Acquiring And Function Study Of Haemonchus Contortus Arrested Development Related Genes Daf-22and Hsp70

The gastrointestinal nematode Haemonchus contortus is a major blood feeding pathogen of sheep and goats, which inhabits the mucosal surface of the abomasum in these ruminants and causes haemonchosis. Its infections may be manifested as anemia, weight loss or even death in some cases, causing a great economic loss to the agricultural industry worldwide. The disease caused by infective third larval was seasonality, and the eggs and larval cannot live when the temperature is too low. The dauer formation is the only way for the larval to overcome the winter which is the reason for the outbreak in spring. Therefore, controlling, investing the mechanism of the dauer formation is crucial to resolving the problem. The studies carried were focused on the Haemonchus contortus, taking Caenorhabditis elegans as a model, the different expression genes between dauer and negative control worms were harvested by DDRT-PCR. The full length of Hc-daf-22and Hc-hsp70were obtained using RACE and genome walking. The impacts on dauer formation were verified by RNAi and rescue experiments. The results will be to play the foundation for clarifying the mechanism of arrest formation.1A new method for culture of Haemonchus contortus larvaeA study was carried out in order to culture Haemonchus contortus third stage larvae according to the published data which was mainly used to culture Trichostrongylus colubriformis and Ostertagia circumcincta. The eggs were extracted from intrauterine of femal from infected sheeps and the larvae were grown in a more defined medium (agar plate fortified with Earle’s medium and yeast extract). All stages of the larvaes were observed; meanwhile the sheep were infected by L3larvaes to verify the L3larvaes were infective. A lot of Haemonchus contortus every stage larvae in free living stage were gotten through this method.2Specific genes from dauers of Haemonchus contortus were acquired by mRNA DDRT-PCR, and analyzed by comparing with genes from normal wormsSheep free of parasitic nematodes infective were challenged with infective third larvae which were treated in tap water for5weeks according to the paper published by Kooyman, and sacrificed in35days to harvest the dauer worms in abomasums which were identified by its morphology. The fourth larval were cultured in vitro as negative control. The RNA from dauer worms and control worms were used to make cDNA by using random primers and anchorage primers. Specific cDNA were harvested and ligated into pMD18-T vector for sequencing.74pieces of genes were compared with the data in NCBI and parasite EST library.14pieces showed similarity with sequences of parasitic nematode having been published, and29of them showed high similarity with the genome of Haemonchus contortus, and the rest may be the new genes.3Screening of specific genes by RNAi using Caenorhabditis elegans as a modelSequences which showed similarity with other species were digested by proper enzymes for ligation into L4440and transformed into HT115after they were identified. The recombinant bacilli were coated on the NGM plates which contained IPTG and placed overnight. The hermaphrodites Caenorhabditis elegans were feeding on the recombinant vectors and hsf-1of Caenorhabditis elegans was used as a positive control and L4440as negative control. After RNAi, by observing phenotypes, brood size and dauer formation, the effective genes can be isolated. The results showed that:A342showed obvious phenotype; the brood size of A182and E181was significantly lower than that of negative control L4440; A342、A382and E211showed obvious effect in dauer formation.4Characterization of daf-22gene from Haemonchus contortus and function analysis and promoter analysis using Caenorhabditis elegansThe complete sequence of A382was identified employing3’RACE and5’RACE using primers designed based on the sequence A382(541bp). The open reading frame of the gene encodes533a.a, which had80.3%similarity with the Caenorhabditis elegans daf-22, designed as Hc-daf-22. The full length of the genome is6939bp,16exons and15introns were included. The5’flanking region was acquired employing genome walking. The1548-bp5’-flanking region of Hc-daf-22was cloned upstream of GFP gene in ppd95.77vector and microinjected into the gonads of Caenorhabditis elegans. The results showed that the transformed animals exhibited fluorescence in the pharynx, intestine, in all the larvae stages and adult worms.2061-bp5’-flanking of Ce-daf-22was cloned to the upstream of GFP gene in ppd95.77vector and microinjected into the gonads of Caenorhabditis elegans again. The result indicated that the transformed worms expressed fluorescence in the intestine, hypodermis and body wall muscle at all development stages. In order to determine whether the Hc-daf-22could be translated in Caenorhabditis elegans, the specific primers were designed based on the cDNA sequence of Hc-daf-22which all of the functional regions were included to obtain expression vector Cedaf22P-pPD95.77-Hc-daf-22. The recombinant vector was microinjected into Caenorhabditis elegans N2strain and showed fluorescence in the intestine in all stages. Next, the vector was injected into the Caenorhabditis elegans mutant (OK693), and the construct was expressed in daf-22(OK693) worms in intestine. Oil-Red-O staining, body size and brood size measurement, growth rate were carried out to ascertain whether Hc-daf-22can be used to rescue C. elegans mutant. The results showed transgenic worm conferred partial rescue of the mutant.The construct containing805bp of Hc-daf-22(RNAi) experiment was carried out to assess whether the endogenous mRNA can be rescued by parasite gene in N2C. elegans worms. The worms grown on Hc-daf-22(RNAi) plates had more fat storage compared with worms grown on control plates.5Characterization of heat shock protein70gene from Haemonchus contortus and its expression and promoter analysis using Caenorhabditis elegansIn the present study, the complete genome sequence of A342of Haemonchus contortus was identified by genome walking and long-PCR, and a cDNA sequence was obtained by reverse transcriptase PCR. Bioinformatic analysis revealed that the open reading frame of this gene the cDNA encoded a646-amino acid peptide, which had a high degree of sequence similarity with the Caenorhabditis elegans hsp-land hsp70with other species (designated as Hc-hsp70). Genome analysis indicated that7exons and6introns were contained. The5’-flanking sequence of the Haemonchus contortus hsp70was analyzed and3putative heat shock factors (HSF) and several transcriptional elements were identified. The5’-flanking region was subcloned into the vector upstream of green fluorescence protein (GFP) reporter gene and microinjected into the Caenorhabditis elegans. The fluorescence was expressed in the intestine in all larvae stages and adult with two expression patterns.Recombinant Hc-hsp70(designed as pET28b-hsp70) with6-His-tag at the C-terminal was expressed in Escherichia coli and was verified by analyses using SDS-PAGE and western blot. The recombinant Hc-hsp70(designed pPD49.78/49.83-hsp70) with6-His-tag at the C-terminal could also expressed in Caenorhabditis elegans. However, its expression induced down-regulation of hsp-1of Caenorhabditis elegans in mRNA level. These results suggested that the H. contortus hsp70might have similar function to that of C. elegans hsp-1.In summary, specific genes from dauers of Haemonchus contortus were firstly acquired, and specific genes which could induce phenotype and influence the dauer formation by bioinformatics analysis and RNAi in C. elegans were screened; the full length of two specific genes Hc-daf-22and Hc-hsp70were firstly obtained by RACE and genome walking respectively; the5’flanking sequence was analyzed in C. elegans and showed the promoter was included; Hc-daf-22can be expressed in N2and daf-22mutant (OK693) and conferred partial rescue of the mutant, meanwhile, Hc-daf-22induced RNAi in C. elegans successfully; the transcriptional activity of Hc-hsp70was confirmed, and it can be expressed in C. elegans and induced the down-regulation of hsp-1.This research will be particularly important for exploring the mechanism of dauer formation of parasitic nematodes.