貝氏貝諾孢子蟲遺傳進(jìn)化分析及巢氏PCR檢測(cè)方法的建立
發(fā)布時(shí)間：2018-08-08 13:04
【摘要】：貝氏貝諾孢子蟲（Besnoitia besnoiti）主要寄生于牛的眼、皮膚和生殖系統(tǒng)等部位，引起牛貝諾孢子蟲�。˙esnoitiosis）。牛感染后會(huì)出現(xiàn)消瘦、貧血、水腫及消化功能障礙，導(dǎo)致母牛流產(chǎn)、產(chǎn)奶量下降、公牛精液質(zhì)量下降，嚴(yán)重感染時(shí)可引起死亡，給養(yǎng)牛業(yè)帶來(lái)重大的經(jīng)濟(jì)損失。 目前對(duì)貝諾孢子蟲的研究主要集中在貝諾孢子蟲病的流行病學(xué)、診斷和綜合防治等傳統(tǒng)寄生蟲學(xué)方面，在分子水平上僅對(duì)核糖體內(nèi)轉(zhuǎn)錄間隔區(qū)1（internal transcribed spacer ITS1），進(jìn)行了研究，對(duì)線粒體全基因組的研究還未見報(bào)道。因此，本研究的第一部分內(nèi)容是利用分子生物學(xué)的方法，擴(kuò)增貝氏貝諾孢子蟲的線粒體全基因組和貝氏貝諾孢子蟲中國(guó)株的ITS-1序列，對(duì)獲得的整個(gè)線粒體基因組序列和ITS-1進(jìn)行測(cè)序和序列分析，并基于線粒體基因組蛋白質(zhì)編碼基因序列和核糖體rDNA的ITS-1構(gòu)建系統(tǒng)發(fā)生樹，探討貝氏貝諾孢子蟲與孢子蟲科其他原蟲的進(jìn)化關(guān)系，確定其在原蟲中的分類地位。結(jié)果，獲得的貝氏貝諾孢子蟲線粒體全基因組由2個(gè)蛋白質(zhì)編碼基因和7個(gè)tRNA基因組成，大小為3679bp。整個(gè)線粒體基因組中核酸組成為A=29.14%，C=19.30%，G=18.48%，T=33.08%，A+T占62.22%，G+C占37.78%，基因組堿基組成明顯偏好堿基A和T。以串聯(lián)的線粒體2個(gè)蛋白質(zhì)編碼基因?yàn)榛驑?biāo)記，采用BI、MP和ML法構(gòu)建系統(tǒng)發(fā)生樹的結(jié)果相似，進(jìn)化樹分為二個(gè)大的分支，葉足亞綱和球蟲亞綱各為一支。在球蟲亞綱中，真球蟲目和梨形蟲目各為一獨(dú)立分支。在真球蟲目中，艾美耳亞目和血孢子亞目各為一個(gè)大的分支。在艾美耳亞目中，艾美耳科為一個(gè)大的分支，其他科共同位于一個(gè)分支上。而貝氏貝諾孢子蟲與剛地弓形蟲、新孢子蟲和哈蒙球蟲在一個(gè)姐妹分支上，是親緣關(guān)系最近的蟲種。此結(jié)果與傳統(tǒng)形態(tài)學(xué)分類一致，，說(shuō)明線粒體全基因序列是研究寄生蟲分子種系發(fā)生的良好基因標(biāo)記。以所獲得的ITS-1rDNA為基因標(biāo)記，采用MP、ML和BI三種方法構(gòu)建系統(tǒng)發(fā)生樹的結(jié)果相似，貝氏貝諾孢子蟲與剛地弓形蟲、新孢子蟲和哈蒙球蟲在一個(gè)姐妹分支上。此結(jié)果與以線粒體全基因序列為標(biāo)記基因構(gòu)建的進(jìn)化樹結(jié)果相似，同時(shí)貝氏貝諾孢子蟲中國(guó)株和美國(guó)、德國(guó)、澳大利亞、以色列等國(guó)家檢出的病原一致。 貝氏貝諾孢子蟲病診斷方法的研究一直還停留在最原始的臨床觀察診斷方面，但是對(duì)感染貝氏貝諾孢子蟲的病牛來(lái)說(shuō)，在感染初期，病牛臨床表現(xiàn)不明顯，出現(xiàn)臨床癥狀的時(shí)候已經(jīng)到了晚期，只能選擇淘汰，給畜牧業(yè)造成重大的經(jīng)濟(jì)損失。因此本研究的第二部分內(nèi)容是在分子水平上，建立一種快速、準(zhǔn)確的診斷方法，對(duì)疾病進(jìn)行早期診斷，以減少經(jīng)濟(jì)損失。根據(jù)已發(fā)表的貝諾孢子蟲rDNA序列，設(shè)計(jì)貝氏貝諾孢子蟲rDNAITS巢氏引物。提取病牛含有貝氏貝諾孢子蟲血液的基因組總DNA，進(jìn)行巢氏PCR擴(kuò)增和測(cè)序，建立一種快速、特異、敏感的巢氏PCR診斷方法，并應(yīng)用該方法對(duì)黑龍江省部分地區(qū)196份奶牛血液樣品進(jìn)行感染情況檢查。結(jié)果表明，建立的貝諾孢子蟲巢式PCR方法具有敏感、特異的優(yōu)點(diǎn)；臨床檢測(cè)出陽(yáng)性血樣3份，陽(yáng)性率為1.53%。 本研究首次獲得了貝氏貝諾孢子蟲的線粒體全基因組序列，并對(duì)其特征進(jìn)行了分析，豐富了原蟲線粒體全基因組資料，基于線粒體基因組蛋白編碼序列和ITS-1序列構(gòu)建的系統(tǒng)發(fā)生樹從分子水平上明確了貝氏貝諾孢子蟲及其他原蟲的進(jìn)化關(guān)系，為其它寄生原蟲群體遺傳學(xué)、系統(tǒng)發(fā)生學(xué)的研究奠定了基礎(chǔ)。首次建立了奶牛貝諾孢子蟲巢氏PCR的診斷方法，并對(duì)黑龍江省部分地區(qū)奶牛感染情況進(jìn)行初步調(diào)查，為該病進(jìn)一步的防控提供可靠依據(jù)。
[Abstract]:Besnoitia besnoiti mainly parasitic on the eyes, skin, and reproductive systems of cattle, causing bovine bainosporidiosis (Besnoitiosis). After cattle infection, emaciation, anemia, edema and digestive dysfunction may result in abortion, lower milk production and the quality of cow semen, death and supply can be caused in severe infection. The cattle industry has brought great economic losses.
At present, the study of Sporozoa Benno mainly focuses on the epidemiology, diagnosis and comprehensive prevention and control of the disease of Benno Sporozoa. At the molecular level, only the transcriptional spacer zone 1 (internal transcribed spacer ITS1) of ribose is studied. The study of the whole genome of the nematome has not been reported. The first part is to amplify the ITS-1 sequence of the whole genome of the mitochondria and the Chinese strain of the Chinese strain of baineno beetle by molecular biology. The sequence and sequence analysis of the whole