發(fā)布時(shí)間：2018-05-21 14:42

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

【摘要】：微孢子蟲(chóng)(Microsporidia)是一類寄主域廣泛的真核生物,從脊椎動(dòng)物到無(wú)脊椎動(dòng)物都會(huì)被微孢子蟲(chóng)感染。微孢子蟲(chóng)作為魚(yú)類、甲殼類和許多經(jīng)濟(jì)類昆蟲(chóng)的病原體,常給農(nóng)業(yè)生產(chǎn)帶來(lái)巨大損失。由家蠶微孢子蟲(chóng)(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(shí)會(huì)給蠶業(yè)生產(chǎn)造成嚴(yán)重影響。迄今為止,雖然在家蠶微孢子蟲(chóng)的傳染規(guī)律和防治技術(shù)方面取得了不少研究進(jìn)展,但在其侵染機(jī)制方面還沒(méi)有完全弄清楚。微孢子蟲(chóng)對(duì)宿主細(xì)胞的侵染,往往是通過(guò)孢子萌發(fā)將孢子內(nèi)的孢原質(zhì)注射到宿主細(xì)胞中來(lái)實(shí)現(xiàn)的。目前普遍認(rèn)為的微孢子蟲(chóng)萌發(fā)感染宿主經(jīng)歷了以下幾個(gè)事件:(1)孢子與宿主細(xì)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(guò)彈出的極絲進(jìn)入宿主細(xì)胞。孢子的發(fā)芽是微孢子蟲(chóng)感染宿主體的首要前提,研究其發(fā)芽機(jī)制對(duì)防治微孢子蟲(chóng)病有著重要的作用。有觀點(diǎn)認(rèn)為微孢子蟲(chóng)中可能有水通道蛋白的存在,將孢外的水分運(yùn)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(dòng)力。因此,本文希望通過(guò)研究,探索家蠶微孢子蟲(chóng)中是否存在水通道蛋白以及該蛋白參與孢子發(fā)芽的機(jī)制。本研究根據(jù)微孢子蟲(chóng)數(shù)據(jù)庫(kù)和MIP數(shù)據(jù)庫(kù)交叉比較分析篩選出可能的家蠶微孢子蟲(chóng)水通道蛋白基因,成功克隆出家蠶微孢子蟲(chóng)NbAQP序列,該序列具有完整的開(kāi)放閱讀框ORF,長(zhǎng)750bp,共編碼249個(gè)氨基酸,蛋白質(zhì)分子質(zhì)量約為26.66kD,等電點(diǎn)為5.12,無(wú)信號(hào)肽。NbAQP氨基酸序列與其他5種微孢子蟲(chóng)的水通道蛋白序列同源性為50%以上,含有6個(gè)跨膜結(jié)構(gòu)域,三級(jí)結(jié)構(gòu)預(yù)測(cè)分析顯示有6個(gè)長(zhǎng)螺旋和2個(gè)短螺旋,分別以兩個(gè)螺旋為起點(diǎn),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(diǎn)處。用半定量PCR的方法檢測(cè)家蠶微孢子蟲(chóng)感染家蠶后NbAQP的轉(zhuǎn)錄情況。利用釀酒酵母真核表達(dá)系統(tǒng)對(duì)家蠶微孢子蟲(chóng)NbAQP蛋白的體外表達(dá)進(jìn)行了研究。首先設(shè)計(jì)帶有酶切位點(diǎn)的引物,將NbAQP基因克隆到真核表達(dá)載體pYES2-NTC中,構(gòu)建重組表達(dá)質(zhì)粒并轉(zhuǎn)化酵母感受態(tài)細(xì)胞。隨后在INVSc1菌株中使用β-半乳糖誘導(dǎo)帶His標(biāo)簽的融合蛋白的表達(dá)以及通過(guò)Western blot免疫印跡鑒定,免疫印跡檢測(cè)到大小為31kD的His-NbAQP融合蛋白條帶,說(shuō)明家蠶微孢子蟲(chóng)NbAQP蛋白可在真核表達(dá)系統(tǒng)中表達(dá)。預(yù)測(cè)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(duì)NbAQP蛋白在家蠶微孢子蟲(chóng)中進(jìn)行定位分析。免疫電鏡結(jié)果顯示,在家蠶微孢子蟲(chóng)的孢子壁上能夠看到膠體金顆粒分布,而對(duì)照組中相應(yīng)位置則沒(méi)有膠體金的分布,說(shuō)明家蠶微孢子蟲(chóng)NbAQP蛋白主要定位于家蠶微孢子蟲(chóng)的孢子壁上,對(duì)其功能的行使有著重要意義。為了研究家蠶微孢子蟲(chóng)NbAQP蛋白是否能夠參與孢子發(fā)芽的過(guò)程,進(jìn)而在微孢子蟲(chóng)感染家蠶的事件中發(fā)揮作用,對(duì)NbAQP蛋白做初步功能研究。通過(guò)抗體封閉實(shí)驗(yàn)檢測(cè)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(duì)照組的發(fā)芽率(23.05%)相比,差異性顯著(P0.05)�？贵w封閉對(duì)孢子發(fā)芽的抑制率約為28%。說(shuō)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(chóng)發(fā)芽的過(guò)程。本研究結(jié)果表明,家蠶微孢子蟲(chóng)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(chóng)感染家蠶、增殖以及孢子形成的整個(gè)生活史均有轉(zhuǎn)錄表達(dá),且在孢子的發(fā)芽過(guò)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(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S884

