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  本文選題：日本血吸蟲　切入點：湖北釘螺　出處：《中南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 1.采用人工方法建立釘螺室內(nèi)外生存的條件,獲得日本血吸蟲感染的湖北釘螺,以實現(xiàn)日本血吸蟲的室內(nèi)傳代與保種。 2.研究湖南省日本血吸蟲不同地域自然隔離群線粒體ATP合成酶FO亞單位6(atp6)基因部分序列的遺傳多態(tài)性,為湖南不同地域自然隔離群日本血吸蟲的遺傳特性研究提供實驗依據(jù)。 方法 1.用尼龍絹篩收集日本血吸蟲成熟蟲卵,在適當(dāng)?shù)臈l件下進行毛蚴孵化。將釘螺與毛蚴按1：15～1：20的比例進行感染,以獲得第一代人工感染性釘螺,再以第一代(F1代)人工感染性釘螺逸出的尾蚴感染實驗動物,獲取成熟蟲卵并孵化毛蚴,然后繼續(xù)感染釘螺,獲得第二代(F2代)人工感染性釘螺、同樣方法完成第三代(F3代)人工感染性釘螺、第四代(F4代)人工感染性釘螺的傳代、…,在實驗室內(nèi)建立血吸蟲的傳代和保種技術(shù)與方法。同時模擬釘螺野外生存環(huán)境,建立斜坡型、秧田型和溝渠型三種自然生態(tài)養(yǎng)螺池,進行釘螺生存、養(yǎng)殖條件的觀察、比較。 2.提取日本血吸蟲基因組總DNA,以特異性引物對線粒體atp6基因進行PCR擴增,通過單鏈構(gòu)象多態(tài)性技術(shù)(SSCP)篩選出差異帶型并進行測序,用DNA Star 5.0及Mega 4.0軟件進行比對分析。 結(jié)果 1.成功模擬釘螺野外生存的環(huán)境,建立了斜坡型、秧田型、溝渠型三種類型的養(yǎng)螺池,分別投放釘螺3萬、2萬、4萬只,三種類型的螺池中的釘螺均成功越冬并出現(xiàn)了新生幼螺,其中以斜坡型螺池生存條件最佳,其平均幼螺率達到17.17%,實現(xiàn)了人工模擬條件下釘螺的自身繁殖。室內(nèi)人工傳代研究共獲得Fl代陽性釘螺796只,F2代陽性釘螺272只, F3代陽性釘螺362只,成功實現(xiàn)了日本血吸蟲的室內(nèi)傳代與種質(zhì)釘螺的保種。 2.湖南省5個流行區(qū)的日本血吸蟲PCR擴增后獲得了483 bpatp6部分序列,檢測出17個變異位點,變異率為3.52%,雌雄蟲之間的差異為0.0～1．3%,不同地理來源蟲株間的差異率為0.0～1.5%。聚類分析結(jié)果表明：湖南省不同地域自然隔離群日本血吸蟲分離株線粒體atp6基因的個體遺傳差異明顯,尤以淚羅和岳陽君山兩地分離蟲株表現(xiàn)突出。 結(jié)論 1.人工模擬了野外養(yǎng)殖釘螺條件,建立了三種類型的養(yǎng)螺池,已可以滿足日本血吸蟲室內(nèi)傳代深入研究的需要。成功實現(xiàn)了日本血吸蟲的F1代、F2代、F3代的室內(nèi)傳代,為血吸蟲種群的遺傳變異、溯源研究創(chuàng)造了條件。 2.湖南不同地域自然隔離群日本血吸蟲線粒體atp6基因存在明顯的個體差異,但是否由于生存環(huán)境的選擇壓力造成了同一地理來源日本血吸蟲個體之間atp6基因的遺傳差異,其原因有待于進一步研究。
[Abstract]:Purpose. 1. Artificial methods were used to establish the living conditions of Oncomelania hupensis in and out of the room, and to obtain Schistosoma japonicum infected Oncomelania hupensis in order to realize the indoor passage and species conservation of Schistosoma japonicum. 2. To study the genetic polymorphism of mitochondrial ATP synthase FO subunit (FO) in different regions of Schistosoma japonicum in Hunan Province, and to provide experimental basis for the study of genetic characteristics of Schistosoma japonicum in different regions of Hunan Province. Method. 1. The mature eggs of Schistosoma japonicum were collected by nylon silk screen and hatched under suitable conditions. The first generation of artificial infected snails were obtained by infecting the snails and the cercariae in the proportion of 1: 15 to 1: 20. The experimental animals were infected with cercariae escaped from artificial infected snails in the first generation (F1 generation). Mature eggs were obtained and hatched cercariae were hatched, and then continued infection of Oncomelania hupensis was carried out to obtain the second generation of artificial infected Oncomelania hupensis. Methods the third generation F 3 generation) artificial infected snail, 4th generation F 4 generation) artificial infected snail were subcultured... The breeding and conservation techniques and methods of Schistosoma japonicum were established in laboratory. Meanwhile, the field living environment of snail was simulated. Three kinds of natural ecological snail culture ponds were established, such as slope type, Yangtian type and ditch type. The survival and culture conditions of Oncomelania hupensis were observed and compared. 2. The total genomic DNA of Schistosoma japonicum was extracted, and the mitochondrial atp6 gene was amplified by PCR with specific primers. The differential bands were screened by single strand conformation polymorphism (SSCP) and sequenced. The results were compared with those of DNA Star 5.0 and Mega 4.0. Results. 1. Successfully simulating the living environment of snails in the field, establishing three types of snail culture ponds: slope type, rice field type and ditch type, and placing 30,000, 20,000 and 40,000 snails, respectively. The snails in the three types of snail ponds were successfully overwintering and the newborn snails appeared. Among them, the sloping snail pond has the best living conditions. The average juvenile snail rate was 17.17, and the self-propagation of Oncomelania hupensis was realized under artificial simulated conditions. 796 F1 generation positive snails and 362 F3 generation positive snails were obtained in laboratory. The indoor passage of Schistosoma japonicum and the conservation of germplasm snail were successfully realized. 2. PCR amplification of Schistosoma japonicum from 5 endemic areas of Hunan Province was carried out, and the partial sequence of 483 bpatp6 was obtained, and 17 mutation sites were detected. The variation rate was 3.52, the difference between female and male was 0.01.3 and the difference among different geographical sources was 0.01.5.The results of cluster analysis showed that the individual genetic difference of mitochondrial atp6 gene of Schistosoma japonicum isolates from different regions of Hunan Province was obvious. Especially in Yiluo and Yueyang Junshan isolated insect strains outstanding. Conclusion. 1. Artificial simulation of snail culture conditions in the field and establishment of three types of snail culture ponds can meet the need of further study on the indoor passage of Schistosoma japonicum, and successfully realize the indoor passage of F _ 1 and F _ 2 generation of Schistosoma japonicum. It provides conditions for genetic variation and traceability of Schistosoma japonicum population. 2. There are obvious individual differences in mitochondrial atp6 gene of Schistosoma japonicum in different regions of Hunan Province, but whether the genetic difference of atp6 gene among individuals from the same geographical origin is caused by the selection pressure of living environment. The reasons need to be further studied.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R184

