Establishment Of PCR-RFLP Technique For Identifitication And Genetic Variation Studies Of Nematodes In Digestive Canal Of Yaks

Nematodes in digestive canal mainly inhabit the alimentary canal of ruminant animals,such as cattle,sheep,yaks and camels.It's characterized by mixed infection with variety of parasitic nematodes,which can cause the digestive tract mechanical trauma and is complicated with gastrointestinal inflammation manifested anorexia,anemia and emaciation in host.Infested with large amount of parasitic nematodes may result in the blockage of host's digestive tract,even lead to death,causing enormous economic losses in worldwide animal husbandry.Therefore,identifying the specie of nematodes in digestive canal accurately and studying its genetic relationship and population characteristics comprehensively have important significance to the treatment,prevention and etiology of nematodes in digestive canal.Taking three different species of nematode found in yaks' abomasums for research object,this study is mainly about researching and analyzing the molecular identification and genetic diversity within these three species.1.Using RNA transcribed spacer sequence amplification and sequencing methods,the collected from gansu province tianzhu county national animals survive the white yak digestive tract of three species of nematodes were studied..And the result showed that these three nematodes are Cooperia oncophora,Teladorsagia circumcincta,and Ostertagia sp,which ITS sequences are 779 bp,791bp and 765 bp respectively.The intra-specific variation of these three nematodes was lower than 0.5%.As for the inter-specific variation,there were considerable genetic differences among different species of nematodes,which the difference between Cooperia oncophora and Ostertagia sp.was 13.6%-13.9%,the difference between Cooperia oncophora and Teladorsagia circumcincta was 12.9%-13.1%,and the difference between Ostertagia sp.and Teladorsagia circumcincta was 4.3%-4.8%.By analyzing the ITS sequence of these three nematodes,it could be concluded that the ITS sequence can be used as the molecular marker to distinguish species of nematodes.2.According to the difference of the internal transcribed spacer sequence and using PCR-RFLP technology established a molecular biology method successfully to distinguish the three species of nematodes.the result show that the technology can identity three species of nematodes successfully.Used two restriction enzymes were Nde I and Eco R V.Three species of nematodes were identitied by two enzyme digestion reaction.Cooperia oncophora was digested by both restriction enzymes,Teladorsagia circumcincta was only digested by Nde I,and Ostertagia sp.remain undigested by both restriction enzymes.Compared with traditional morphological method,molecular biological method has better maneuverability and higher accuracy,and it also develops new ideas of distinguishing these three species of nematodes.3.Amplifyed and sequenced the nad6 sequences of 14 Teladorsagia circumcincta and 15 Cooperia oncophora,which were found in alimentary canal of yaks from Tianzhu county in GANSU province.The complete nad6 genome sequences of Teladorsagia circumcincta was 438 bp.The difference of intra-specific nucleotide was 1.1-2.3%,and the difference of intra-specific amino acids was 0.7-2.7%.Evolutionary tree showed that Teladorsagia circumcincta Tianzhu strain has no apparent branch with Japanese strain and Indian strain.The complete nad6 genome sequences of Cooperia oncophora was 438 bp.The difference of intra-specific nucleotide was 0.9-1.8%,and the difference of intra-specific amino acids was 0.7-1.4%.Evolutionary tree showed that Cooperia oncophora Tianzhu strain has obvious branch with Australia strain.4.Determined the complete sequences and structures of mitochondrial genomes of Cooperia oncophora Tianzhu strain.The complete circular mitochondrial genome sequences of Cooperia oncophora Tianzhu strain was 13583 bp,and had 36 genes including 12 protein genes,22 t RNA genes and 2 r RNA genes,lacking of atp8 gene.By analyzing ITS sequences with BLAST program,the difference between Cooperia oncophora Tianzhu strain and Cooperia oncophora Australia strain was less than 0.5%,while it was 12.2% by analyzing mitochondrial genome.Thus Cooperia oncophora Tianzhu strain can be regarded as Cooperia oncophora Australia strain's cryptic specie.This finding enriched the information of Cooperia oncophora's dataset in genetic evolution.In conclusion,this study amplified and sequenced the ITS sequences of Cooperia oncophora,Teladorsagia circumcincta,and Ostertagia sp,and the nad6 sequences of Cooperia oncophora,Teladorsagia circumcincta,determined the complete sequences and structures of mitochondrial genomes of Cooperia oncophora Tianzhu strain.,established a molecular biological method to identify these three nematodes,analyzed their genetic variation and population structures,which supplied data support for parasitic nematodes' research and provided a theoretical basis for controlling parasitic disease.