本文選題：家蠶微孢子蟲 + 水通道蛋白��； 參考：《江蘇科技大學》2016年碩士論文

【摘要】：微孢子蟲(Microsporidia)是一類寄主域廣泛的真核生物,從脊椎動物到無脊椎動物都會被微孢子蟲感染。微孢子蟲作為魚類、甲殼類和許多經(jīng)濟類昆蟲的病原體,常給農(nóng)業(yè)生產(chǎn)帶來巨大損失。由家蠶微孢子蟲(Nosema bombycis)引起的家蠶微粒子病是蠶業(yè)生產(chǎn)中一種重要的蠶病,不僅能夠水平傳播,還能經(jīng)由卵垂直傳播給后代,有時會給蠶業(yè)生產(chǎn)造成嚴重影響。迄今為止,雖然在家蠶微孢子蟲的傳染規(guī)律和防治技術(shù)方面取得了不少研究進展,但在其侵染機制方面還沒有完全弄清楚。微孢子蟲對宿主細胞的侵染,往往是通過孢子萌發(fā)將孢子內(nèi)的孢原質(zhì)注射到宿主細胞中來實現(xiàn)的。目前普遍認為的微孢子蟲萌發(fā)感染宿主經(jīng)歷了以下幾個事件:(1)孢子與宿主細胞的粘附;(2)孢子被激活;(3)孢子內(nèi)的滲透壓升高;(4)極絲外翻彈出;(5)孢原質(zhì)通過彈出的極絲進入宿主細胞。孢子的發(fā)芽是微孢子蟲感染宿主體的首要前提,研究其發(fā)芽機制對防治微孢子蟲病有著重要的作用。有觀點認為微孢子蟲中可能有水通道蛋白的存在,將孢外的水分運輸?shù)芥咦觾?nèi)部,造成孢子內(nèi)部滲透壓的升高,這種升高的內(nèi)壓便成為孢子發(fā)芽的動力。因此,本文希望通過研究,探索家蠶微孢子蟲中是否存在水通道蛋白以及該蛋白參與孢子發(fā)芽的機制。本研究根據(jù)微孢子蟲數(shù)據(jù)庫和MIP數(shù)據(jù)庫交叉比較分析篩選出可能的家蠶微孢子蟲水通道蛋白基因,成功克隆出家蠶微孢子蟲NbAQP序列,該序列具有完整的開放閱讀框ORF,長750bp,共編碼249個氨基酸,蛋白質(zhì)分子質(zhì)量約為26.66kD,等電點為5.12,無信號肽。NbAQP氨基酸序列與其他5種微孢子蟲的水通道蛋白序列同源性為50%以上,含有6個跨膜結(jié)構(gòu)域,三級結(jié)構(gòu)預測分析顯示有6個長螺旋和2個短螺旋,分別以兩個螺旋為起點,各自延伸出一條氨基酸鏈,氨基酸鏈在外部環(huán)繞后返回起點處。用半定量PCR的方法檢測家蠶微孢子蟲感染家蠶后NbAQP的轉(zhuǎn)錄情況。利用釀酒酵母真核表達系統(tǒng)對家蠶微孢子蟲NbAQP蛋白的體外表達進行了研究。首先設計帶有酶切位點的引物,將NbAQP基因克隆到真核表達載體pYES2-NTC中,構(gòu)建重組表達質(zhì)粒并轉(zhuǎn)化酵母感受態(tài)細胞。隨后在INVSc1菌株中使用β-半乳糖誘導帶His標簽的融合蛋白的表達以及通過Western blot免疫印跡鑒定,免疫印跡檢測到大小為31kD的His-NbAQP融合蛋白條帶,說明家蠶微孢子蟲NbAQP蛋白可在真核表達系統(tǒng)中表達。預測蛋白抗原表位并合成多肽,以該條多肽作為抗原免疫新西蘭兔制備多克隆抗體,利用免疫膠體金電鏡技術(shù)對NbAQP蛋白在家蠶微孢子蟲中進行定位分析。免疫電鏡結(jié)果顯示,在家蠶微孢子蟲的孢子壁上能夠看到膠體金顆粒分布,而對照組中相應位置則沒有膠體金的分布,說明家蠶微孢子蟲NbAQP蛋白主要定位于家蠶微孢子蟲的孢子壁上,對其功能的行使有著重要意義。為了研究家蠶微孢子蟲NbAQP蛋白是否能夠參與孢子發(fā)芽的過程,進而在微孢子蟲感染家蠶的事件中發(fā)揮作用,對NbAQP蛋白做初步功能研究。通過抗體封閉實驗檢測抗體封閉處理后孢子發(fā)芽率的變化。結(jié)果顯示,使用多克隆抗體孵育封閉1h的處理組的發(fā)芽率為16.58%,與空白對照組的發(fā)芽率(23.05%)相比,差異性顯著(P0.05)�？贵w封閉對孢子發(fā)芽的抑制率約為28%。說明封閉NbAQP蛋白后能夠降低孢子的發(fā)芽率,NbAQP蛋白可能參與了家蠶微孢子蟲發(fā)芽的過程。本研究結(jié)果表明,家蠶微孢子蟲中存在水通道蛋白,該蛋白主要存在于孢子壁和原生質(zhì)膜,在微孢子蟲感染家蠶、增殖以及孢子形成的整個生活史均有轉(zhuǎn)錄表達,且在孢子的發(fā)芽過程中起到重要作用。
[Abstract]:Microspore (Microsporidia) is a wide range of eukaryotes in the host region. Both vertebrates and invertebrates are infected by microspore. Microspore, as the pathogen of fish, crustaceans and many economic insects, often causes huge losses in agricultural production. The silkworm particles caused by the Nosema bombycis of the silkworm (silkworm, microspore) Disease is an important sericulture disease in the production of sericulture, which not only can spread horizontally, but also propagate vertically to the offspring, sometimes causing serious influence on the production of sericulture. So far, although a lot of research progress has been made in the infection laws and control techniques of the silkworm microspore worm, the infection mechanism has not yet been completely made. It is clear that the infection of microspore to host cells is often achieved by injecting the sporozoite into the host cell by spore germination. At present, the commonly believed microspore germinating host has experienced several events: (1) the conidium and host cell adhesion; (2) the spores are activated; (3) the osmotic pressure in the spores is increased; (4) Polar filament echinovalgus pops out; (5) spores enter the host cells through the pop-up pole. Spore germination is the primary prerequisite for microspore infection, and its germination mechanism plays an important role in the prevention and control of microspore disease. It is believed that the presence of water channel protein in microspore is considered to be transported to the spores. The internal pressure of the internal osmotic pressure of the spores is increased, and the internal pressure of this increase becomes the motive force of spore germination. Therefore, this paper hopes to explore the existence of aquaporin in the microspore of silkworm and the