mitochondrial genome sequence and ITS-1 are carried out, based on the sequence of the gene encoding gene of the mitochondrial genome and the ribosome R. DNA's ITS-1 constructs a phylogenetic tree to explore the evolutionary relationship between the Sporozoa baino spore and other protozoa, and determine its classification status in the protozoa. Results, the complete genome of the mitochondrial genome of the Sporozoa baineno sporozoite consists of 2 protein encoding genes and 7 tRNA genes, and the size is nucleic acid in the whole mitochondrial genome of 3679bp.. The composition of A=29.14%, C=19.30%, G=18.48%, T=33.08%, A+T accounted for 62.22%, G+C accounted for 37.78%. The genome base composition obviously preferred the base A and T. in series of mitochondrial 2 protein coding genes as gene markers. The results were similar with BI, MP and ML method for constructing phylogeny tree, and the intake tree was divided into two large branches, leaf subclass and coccidiosis. In the subclass of coccidia, the Eimeria and the suborder of the genus pyriform are an independent branch. In the order Eimeria, the amieriera and the suborder of the blood spores are a large branch. In the amieriera, eimerid is a large branch and the other families are located on a branch. In a sister branch of a sister branch, the parasite and the Harmon coccidia are the closest species of the relationship. This result is consistent with the traditional morphological classification, indicating that the mitochondrial whole gene sequence is a good genetic marker for the study of the parasite molecular phylogeny. The ITS-1rDNA was used as the gene marker to construct the phylogeny tree with three methods, MP, ML and BI. The results were similar. The results were similar to those of the phylogenetic tree constructed with the sequence of mitochondrial gene sequences. The results were similar to those in the United States, Germany, Australia and Israel.
The study of the diagnosis method of bainobo sporozoite has always stayed in the most primitive clinical observation, but for the infected cattle infected with bainobella spore, the clinical symptoms of the infected cattle were not obvious in the early stage of infection. When the symptoms appeared, they had come to the late stage and could only choose and eliminate them, causing major economic damage to the animal husbandry. Therefore, the second part of this study is to establish a rapid and accurate diagnostic method at the molecular level, to diagnose the disease early in order to reduce the economic loss. According to the published rDNA sequence of the sporozoite of Benno, the rDNAITS nesting primers of the Sporozoa bainite were designed. The extraction of the genes of the infected cattle containing the blood of the Benno Sporozoa was extracted. Group DNA, nest PCR amplification and sequencing, a rapid, specific and sensitive nesting PCR diagnosis method was established, and the method was used to detect the infection of blood samples of 196 dairy cows in parts of Heilongjiang province. The results showed that the established nested PCR recipe was sensitive and specific, and the positive blood was detected in clinical. 3 samples with a positive rate of 1.53%.