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 吳正理;譚小輝;潘國(guó)慶;李艷紅;周澤揚(yáng);;家蠶微孢子蟲(chóng)(Nosema bombycis)孢壁蛋白提取方法的優(yōu)化研究[J];蠶業(yè)科學(xué);2007年01期

2 劉鐵;胡軍華;潘國(guó)慶;周澤揚(yáng);;家蠶微孢子蟲(chóng)幾丁質(zhì)酶基因的比較基因組學(xué)分析[J];蠶學(xué)通訊;2008年01期

3 張小燕;蔡紅英;周興建;肖宇;黃蕾;;家蠶微孢子蟲(chóng)轉(zhuǎn)宿主果蠅的研究初探[J];安徽農(nóng)業(yè)科學(xué);2009年23期

4 張小燕;蔡紅英;周興建;肖宇;黃蕾;;家蠶微孢子蟲(chóng)轉(zhuǎn)宿主果蠅的研究[J];Agricultural Science & Technology;2009年04期

5 吳正理;李艷紅;宋元達(dá);;微孢子蟲(chóng)蛋白質(zhì)及其與宿主互作的研究進(jìn)展[J];蠶業(yè)科學(xué);2009年04期

6 劉吉平;李進(jìn);呂思行;魏建影;楊吉龍;;微孢子蟲(chóng)基因組學(xué)研究新進(jìn)展[J];廣東蠶業(yè);2011年02期

7 劉吉平;徐興耀;;家蠶微孢子蟲(chóng)分子生物學(xué)研究進(jìn)展[J];廣東蠶業(yè);1997年04期

8 邱寶利;徐興耀;盧鏗明;周鵬;;家蠶微孢子蟲(chóng)診斷新技術(shù)的研究[J];廣東蠶業(yè);1998年03期

9 向恒;潘國(guó)慶;周澤揚(yáng);;微孢子蟲(chóng)系統(tǒng)進(jìn)化研究的變遷與展望[J];微生物學(xué)通報(bào);2014年04期

10 黃金山,王建芳,李宏,楊桂芳,錢(qián)永華;蠶熏安對(duì)家蠶微孢子蟲(chóng)的殺滅效果[J];北方蠶業(yè);2000年02期

相關(guān)會(huì)議論文 前10條

1 楊瓊;徐興耀;盧鏗明;鄭祥明;方定堅(jiān);;感染東方粉蝶的兩種微孢子蟲(chóng)的研究(摘要)[A];中國(guó)蠶學(xué)會(huì)養(yǎng)蠶與蠶生理病理學(xué)術(shù)討論會(huì)論文匯編[C];2000年

2 楊柳;潘國(guó)慶;劉含登;許金山;李田;劉鐵;張紹鵬;周澤揚(yáng);;家蠶微孢子蟲(chóng)細(xì)菌人工染色體文庫(kù)的構(gòu)建[A];中國(guó)蠶學(xué)會(huì)第六屆青年學(xué)術(shù)研討會(huì)論文集（2）[C];2009年

3 高繪菊;牟志美;王彥文;;幾種家蠶微孢子蟲(chóng)的形態(tài)構(gòu)造觀察(摘要)[A];中國(guó)蠶學(xué)會(huì)養(yǎng)蠶與蠶生理病理學(xué)術(shù)討論會(huì)論文匯編[C];2000年

4 劉吉平;曾玲;;微孢子蟲(chóng)的生物多樣性研究[A];中國(guó)蠶學(xué)會(huì)第七屆二次理事會(huì)暨學(xué)術(shù)年會(huì)論文集[C];2005年

5 王強(qiáng);周婷;代平禮;;微孢子蟲(chóng)研究進(jìn)展[A];中國(guó)養(yǎng)蜂學(xué)會(huì)蜜蜂產(chǎn)品、蜜蜂保護(hù)、蜜源與授粉專業(yè)委員會(huì)第七次學(xué)術(shù)研討會(huì)論文集[C];2006年

6 時(shí)連根;何春波;;壯蠶感染微孢子蟲(chóng)與蛾期毒率關(guān)系的研究(摘要)[A];中國(guó)蠶學(xué)會(huì)養(yǎng)蠶與蠶生理病理學(xué)術(shù)討論會(huì)論文匯編[C];2000年

7 王見(jiàn)楊;黃可威;趙昀;陸長(zhǎng)德;;微孢子蟲(chóng)RNA的制備方法(摘要)[A];中國(guó)蠶學(xué)會(huì)養(yǎng)蠶與蠶生理病理學(xué)術(shù)討論會(huì)論文匯編[C];2000年

8 段艷;潘耀謙;張柳平;;兔腦炎微孢子蟲(chóng)感染對(duì)家兔細(xì)胞免疫功能的影響[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)獸醫(yī)病理學(xué)分第12次暨中國(guó)動(dòng)物病理生理學(xué)專業(yè)委員會(huì)第11次學(xué)術(shù)討論會(huì)論文集[C];2003年