【參考文獻】

相關(guān)期刊論文 前10條

1 樊晉江;曼氏血吸蟲發(fā)育過程中的基因組DNA變異[J];動物學(xué)研究;1994年04期

2 牛安歐,馮友仁,方正明;中國日本血吸蟲DNA遺傳變異及聚類分析[J];寄生蟲與醫(yī)學(xué)昆蟲學(xué)報;2003年02期

3 郭凱文,牛安歐;日本血吸蟲線粒體DNA的遺傳變異[J];熱帶醫(yī)學(xué)雜志;2004年04期

4 胡忠勤,周賢坤,王文霓;四川日本血吸蟲在安徽釘螺體內(nèi)傳代的研究[J];四川動物;1988年04期

5 樊晉江，，宋昌存;曼氏血吸蟲核糖體RNA基因(rDNA)的重組和亞克隆[J];實用寄生蟲病雜志;1994年04期

6 石孟芝,余冬保,魏望遠,張楚霜,賀宏斌,陽桂芬,李廣平,任茂元;野外自然與室內(nèi)人工血吸蟲感染性釘螺尾蚴感染小鼠的觀察[J];實用預(yù)防醫(yī)學(xué);2003年06期

7 黃希寶,夏萍鳳,楊麗華,蔣ng;日本血吸蟲對不同地區(qū)釘螺的感染性及其致病力的研究[J];中國血吸蟲病防治雜志;2002年01期

8 錢寶珍,Jaya Shrivastava,Joanne P.Webster,賀晨;中國大陸日本血吸蟲微衛(wèi)星DNA的初步研究[J];中國血吸蟲病防治雜志;2004年04期

9 李娟;趙光輝;莫細好;林瑞慶;袁子國;翁亞彪;朱興全;;基于細胞色素b氧化酶基因序列的中國大陸日本血吸蟲蟲株種系發(fā)育分析[J];中國獸醫(yī)科學(xué);2009年02期

10 周曉農(nóng),汪天平,王立英,郭家鋼,余晴,許靜,王汝波,陳朝,賈鐵武;中國血吸蟲病流行現(xiàn)狀分析[J];中華流行病學(xué)雜志;2004年07期

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