mechanism of its participation in spore germination. This study is based on the cross comparison and analysis of microspore database and MIP database. The NbAQP sequence of microspore microsporworm, silkworm, was successfully cloned. The sequence has a complete open reading frame ORF, long 750bp, a total of 249 amino acids, protein molecular mass of about 26.66kD, isoelectric point 5.12, no signal peptide.NbAQP amino acid sequence and water channel eggs of other 5 microspore insects. The homology of the white sequence is over 50% and contains 6 transmembrane domains. The three stage structure prediction analysis shows that there are 6 long helix and 2 short helix, respectively, with two helices, respectively extending an amino acid chain, and returning the amino acid chain to the starting point after the external surround. A semi quantitative PCR method is used to detect the NbAQP infection of silkworm after silkworm infection. By using the eukaryotic expression system of Saccharomyces cerevisiae, the expression of NbAQP protein in the silkworm microspore was studied in vitro. First, the primers with the enzyme cut site were designed, the NbAQP gene was cloned into the eukaryotic expression vector pYES2-NTC, and the recombinant expression plasmid was constructed and transformed into the yeast susceptible cell. Then the beta strain was used in the INVSc1 strain. The expression of fusion protein with His label with galactose was induced and identified by Western blot immunoblotting. The His-NbAQP fusion protein band of 31kD was detected by Western blot. It indicated that the NbAQP protein of the silkworm microspore can be expressed in the eukaryotic expression system. The epitope of the protein antigen was predicted and the polypeptide was synthesized, and the polypeptide was used as the antigen free of the antigen. The polyclonal antibody of New Zealand rabbits was prepared. The NbAQP protein was located in the microspore of silkworm by immuno colloid gold electron microscopy. The results of immuno electron microscopy showed that the colloid gold particles could be found on the spore wall of the microspore worm, while the distribution of colloid gold was not found in the corresponding position of the control group, indicating the microspore of the silkworm. The insect NbAQP protein is mainly located on the spore wall of the microspore worm of the silkworm, which plays an important role in the exercise of its function. In order to study whether the NbAQP protein of the microspore worm can participate in the germination of the spores, and then play a role in the events of the microspore worm infection of the silkworm, the preliminary function of the NbAQP protein is studied. The results showed that the germination rate of the treated group which was incubated with 1H with polyclonal antibody was 16.58%, and the difference was significant compared with that of the blank control group (23.05%). The inhibition rate of antibody closure on spore germination was about 28%. indicating that the spores could reduce the spores after the closed NbAQP protein was closed. The germination rate, NbAQP protein may be involved in the process of germination of microspore of silkworm. The results of this study showed that there was aquaporin in the microspore of silkworm, which mainly existed in the spore wall and protoplasm membrane. The whole life history of microspore infecting silkworm, proliferation and spores formation were all transcriptional and germinated in the spores. It plays an important role in the process.
【學位授予單位】：江蘇科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S884

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