虫基因组进化上具有保守性。【结论】首次鉴定到家蚕微孢子虫的转座子相关性小RNAs,暗示小RNAs在家蚕微孢子虫基因组对转座子防御过程中起到作用,31个潜在的miRNAs为家蚕微孢子虫miRNAs的功能验证提供了后续靶标。     

家蚕微孢子虫丝氨酸蛋白酶抑制剂蛋白serpin基因NbSPN106的鉴定

摘要：【目的】Serpin在病原与宿主互作中起着重要的作用。本研究旨在分析在家蚕微孢子虫中的丝氨酸蛋白酶抑制剂蛋白（Serpin）的结构特征,原核克隆表达以及Western blotting检测。【方法】 基于家蚕微孢子虫全基因组序列,同源序列比对搜索获得serpin基因序列。利用在线软件分析基因的序列特征,ClustalX对氨基酸序列进行多重序列比对。构建含有GST标签的pGEX4T1-NbSPN106原核重组表达载体,在大肠杆菌BL21（DE3）诱导表达并进行纯化。纯化的重组蛋白免疫小鼠,制备抗体,并与家蚕微孢子虫总蛋白进行Western blotting免疫杂交。【结果】比对搜索在家蚕微孢子虫基因组中发现一个新的serpin基因NbSPN106。NbSPN106蛋白序列长度为384 aa,N端具有信号肽,编码一个42 kDa左右的成熟蛋白。多重序列比对说明NbSPN106具有保守的serpin位点,可能具有抑制功能。免疫杂交在家蚕微孢子虫总蛋白检测到一条45 kDa左右的特异条带。【讨论】生物信息学分析以及免疫杂交结果说明在家蚕微孢子虫中存在NbSPN106。这是在微孢子虫中首次报道发现serpin基因。对研究家蚕微孢子虫与宿主家蚕的互作具有一定的参考价值。     

家蚕微孢子虫在家蚕胚胎细胞系BmE-SWU1中的极丝弹出情况

本文借助扫描电镜(Scanning Electron Microscope,SEM)对在家蚕胚胎细胞(Bombyx mori-SWU1Embryoniccell line,BmE-SWU1)中增殖的家蚕微孢子虫(Nosema bombycis)进行了观察;同时,利用间接免疫荧光试验(Indirect Immunofluorescence Test,IFAT)对家蚕微孢子虫极丝弹出情况进行了研究。结果发现:在家蚕微孢子虫体外培养体系中观察到了二型孢子、发芽后的孢子空壳以及未成熟孢子等;此外,在接种后10d的细胞爬片中,发现了大量孢子弹出极丝的情况,其极丝的长度在58μm-120μm之间,其中100μm以上的居多,且其极丝的扭曲程度、方向性、粗细也大都不同。本研究表明了微孢子虫在培养细胞体系中自动发芽可以以长极丝孢子的形式来完成。     

Phylogenetic analysis of Nosema antheraeae (Microsporidia) isolated from Chinese oak silkworm, Antheraea pernyi

The microsporidian Nosema antheraeae is a pathogen that infects the Chinese oak silkworm, Antheraea pernyi. We sequenced the complete small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) of N. antheraeae, and compared the SSU rRNA sequences in other microsporidia. The results indicated that Nosema species, including N. antheraeae, formed two distinct clades, consistent with previous observations. Furthermore, N. antheraeae is clustered with N. bombycis with high bootstrap support. The organization of the rRNA gene of N. antheraeae is LSU-ITS1-SSU-ITS2-5S, also following a pattern similar to the Nosema type species, N. bombycis. Thus, N. antheraeae is a Nosema species and has a close relationship to N. bombycis.     

A complete Sec61 complex in Nosema bombycis and its comparative genomics analyses

We characterized a complete Sec61 complex in Nosema bombycis, which has been shown to consist of Sec61alpha, Sec61beta, and Sec61gamma genes. Comparing the genomic regions that harbor the respective subunit genes between N. bombycis, Encephalitozoon cuniculi, and Antonospora locustae, we found that microsporidian genomes have high degree of synteny in short genomic fragment, and that this gene synteny in general might exist throughout microsporidian genomes.     

