Analysis Of UGT Gene And Study On Pcr Detection Method For Ivermectin Resistance In Haemonchus Contortus

With the development of large-scale animal husbandry and the change of breeding methods,the harm of parasitic diseases has become increasingly significant,especially the harm of Haemonchus contortus.Therefore,farmers and herdsmen have to use a large amount of deworming drugs to control the harm,but this frequent and long-term use of drugs also leads to the generation and increase of drug resistance.Due to the progressive development of drug resistance,gastrointestinal nematode infections in ruminants are becoming more and more difficult to control.At present,there is a lack of in-depth systematic research on the problem of drug resistance,especially the lack of rapid and specific diagnostic methods.In view of this,in this study,ivermectin-resistant Haemonchus contortus was selected as the research object,and UDP-glycosyltransferase（UGTs）protein that were screened earlier and related to drug resistance were selected.Based on bioinformatics analysis,the differential expression of UGT gene in the eggs and stage III larvae of Haemonchus contortus was analyzed by fluorescence quantitative PCR method.Then,the UGT gene sequences of drug-resistant and sensitive H.contortus strains were analyzed and compared by Megalign biological software in DNAstar,which laid the foundation for the establishment of PCR-RFLP method.The results are as follows:（1）UDP-glycosyltransferase（UGTs）protein was composed of 170 amino acids,of which serine（S,15.3%）was the highest.Its molecular formula is C₈₈₃H₁₃₄₉N₂₂₃O₂₄₉S₈ and its molecular weight is 19329.25.All the domains of this protein are located outside the membrane,and there is no transmembrane domain.It is a hydrophilic protein,which mainly plays a biological role in the plasma membrane and has multiple antigenic sites.Moreover,UGT gene of H.contortus has certain conserved properties,which provides basic data for the subsequent molecular detection of UGTs gene.（2）Quantitative PCR analysis showed that the expression level of UGT gene in drug-resistant eggs and stage III larvae was significantly different from that of sensitive eggs and stage III larvae.The expression level of UGT gene in drug-resistant eggs of H.contortus was higher than that of sensitive eggs.The expression level in the third stage larvae of drug-resistant strains was higher than that in the third stage larvae of sensitive strains,and the expression level in the egg stage was higher than that in the larval stage.Ivermectin resistance was related to the expression of UGT gene.Based on this,a fluorescence quantitative PCR detection method based on UGT gene was established.（3）According to the sequencing results,the gene sequence of UGT gene exon region was selected,and the length of the fragment was 522 bp.Compared with the standard sensitive strain of H.contortus,8 base sites were mutated in the UGT gene fragment of H.contortus standard drug-resistant strain,and the mutation sites could be used as potential molecular markers for drug resistance detection.Based on this,a RFLP analysis method based on the polymorphism of UGT gene was finally established.There was a mutation in the ivermectin sensitive strain sequence,so it was not cut and only three target bands appeared.In contrast,ivermectin resistant strains of H.contortus showed four bands of purpose.These results provide important technical support for the rapid diagnosis of drug resistance of H.contortus,and lay a foundation for the in-depth study and elucidation of drug resistance mechanism.At the same time,they are also of great significance for parasite control and the development of animal husbandry.