本文選題：日本血吸蟲 + 線粒體��； 參考：《昆明醫(yī)學(xué)院》2008年碩士論文

【摘要】： [目的]研究云南省三個(gè)自然隔離群日本血吸蟲線粒體CO1、cytb、ND1、ND6、ND4L基因差異,探討日本血吸蟲種內(nèi)遺傳特點(diǎn),為闡明我國(guó)日本血吸蟲的種群遺傳結(jié)構(gòu)及進(jìn)一步研究日本血吸蟲生物學(xué)特性奠定基礎(chǔ)。 [方法]采集云南省大理古城、大理喜洲、永勝三地陽(yáng)性釘螺及購(gòu)買江蘇省實(shí)驗(yàn)室傳代陽(yáng)性釘螺,逸出尾蚴,經(jīng)腹部攻擊感染健康家兔(雄性),每兔感染尾蚴200條～250條,攻擊感染后42天剖殺家兔,利用灌注法取成蟲。試劑盒抽提基因組DNA,以NC_002544為參考序列設(shè)計(jì)引物,采用PCR技術(shù)對(duì)云南三地以及江蘇實(shí)驗(yàn)室傳代的日本血吸蟲的CO1、Cytb、tb、ND1、ND6、ND4L全基因進(jìn)行擴(kuò)增、測(cè)序。用DNAStar軟件及Mega3.1軟件進(jìn)行相似性比較和進(jìn)化樹分析。 [結(jié)果]分別獲得云南三地及江蘇省實(shí)驗(yàn)室傳代日本血吸蟲全長(zhǎng)CO1、Cytb,ND1、ND6、ND4L基因片段,全長(zhǎng)分別為1527bp、1116bp、891bp、459bp、264bp。相似性分析,各序列有微小差異,分別有10、6、6、6、2個(gè)堿基位點(diǎn)不同,同源性分別為:99.5%～100.0%、99.6%～100%、99.5%～100.0%、99.1%～100%、99.2%～100%。除ND4L基因外其CO1、Cytb均有一個(gè)錯(cuò)義突變,ND1、ND6各有兩個(gè)錯(cuò)義突變。進(jìn)化樹顯示云南三地及江蘇省實(shí)驗(yàn)室傳代日本血吸蟲明顯聚集在一起,同曼氏血吸蟲、埃及血吸蟲、湄公血吸蟲、馬來血吸蟲有明顯的分支,但與所檢獲的以往發(fā)表的日本血吸蟲線粒體相同基因序列比較不構(gòu)成獨(dú)立的分支。 [結(jié)論]各序列有微小差異,同源性較高;CO1、cytb均有一個(gè)錯(cuò)義突變,ND1、ND6各有兩個(gè)錯(cuò)義突變,ND4L無(wú)錯(cuò)義突變;本研究所選云南三地及一江蘇省實(shí)驗(yàn)室傳代的樣本均屬于日本血吸蟲,且親緣關(guān)系密切,無(wú)明顯分支,不能構(gòu)成種內(nèi)分類。
[Abstract]:[objective] to study the difference of mitochondrial CO1Cyt bND1 (ND1) ND6Nd4L gene in three natural isolated populations of Schistosoma japonicum in Yunnan Province, and to explore the genetic characteristics of Schistosoma japonicum. In order to clarify the genetic structure of Schistosoma japonicum population and further study the biological characteristics of Schistosoma japonicum in China. [methods] the ancient city of Dali, Yunnan Province, Dali Xizhou, was collected. Oncomelania hupensis was positive in Yongsheng three places and purchased from Jiangsu laboratory, and cercariae escaped. The rabbits were infected with 250 cercariae by abdominal attack (male, 200 cercariae per rabbit). The rabbits were killed 42 days after attack, and the adult worms were harvested by perfusion method. Genomic DNA was extracted by the kit and primers were designed using NC002544 as reference sequence. The whole gene of Schistosoma japonicum was amplified by PCR and sequenced. DNA Star software and Mega3.1 software were used to compare the similarity and analyze the phylogenetic tree. [results] the full-length gene fragment of Schistosoma japonicum was subcultured in three places of Yunnan Province and Jiangsu Province, respectively. The total length of the gene was 1527bp1 1116bpn891bp459bp264bp. In similarity analysis, there were slight differences in each sequence. There were two different base loci. The homology was 10. 99. 5 and 10. 0. 0. 10. 0. 0. 0. 99. 6 and 100. 99. 5 and 99. 0. 0. 99. 2. With the exception of ND4L gene, there was one missense mutation in all of the CO1C Cytb and two missense mutations in Nd1 and ND6. The phylogenetic tree shows that Schistosoma japonicum from three places in Yunnan and Jiangsu Province obviously gathered together, and had obvious branches with Schistosoma mansoni, Schistosoma japonicum, Schistosoma Mekong, Schistosoma Malay. But compared with the previously reported mitochondrial gene sequence of Schistosoma japonicum, it does not constitute an independent branch. [conclusion] there are slight differences among the sequences. Two missense mutations were found in each of the two missense mutants (Nd4L), and the samples collected from three places of Yunnan and one Jiangsu Province were all Schistosoma japonicum, and were closely related to each other and had no obvious branches. It does not constitute an intraspecific classification.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R383

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