Action of carbendazim on the development of Nosema bombycis Naegeli in silkworm Bombyx mori L.

Abstract:  Carbendazim, a benzimidazole compound is effective in curing pebrine disease in silkworm, Bombyx mori , caused by Nosema bombycis , if treatment is given within 48 h post-infection or before the parasite establishes itself in the host tissue. Ultrastructural evidence of the action of carbendazim showed an adverse effect of the drug on both merogonic and sporogonic stages of the parasite in the midgut and silk gland of the silkworm. The drug caused elongation, vacuolation and depletion of cytoplasmic contents of the meront, sporont, sporoblast and spore stages of N. bombycis .     

不同DNA抽提方法对普通PCR和实时荧光定量PCR方法检测家蚕微孢子虫的影响

为了优选快速、 灵敏、 特异的家蚕微孢子虫Nosema bombycis分子检测方法和DNA抽提方法, 本文通过对家蚕微孢子虫TaqMan探针荧光定量PCR检测方法和SYBR Green荧光定量PCR检测方法的建立以及反应体系优化, 并与普通PCR方法进行比较; 再采用4种不同DNA抽提方法分别对PCR和实时荧光定量PCR方法检测家蚕微孢子虫悬浮液的效果评价。结果显示: 不经过DNA抽提, 直接将家蚕微孢子虫发芽液进行PCR反应的效果优于其他方法, 检测灵敏度由高到低依次为直接法、 酚/氯仿抽提法、 动物组织DNA试剂盒抽提法和植物组织DNA试剂盒抽提法; TaqMan探针法检测家蚕微孢子虫发芽液的灵敏度和SYBR Green法相近, 达到微孢子10²个/mL, 两者均优于普通PCR方法。实验表明, 直接采用发芽液结合荧光定量PCR方法检测家蚕微孢子虫最为简便、 快速、 灵敏。该研究结果将有助于提高家蚕微粒子病监控技术和检疫能力, 对家蚕微粒子病的检疫和防治具有积极意义。     

家蚕微孢子虫孢壁蛋白与其发芽的相关性

为了研究孢壁蛋白与家蚕微孢子虫发芽（孢原质弹出）的相关性,我们采用碳酸钾诱导微孢子虫体外发芽结合密度梯度离心的方法（简称GDGC法）,收集纯化发芽后的孢子空壳（简称孢壳）,对发芽液、纯化的孢壳及成熟孢子的孢壁蛋白组分进行了分析。结果表明：GDGC法可以获得高纯度孢壳,计算出其密度为1．130g/cm^3;与发芽前成熟孢子提取的孢壁蛋白相比,空孢壳可以提取到主要孢壁蛋白SWP32、SWP30、SWP25,同时发现SWP32、SWP25丰度有所降低;结合碳酸钾发芽液的蛋白电泳分析,发现孢壳上丰度降低的SWP32在发芽液蛋白样品中存在,LC—MS／MS数据分析也发现SWP32、SWP30、SWP25在碳酸钾处理液中都有存在;而用碳酸钾溶液处理冷冻孢子时,未观察到发芽现象,电泳结果显示此时K2CO,溶液中只有SWP30条带,说明在碳酸钾溶液诱导的发芽过程中SWP32和SWP25从孢壳上脱落可能与发芽相关而不是被碱性的碳酸钾溶解下来的[动物学报54（6）：1068—1074,2008]。     

家蚕微孢子虫极管蛋白1 (NbPTP1) 在果蝇S2细胞中的表达与糖基化修饰

极管蛋白(Polar tube protein)是极管的主要成分,能特异性定位于微孢子虫极管,在微孢子虫侵染宿主过程中发挥重要作用。文中分析了家蚕微孢子虫极管蛋白1中潜在的O-、N-糖基化修饰位点,克隆了家蚕微孢子虫极管蛋白1全基因序列,并将其插入带有V5和His标签的真核表达载体pMT/Bip/V5-His A中,成功构建了pMT/Bip/V5-His A-NbPTP1重组质粒,经转染果蝇S2细胞后,发现NbPTP1基因能在果蝇细胞中高效表达。此外,Lectin blotting和β-消除反应分析结果表明:果蝇S2细胞内表达的NbPTP1具有O-糖基化修饰特征。以上结果为研究NbPTP1的糖基化修饰特征与其功能之间的关系提供了基础,有助于揭示微孢子虫侵染机制,建立可行有效的微孢子虫病诊断和防治措施。     

