本文選題：腫孔古柏線蟲 + 環(huán)紋背帶線蟲　； 參考：《中國寄生蟲學(xué)與寄生蟲病雜志》2017年02期

【摘要】：目的基于內(nèi)轉(zhuǎn)錄間隔區(qū)(ITS)序列,建立鑒別3種感染白牦牛線蟲的PCR-限制性片段長度多態(tài)性(PCR-RFLP)方法。方法將從甘肅省天祝藏族自治縣1頭白牦牛小腸中分離的12條線蟲進(jìn)行消化并提取DNA,PCR擴(kuò)增ITS序列并送測序,測序結(jié)果在GenBank數(shù)據(jù)庫進(jìn)行同源性分析。用DNAstar7.1軟件分析序列之間的差異,用鄰接(NJ)法構(gòu)建系統(tǒng)進(jìn)化樹。根據(jù)序列選擇特異的酶切位點(diǎn),對PCR產(chǎn)物分別進(jìn)行酶切反應(yīng),建立PCR-RFLP方法。結(jié)果 12條線蟲均擴(kuò)增出長度約900 bp的ITS序列片段,與預(yù)期結(jié)果一致。測序結(jié)果同源性分析顯示,其中3種各4條分別與已報(bào)道的腫孔古柏線蟲(Cooperia oncophora,GenBank登錄號為AB534601)、環(huán)紋背帶線蟲(Teladorsagia circumcincta,GenBank登錄號為JF680984)、奧斯特線蟲(Ostertagia sp.,Gen Bank登錄號為HQ844228)的序列相似性高于99%。ITS序列比較發(fā)現(xiàn),3種線蟲種間差異為4.3%~13.9%,種內(nèi)差異均低于1%。其中腫孔古柏線蟲與奧斯特線蟲的ITS序列差異最大,為13.6%~13.9%;腫孔古柏線蟲與環(huán)紋背帶線蟲ITS序列差異為12.9%~13.1%;環(huán)紋背帶線蟲和奧斯特線蟲ITS序列的差異最小,為4.3%~4.8%。系統(tǒng)進(jìn)化樹結(jié)果顯示,3種線蟲被分為明顯的3支,且環(huán)紋背帶線蟲和奧斯特線蟲有更近的親緣性。ITS序列酶切結(jié)果顯示,NdeⅠ酶可將環(huán)背帶線蟲和腫孔古柏線蟲的ITS序列酶切成約650 bp和250 bp的片段,Eco RⅤ酶僅將腫孔古柏線蟲酶切成約700 bp和200 bp的片段,2種內(nèi)切酶均不能酶切奧斯特線蟲的ITS序列。結(jié)論建立的基于ITS序列的PCR-RFLP方法可鑒別感染白牦牛的腫孔古柏線蟲、環(huán)紋背帶線蟲、奧斯特線蟲。
[Abstract]:Objective to establish a PCR- restriction fragment length polymorphism (PCR-RFLP) method for identification of three species of infected yak nematode based on its sequence. Methods 12 nematodes isolated from the small intestine of a white yak in Tianzhu Tibetan Autonomous County of Gansu Province were digested, their DNA sequence was amplified by PCR and sequenced. The sequence was analyzed in GenBank database. DNA star7.1 software was used to analyze the differences between sequences, and NJ method was used to construct phylogenetic tree. The PCR products were digested with specific restriction endonuclease sites according to the sequence and the PCR-RFLP method was established. Results all 12 nematodes amplified its sequences of about 900 BP, which was consistent with the expected results. Homology analysis of sequencing results showed that, The sequence similarity of 3 species (Ostertagia sp. GenBank accession number is AB534601), Teladorsagia circumcinctata GenBank accession number (JF680984) and Ostertagia sp. GenBank accession number (HQ844228) is higher than that of 99.ITS sequence comparison of 3 species of nematodes. The sequence similarity of each species is higher than that of reported Cooperia oncophora GenBank accession number AB534601, Teladorsagia circumcincta-GenBank accession number JF680984, Ostertagia sp. GenBank accession number HQ844228 and Ostertagia sp. GenBank accession number HQ844228. The interspecific difference of nematodes was 4.3 and 13.9.The intraspecific difference was lower than 1. Among them, the difference of its sequence was the biggest (13.6m) between Cymopterus tumefaciens and Orsteria, the difference between them was 12.9% and 13.1, and the difference between them was 4.34.8 (4.34.8), the difference of its sequence was 12.9% (P < 0.05), and the difference of its sequence was 12.9% (P < 0.05), and the difference of its sequence was (4.34.8) (P < 0.05) between A. annularis and C. oryzae (P < 0.05). Phylogenetic tree results showed that the three species of nematodes were divided into three distinct branches. The results of its sequencing showed that Nde 鈪，

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