| With the development of animal husbandry intensification and industrialization,farmers have sought to breeding more animals in smallest space.In order to maximize the performance of animals production,reduce labor costs and increase economic benefits,farmers are often overlooked some adverse external factors in the breeding process,which affect the growth of animal,reduce feed conversion,and even cause disease or death,result in huge losses to the livestock industry.These factors include high temperature,anoxia,high pressure,UV,tissue infections,heavy metals et.al,in which the adverse effects of heat stress caused especially significant.Livestock and poultry are warm-blooded animals,which has a zone of thermoneutrality,when the ambient temperature and other conditions are suitable,the physiology of the body would normally be able to give full play to its performance.When the ambient temperature exceeds the zone of thermoneutrality limits,the body will occurs a variety of physiological functions of disorder,produce a variety of non-specific defense responses.In this experiment,we selected Pekin ducks which grow up in the north of China and Muscovy ducks in south of China for the study,focused on the effects of heat stress on the duck liver metabolism of this two species;We also studied the effects of heat stress on the duck liver damage and oxidation function by histological examination and determination of antioxidant indicators;We analysed the effects of heat stress on the mRNA expression of heat shock protein and inflammatory factors genes by quantitative PCR;Besides,de novo transcriptome assembly and gene expression analysis of heat stress using Illumina sequencing technology were performed;We used the method of in vitro cultured duck hepatocyte,discussed the effects of HSP70-siRNA on the expression of duck hepatocyte apoptosis signaling pathway genes.The use of in vitro methods to study the HSP70-siRNA on apoptosis duck cell signaling pathways expression of key genes;Moreover,we used comparative proteomic approach to uncover the temperature-dependent proteins of two duck species;At the same time,we cloned heat-related genes according to the proteomic results,and analysed the gene sequences and structure differences between this two duck species.1.Effects of heat stress on antioxidant defense system,inflammatory injury,and heat shock proteins of Muscovy and Pekin ducksRising temperatures are severely affecting the mortality,laying performance,and meat quality of duck.Our aim was to investigate the effect of acute heat stress on histopathological,antioxidant enzyme activity,inflammatory factors and heat shock proteins in livers of ducks and to compare the thermal tolerance of Pekin and Muscovy ducks exposed to acute heat stress.Ducks were exposed to heat at 39±0.5℃ for 1 h and then returned to 20℃ for 1 h followed by a 3-h recovery period.The liver and other tissues were collected from each individual for analysis.Morphological analysis indicated that heat stress induced tissue injury in both species,and the liver of Pekin ducks were severely damaged.The activities of several antioxidant enzymes increased in Muscovy duck liver,but decreased in Pekin duck.The mRNA levels of HSPs(70,60,and 40)increased in both species,except for HSP10,which was upregulated in Muscovy ducks and had no difference in Pekin ducks after heat stress.Simultaneously,the mRNA level of HSP90 decreased in the stress group in both species.The mRNA levels of inflammatory factors were increased after heat stress in both duck species.These results suggested that heat stress could influence the activities of antioxidant enzymes,HSPs and inflammatory factors expression.Moreover,the differential response to heat stress indicated that the Muscovy duck has a better thermal tolerance than does the Pekin duck.2.De novo assembly and characterization of Muscovy duck liver transcriptome and analysis of differentially regulated genes in response to heat stressIn this study,de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed.More than 225 million clean reads were generated and assembled into 36,903 unique transcripts with an average length of 1,135 bp.A total of 21,221(57.50%)Unigenes were annotated.Gene Ontology(GO)analysis of the annotated Unigenes revealed that the majority of sequenced genes were associated with transcription,signal transduction and apoptosis.We also performed gene expression profiling analysis upon heat treatment in Muscovy ducks and identified 470 unique heat-response unique transcripts.GO term enrichment showed that protein folding and chaperone binding were significant enrichment,whereas KEGG pathway analyses showed that Ras and MAPKS were activated after heat stress in Muscovy ducks.Our research enriched sequences information of Muscovy duck,provided novel insights into responses to heat stress in these ducks,and serve as candidate genes or markers that can be used to guide future efforts to breed heat-tolerance duck strains.3.Effects of HSP70 knockdown on the expression of duck hepatocyte apoptosis related genesHSP can not only block protein denaturation and aggregation of cells to protect cells from stress and other adverse factors,can also act directly on the anti-apoptotic mechanisms of cells.The research used the method of in vitro cultured duck hepatocyte,discussing the effects of HSP70-siRNA on the expression of duck hepatocyte apoptosis signaling pathway genes.The results showed that according to the rat liver cell isolation and culture method which using the classic two-step perfusion,we isolated high vitality duck liver cells.We added some growth factors like insulin and dexamethasone,making better duck liver cells in vitro culture for some time.When used HSP70-siRNA treated cells,the level of CCO gene increased 1156.7%(P<0.01),SAPK gene expression levels increased 733.3%(P<0.01),p38MAPK gene expression level decreased 70.0%(P<0.01),RASA1 gene expression was reduced 83.4%(P<0.01).4.Comparative proteomic analysis of the hepatic response to heat stress in Muscovy and Pekin ducks:insight into thermal tolerance related to energy metabolismThis study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach.Duck was cultured under 39℃±0.5℃for 1 h,and then immediately returned to 20℃for a 3 h recovery period,the liver proteins were extracted and electrophoresed in two-dimensional mode.After analysis of gel images,61 differentially expressed proteins were detected,54 were clearly identified by MALDI TOF/TOF MS.Of the 54 differentially expressed protein spots identified,7 were found in both species,whereas 47 were species specific(25 in Muscovy duck and 22 in Pekin duck).As is well known,chaperone proteins,such as heat shock protein(HSP)70 and HSP 10,were abundantly up-regulated in both species in response to heat stress.However,we also found that several proteins,such asα-enolase,and S-adenosylmethionine synthetase,showed different expression patterns in the 2 duck species.The enriched biological processes were grouped into 3 main categories according to gene ontology analysis:cell death and apoptosis(20.93%),amino acid metabolism(13.95%)and oxidation reduction(20.93%).The mRNA levels of several differentially expressed proteins were investigated by real-time RT-PCR.To our knowledge,this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals.5.Cloning and structure analysis of Muscovy duck ENO1 gene cdsTemperature affects stabilities of protein 3D structures,causing protein functions like ligand recognition and binding to be thermally sensitive.Therefore,natural selection should favor amino acid sequences that optimize protein function in a particular thermal environment,for example,by conserving ligand-binding abilities within norrow ranges.In our study,we selected α-enolase(ENO1)depend on the proteomic data before,and then analysed the ENO1 gene sequence and structure differences between Pekin duck and Muscovy duck.The results showed that the cds of ENO1 gene contains 1305 bases which encodes 434 amino acids.There were 21 bases differences between Pekin duck and Muscovy duck ENO1gene,5 amino acids differences between protein sequences.Site 171 and 173 were polymorphic for Pro/Cys in Pekin duck a-enolase but Leu/Val in Muscody duck.Cys was polar amino acids while Val was nonpolar amino acids.Meanwhile,site 171 and 173 were located in the top of β-sheet depend on the 3D structure of a-enolase,which may have a role in guiding the substrate enter protein catalytic domain. |
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