不同消毒方法对家蚕微粒子的存活和侵染力影响

家蚕微粒子病是家蚕Bombyx mori的重要病害,探索其消毒杀灭方法对蚕业生产具有重要意义。本研究采用微粒子染色法和添食家蚕侵染法测试了温度、紫外线和消毒剂处理108个/mL家蚕微粒子后对家蚕微粒子的消杀作用,结果表明：温度对家蚕微粒子虫灭活温度为60℃处理30 min以上;使用20 wx功率紫外灯距离50 cm照射12 min及以上时,微粒子死亡率为47.70%,对家蚕侵染率为0;三氯异氰脲酸800 mg/L及以上浓度处理6 min以上家蚕微粒子死亡率为100%,对家蚕的侵染率为0;戊二醛癸甲溴铵200 mg/L及以上浓度处理6 min以上家蚕微粒子死亡率为100%,对家蚕的侵染率为0。3种消毒方法中温度法主要用于小型蚕具消杀微粒子;紫外线用于蚕业辅助设施的表面微粒子的消杀;三氯异氰脲酸和戊二醛癸甲溴铵均表现出较好的消杀效果,其中戊二醛癸甲溴铵较含氯的消毒剂三氯异氰脲酸稳定性强,刺激性小,对养蚕的金属腐蚀性小,可作为含氯消毒剂的部分替代使用。本研究结果可为蚕业生产中消杀微粒子提供参考。     

Calcofluor White M2R荧光染色法识别家蚕微孢子虫

本研究探讨应用荧光染色试剂Calcofluor White M2R染色鉴别家蚕微孢子虫Nosema bombycis。结果表明：在荧光显微镜下可见家蚕微孢子虫孢子被染上强烈的青蓝色荧光, 而寄主组织碎片、病毒、细菌等不被染色。该法是一种快速有效鉴别微孢子虫的方法。     

The nuclear apparatus and chromosomal DNA of the microsporidian Nosema antheraeae

The microsporidian Nosema antheraeae is a pathogen of the Chinese oak silkmoth Antheraea pernyi, the molecular karyotype of which is still poorly understood. Here the diplokaryon of N. antheraeae strain NP-YY has been visualized both by fluorescence and electron microscopy. In addition, pulsed-field gel electrophoresis (PFGE) showed that the haploid genome of N. antheraeae is approximately 9.3-9.5 million base pairs organized into 15 chromosomal bands. The mean fluorescence intensity of N. antheraeae and Nosema bombycis DNA measured by flow cytometry confirmed that the genome size of these two species was congruent with measurements obtained by PFGE. These initial results on the chromosome organization of N. antheraeae provide a foundation for the comparative genomics of N. antheraeae with other species of Nosema.     

Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.     

