廣西兩地異盤并殖吸蟲分子系統(tǒng)發(fā)生研究
發(fā)布時間：2018-11-06 10:32
【摘要】： 目的:通過研究廣西兩地(靈川、那坡)異盤并殖吸蟲分離株的核糖體DNA第二內(nèi)部轉(zhuǎn)錄間隔區(qū)基因(ITS2)和細(xì)胞色素c氧化酶亞單位Ⅰ基因(CO1)序列,比較分析兩地蟲體的基因差異,揭示它們之間的親緣關(guān)系和分類學(xué)地位。 方法:采集靈川和那坡兩地的異盤并殖吸蟲囊蚴接種適宜終宿主大鼠,獲得成蟲后提取基因組DNA,PCR擴(kuò)增其ITS2和CO1基因序列并測序。從Genebank檢索到與它們同源性相近的并殖吸蟲基因序列,應(yīng)用Megalign軟件對基因序列進(jìn)行比較和同源性分析,然后用Mega及Phylip軟件以最小進(jìn)化法(ME)、鄰接法(N-J)、最大似然法(ML)、最大簡約法(MP)構(gòu)建系統(tǒng)發(fā)生樹,其中ME和N-J法用Kimura雙參數(shù)法計(jì)算遺傳距離,自舉檢驗(yàn)副本數(shù)為1000,ML和MP法自舉檢驗(yàn)副本數(shù)為500。 結(jié)果:那坡異盤并殖吸蟲分離株與靈川異盤并殖吸蟲分離株的ITS2基因序列相似度為100%,CO1基因序列相似度為97.5%,同源性較高;而那坡與泰國一個分離株的ITS2和CO1基因序列相似度高達(dá)100%,具有高度同源性。 從ITS2序列系統(tǒng)發(fā)生樹來看,來自那坡、靈川、越南、泰國和印度的分離株均處于異盤并殖吸蟲的種群下,但印度分離株是個相對獨(dú)立的分枝,親緣關(guān)系較遠(yuǎn)。從CO1序列系統(tǒng)發(fā)生樹來看,不同的發(fā)生樹中那坡分離株均與泰國、越南分離株的親緣關(guān)系較近,以較高的自舉檢驗(yàn)置信值形成單系群,而靈川分離株的分枝均處在這個群之外,印度分離株的親緣關(guān)系則更遠(yuǎn)一些。 結(jié)論:那坡分離株與靈川分離株的ITS2基因和CO1基因序列的同源性比較高,而那坡分離株與泰國分離株的2個基因序列的同源性更高。雖然那坡和靈川分離株在形態(tài)學(xué)上有一定差異,但在不同方法構(gòu)建的系統(tǒng)發(fā)生樹中都位于異盤并殖吸蟲的種群分枝下。CO1系統(tǒng)發(fā)生樹中那坡分離株均與泰國、越南分離株的親緣關(guān)系較近,形成有共同起源的單系群,而靈川分離株的分枝均處在這個群之外。據(jù)此認(rèn)為,靈川和那坡的分離株可能屬于不同的地理株。
[Abstract]:Objective: to study the sequence of ribosomal DNA second internal transcriptional spacer (ITS2) and cytochrome c oxidase subunit I (CO1) gene of paragonimus xenostroma isolated from Lingchuan, Napo, Guangxi. The genetic differences between the two species were analyzed, and the phylogenetic relationship and taxonomic status between them were revealed. Methods: the metacercariae of Lingchuan and Napo were collected and inoculated into the rats suitable for the final host. The genomic DNA,PCR was extracted from adult worms to amplify the sequence of ITS2 and CO1 genes and sequenced. The homologous gene sequences of paragonimus were retrieved from Genebank. The sequence of paragonimus was compared and homologous analyzed by Megalign software. Then, the sequence of paragonimus gene was analyzed by Mega and Phylip using (ME), adjacency method of minimal evolution (N-J). The phylogenetic tree was constructed by maximum likelihood method (ML),) and maximum reduction method (MP), in which ME and N-J methods were used to calculate genetic distance by Kimura two-parameter method, and the number of bootstrap test replicas was 1000m L and 500 by MP method. Results: the similarity of ITS2 gene sequence between paragonimiasis natapo isolate and Lingchuan paragonimiasis isolate was 97.5, and the similarity of ITS2 gene sequence was 97.5. The sequence similarity of ITS2 and CO1 between Napo and a Thai isolate is as high as 100, with high homology. From the phylogenetic tree of ITS2 sequence, the isolates from Napo, Lingchuan, Vietnam, Thailand and India are all under the population of paragonimus heterodendens, but the Indian isolate is a relatively independent branch, and the genetic relationship is relatively distant. According to the phylogenetic tree of CO1 sequence, Napo isolates in different phylogenetic trees were closely related to Thai and Vietnamese isolates, and monophyletic groups were formed with higher confidence values of bootstrap test, but the branches of Lingchuan isolates were all outside this group. The Indian isolates were more closely related. Conclusion: the homology of ITS2 gene and CO1 gene sequence between Napo isolate and Lingchuan isolate is higher than that between Napo isolate and Thai isolate. Although there are some differences in morphology between Napo and Lingchuan isolates, the phylogenetic trees constructed by different methods are all located under the branches of the population of paragonimus heterophylla. The Napo isolates in the phylogenetic tree of CO1 are all different from those in Thailand. Vietnamese isolates were closely related to each other, forming a monophyletic group with a common origin, while the branches of Lingchuan isolates were all located outside this group. It is concluded that the isolates of Lingchuan and Napo may belong to different geographical strains.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R383.23