9 韋亞?wèn)|;張國(guó)政;陸長(zhǎng)德;;家蠶微孢子蟲(chóng)原位雜交診斷技術(shù)研究[A];中國(guó)蠶學(xué)會(huì)第四屆青年學(xué)術(shù)研討會(huì)會(huì)議論文集[C];2004年

10 王麗英;曹春;任鋼;;蝗蟲(chóng)微孢子蟲(chóng)的研究與應(yīng)用[A];全國(guó)生物防治學(xué)術(shù)討論會(huì)論文集[C];1991年

相關(guān)重要報(bào)紙文章 前4條

1 甘田;微孢子蟲(chóng)生物防治蝗災(zāi)[N];農(nóng)民日?qǐng)?bào);2001年

2 記者 劉霞;科學(xué)家發(fā)現(xiàn)微孢子蟲(chóng)為有性繁殖[N];科技日?qǐng)?bào);2008年

3 姬全;微孢子蟲(chóng)誘發(fā)蝦蟹大批死亡[N];中國(guó)漁業(yè)報(bào);2013年

4 王來(lái);凹凸病導(dǎo)致鰻魚(yú)衰弱[N];中國(guó)漁業(yè)報(bào);2013年

相關(guān)博士學(xué)位論文 前10條

1 黨曉群;家蠶微孢子蟲(chóng)（Nosema bombycis）功能基因組研究[D];西南大學(xué);2012年

2 邱海洪;微孢子蟲(chóng)感染家蠶相關(guān)基因的研究[D];浙江大學(xué);2012年

3 劉鐵;一株寄生于金鳳蝶的微孢子蟲(chóng)的鑒定及其基因組研究[D];西南大學(xué);2015年

4 向恒;家蠶微孢子蟲(chóng)（Nosema bombycis）基因組學(xué)研究[D];西南大學(xué);2010年

5 胡軍華;家蠶微孢子蟲(chóng)（Nosema bombycis）線粒體相關(guān)基因研究[D];西南大學(xué);2008年

6 劉含登;家蠶微孢子蟲(chóng)（Nosema bombycis）基因組學(xué)研究[D];西南大學(xué);2011年

7 李巍;肯尼亞狒狒腸道原蟲(chóng)系統(tǒng)進(jìn)化分析及畢氏腸微孢子蟲(chóng)群體遺傳學(xué)研究[D];吉林大學(xué);2011年

8 蔡順風(fēng);家蠶微孢子蟲(chóng)孢子母細(xì)胞的分離及孢子吞噬作用的研究[D];浙江大學(xué);2012年

9 王營(yíng);家蠶微孢子蟲(chóng)（Nosema bombycis）功能基因組研究[D];西南大學(xué);2015年

10 萬(wàn)永繼;家蠶病原性微孢子蟲(chóng)若干種群的研究與控制[D];中國(guó)農(nóng)業(yè)大學(xué);2005年

相關(guān)碩士學(xué)位論文 前10條

1 張海燕;家蠶微孢子蟲(chóng)（浙江）α、β微管蛋白部分基因的克隆及系統(tǒng)發(fā)育分析[D];西北農(nóng)林科技大學(xué);2007年

2 徐小芳;家蠶微孢子蟲(chóng)與菜粉蝶微孢子蟲(chóng)的形態(tài)學(xué)與系統(tǒng)發(fā)育研究[D];江蘇科技大學(xué);2011年

3 馬成;家蠶微孢子蟲(chóng)（Nosema bombycis）功能基因組研究[D];西南大學(xué);2012年

4 鄧遠(yuǎn)洪;家蠶微孢子蟲(chóng)（Nosema bombycis）功能基因組研究[D];西南大學(xué);2012年

5 周小偉;家蠶微孢子蟲(chóng)（Nosema bombycis）功能基因組研究[D];西南大學(xué);2013年

6 彭湃;家蠶微孢子蟲(chóng)（Nosema bombycis）功能基因組研究[D];西南大學(xué);2015年

7 彭先啟;陜西部分地區(qū)山羊畢氏腸微孢子蟲(chóng)感染情況及其基因型研究[D];西北農(nóng)林科技大學(xué);2016年

8 倪琪;家蠶微孢子蟲(chóng)核酸側(cè)向?qū)游鲈嚰垪l檢測(cè)方法的建立[D];西南大學(xué);2016年

9 萬(wàn)強(qiáng);中國(guó)東北地區(qū)豬兩種腸道原蟲(chóng)遺傳特征分析及豬源畢氏腸微孢子蟲(chóng)群體遺傳學(xué)研究[D];東北農(nóng)業(yè)大學(xué);2016年

10 婁志龍;我國(guó)東部地區(qū)養(yǎng)殖北極狐弓形蟲(chóng)、微孢子蟲(chóng)、隱孢子蟲(chóng)流行病學(xué)調(diào)查和基因型研究[D];吉林農(nóng)業(yè)大學(xué);2016年

，

本文編號(hào)：1919615