家蚕微孢子虫ADP/ATP转运蛋白的原核表达及间接免疫荧光定位分析

【目的】家蚕微孢子虫Nosema bombycis ADP/ATP转运蛋白可能参与搬运宿主细胞的能量。本研究克隆家蚕微孢子虫ADP/ATP转运蛋白基因,并进行原核表达、抗体制备及间接免疫荧光定位,为控制和防治家蚕微粒子病提供理论基础。【方法】通过同源序列比对鉴定家蚕微孢子虫N. bombycis ADP/ATP转运蛋白序列,采用生物合成的方法将编码3段面向膜内侧肽段的核酸序列拼接合成,在其两端引入BglⅡ和SalⅠ酶切位点,克隆至pUC57载体并测序,再亚克隆至含有二氢叶酸还原酶（dihydrofolate reductase,DHFR）标签的表达载体pQE40中,然后利用BamHⅠ和SalⅠ酶切获得含有DHFR标签的重组序列,并连接至pET30a（+）载体中进行诱导表达。通过SDS-PAGE、镍柱亲和层析和免疫印迹法鉴定表达蛋白,利用间接免疫荧光对ADP/ATP转运蛋白的分布进行检测。【结果】家蚕微孢子虫的ADP/ATP转运蛋白编码序列（GenBank登录号为EOB13854.1）全长1 524 bp,编码蛋白含有507个氨基酸残基,预测分子质量为59 kDa,等电点为9.35。具有12个跨膜结构域和TLC结构域,其中TLC结构域含有4个功能保守位点。与蜜蜂微孢子虫的ADP/ATP转运蛋白比较,氨基酸序列一致性达30%。系统进化分析表明微孢子虫ADP/ATP转运蛋白聚为一类,具有共同的起源。成功构建了NbADP/ATP-△TM-DHFR-pET30a原核表达重组质粒,目的基因获得表达,其融合蛋白分子量约为37 kDa,纯化重组蛋白并制备了多克隆抗体。免疫印迹分析表明,成熟微孢子虫中表达ADP/ATP转运蛋白;间接免疫荧光定位结果显示,家蚕微孢子虫孢子ADP/ATP转运蛋白定位于孢子质膜上。【结论】本研究将为阻断微孢子虫能量来源,达到控制和防治家蚕微粒子病提供新的思路。     

家蚕微孢子虫感染对家蚕海龟蛋白(Bmtutl)基因表达水平的影响

【目的】本研究旨在阐明家蚕微孢子虫Nosema bombycis感染不同时间对家蚕Bombyx mori幼虫不同组织中家蚕海龟蛋白(Bombyx Turtle, Bmtutl)基因表达水平的影响,为揭示家蚕微孢子虫的侵染机制奠定基础。【方法】利用生物信息学方法对家蚕海龟蛋白3种亚型Bmtutl-464, Bmtutl-519和Bmtutl-810的序列结构特征进行了分析;利用qPCR检测家蚕微孢子虫感染后12, 24, 48, 72, 96和120 h,家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因表达水平的变化情况。【结果】家蚕海龟蛋白3种亚型的二级结构均主要由无规则卷曲、α螺旋、β转角和延伸链组成,其中无规则卷曲所占比例最高。但是PredictProtein分析发现,Bmtutl-464, Bmtutl-519和Bmtutl-810之间的蛋白/多核苷酸结合位点存在较大差异。qPCR结果表明,感染家蚕微孢子虫后,家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因的整体表达处于被抑制状态,尤其在脂肪体中最为明显：Bmtutl-519和Bmtutl-810基因的表达在家蚕微孢子虫感染家蚕后的72 h开始受到显著抑制,特别是Bmtutl-519基因,其相对表达水平均不到对照的5.0%。【结论】家蚕海龟蛋白这3种亚型的序列结构特征存在较大差异,家蚕微孢子虫感染在一定程度抑制了家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因尤其是Bmtutl-519的表达。结果说明,与其他两种家蚕海龟蛋白亚型相比,Bmtutl-519蛋白可能在家蚕微孢子虫侵染宿主的过程中起主要作用。     

Molecular phylogeny of onygenalean fungi based on small subunit ribosomal DNA (SSU rDNA) sequences

Phylogenetic analysis of nucleotide data from small subunit ribosomal DNA (SSU rDNA) sequences (ca. 1685 bp.) was performed on 19 taxa of the Onygenales and three related mitosporic fungi. Phylogenetic trees were constructed by the neighbor-joining method with the sequence data of related taxa obtained from DNA databases. The species in the Onygenales form two clusters and seven subclusters, and the tree topology reflects the traditional classification by Currah (1985) with some exceptions. The Myxotrichaceae is placed in the different lineage, separate from other plectomycetous taxa and among the Leotiales and the Erysiphales. Furthermore, two separate lineages in the Myxotrichaceae were found. Tree topology suggested the Onygenaceae is polyphyletic and composed of three subgroups; 1) most members of Onygenaceae, 2)Spiromastix warcupii, and 3) pathogenic dimorphic fungi classified inAjellomyces.