In this study, the whole genome sequence of the Sporozoa baineno Sporozoa was first obtained, and its characteristics were analyzed. The whole genome of the protozoa was enriched. The phylogenetic tree based on the sequence of mitochondrial genome protein and the sequence of ITS-1 sequence clearly defined the progress of the baineno bereno Sporozoa and other protozoa. The relationship was the basis for the study of other parasitic protozoa population genetics and phylogenetic studies. The diagnosis of nesting PCR of the dairy cow Benno Sporozoa was established for the first time, and the infection of dairy cows in some areas of Heilongjiang province was preliminarily investigated to provide a reliable basis for the further prevention and control of the disease.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7
本文編號(hào)：2171861
[Abstract]:Besnoitia besnoiti mainly parasitic on the eyes, skin, and reproductive systems of cattle, causing bovine bainosporidiosis (Besnoitiosis). After cattle infection, emaciation, anemia, edema and digestive dysfunction may result in abortion, lower milk production and the quality of cow semen, death and supply can be caused in severe infection. The cattle industry has brought great economic losses.
At present, the study of Sporozoa Benno mainly focuses on the epidemiology, diagnosis and comprehensive prevention and control of the disease of Benno Sporozoa. At the molecular level, only the transcriptional spacer zone 1 (internal transcribed spacer ITS1) of ribose is studied. The study of the whole genome of the nematome has not been reported. The first part is to amplify the ITS-1 sequence of the whole genome of the mitochondria and the Chinese strain of the Chinese strain of baineno beetle by molecular biology. The sequence and sequence analysis of the whole mitochondrial genome sequence and ITS-1 are carried out, based on the sequence of the gene encoding gene of the mitochondrial genome and the ribosome R. DNA's ITS-1 constructs a phylogenetic tree to explore the evolutionary relationship between the Sporozoa baino spore and other protozoa, and determine its classification status in the protozoa. Results, the complete genome of the mitochondrial genome of the Sporozoa baineno sporozoite consists of 2 protein encoding genes and 7 tRNA genes, and the size is nucleic acid in the whole mitochondrial genome of 3679bp.. The composition of A=29.14%, C=19.30%, G=18.48%, T=33.08%, A+T accounted for 62.22%, G+C accounted for 37.78%. The genome base composition obviously preferred the base A and T. in series of mitochondrial 2 protein coding genes as gene markers. The results were similar with BI, MP and ML method for constructing phylogeny tree, and the intake tree was divided into two large branches, leaf subclass and coccidiosis. In the subclass of coccidia, the Eimeria and the suborder of the genus pyriform are an independent branch. In the order Eimeria, the amieriera and the suborder of the blood spores are a large branch. In the amieriera, eimerid is a large branch and the other families are located on a branch. In a sister branch of a sister branch, the parasite and the Harmon coccidia are the closest species of the relationship. This result is consistent with the traditional morphological classification, indicating that the mitochondrial whole gene sequence is a good genetic marker for the study of the parasite molecular phylogeny. The ITS-1rDNA was used as the gene marker to construct the phylogeny tree with three methods, MP, ML and BI. The results were similar. The results were similar to those of the phylogenetic tree constructed with the sequence of mitochondrial gene sequences. The results were similar to those in the United States, Germany, Australia and Israel.
The study of the diagnosis method of bainobo sporozoite has always stayed in the most primitive clinical observation, but for the infected cattle infected with bainobella spore, the clinical symptoms of the infected cattle were not obvious in the early stage of infection. When the symptoms appeared, they had come to the late stage and could only choose and eliminate them, causing major economic damage to the animal husbandry. Therefore, the second part of this study is to establish a rapid and accurate diagnostic method at the molecular level, to diagnose the disease early in order to reduce the economic loss. According to the published rDNA sequence of the sporozoite of Benno, the rDNAITS nesting primers of the Sporozoa bainite were designed. The extraction of the genes of the infected cattle containing the blood of the Benno Sporozoa was extracted. Group DNA, nest PCR amplification and sequencing, a rapid, specific and sensitive nesting PCR diagnosis method was established, and the method was used to detect the infection of blood samples of 196 dairy cows in parts of Heilongjiang province. The results showed that the established nested PCR recipe was sensitive and specific, and the positive blood was detected in clinical. 3 samples with a positive rate of 1.53%.
In this study, the whole genome sequence of the Sporozoa baineno Sporozoa was first obtained, and its characteristics were analyzed. The whole genome of the protozoa was enriched. The phylogenetic tree based on the sequence of mitochondrial genome protein and the sequence of ITS-1 sequence clearly defined the progress of the baineno bereno Sporozoa and other protozoa. The relationship was the basis for the study of other parasitic protozoa population genetics and phylogenetic studies. The diagnosis of nesting PCR of the dairy cow Benno Sporozoa was established for the first time, and the infection of dairy cows in some areas of Heilongjiang province was preliminarily investigated to provide a reliable basis for the further prevention and control of the disease.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7
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