本文編號：2314023
[Abstract]:Objective: to study the sequence of ribosomal DNA second internal transcriptional spacer (ITS2) and cytochrome c oxidase subunit I (CO1) gene of paragonimus xenostroma isolated from Lingchuan, Napo, Guangxi. The genetic differences between the two species were analyzed, and the phylogenetic relationship and taxonomic status between them were revealed. Methods: the metacercariae of Lingchuan and Napo were collected and inoculated into the rats suitable for the final host. The genomic DNA,PCR was extracted from adult worms to amplify the sequence of ITS2 and CO1 genes and sequenced. The homologous gene sequences of paragonimus were retrieved from Genebank. The sequence of paragonimus was compared and homologous analyzed by Megalign software. Then, the sequence of paragonimus gene was analyzed by Mega and Phylip using (ME), adjacency method of minimal evolution (N-J). The phylogenetic tree was constructed by maximum likelihood method (ML),) and maximum reduction method (MP), in which ME and N-J methods were used to calculate genetic distance by Kimura two-parameter method, and the number of bootstrap test replicas was 1000m L and 500 by MP method. Results: the similarity of ITS2 gene sequence between paragonimiasis natapo isolate and Lingchuan paragonimiasis isolate was 97.5, and the similarity of ITS2 gene sequence was 97.5. The sequence similarity of ITS2 and CO1 between Napo and a Thai isolate is as high as 100, with high homology. From the phylogenetic tree of ITS2 sequence, the isolates from Napo, Lingchuan, Vietnam, Thailand and India are all under the population of paragonimus heterodendens, but the Indian isolate is a relatively independent branch, and the genetic relationship is relatively distant. According to the phylogenetic tree of CO1 sequence, Napo isolates in different phylogenetic trees were closely related to Thai and Vietnamese isolates, and monophyletic groups were formed with higher confidence values of bootstrap test, but the branches of Lingchuan isolates were all outside this group. The Indian isolates were more closely related. Conclusion: the homology of ITS2 gene and CO1 gene sequence between Napo isolate and Lingchuan isolate is higher than that between Napo isolate and Thai isolate. Although there are some differences in morphology between Napo and Lingchuan isolates, the phylogenetic trees constructed by different methods are all located under the branches of the population of paragonimus heterophylla. The Napo isolates in the phylogenetic tree of CO1 are all different from those in Thailand. Vietnamese isolates were closely related to each other, forming a monophyletic group with a common origin, while the branches of Lingchuan isolates were all located outside this group. It is concluded that the isolates of Lingchuan and Napo may belong to different geographical strains.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R383.23
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