| Cold stress is seriously restricting the development of animal husbandry as the major stressor in the alpine region. It is generally use hormone levels to study the effect of cold stress on the animals at present. The hormone levels in different animals or at different growth stages of the same animals are not the same. Hormone content was susceptible to impact by external factors such as sampling and capture. Therefore, the results were not very accurate to evaluate the effects of stress on animal by hormone levels.48three-week-old AA broilers were randomly divided into three treatment groups and a control group (N=12), each group with4replicates. Treatment group1:temperature is7℃lower than the room temperature for6hours. Treatment group2:temperature is7℃lower than the room temperature for3hours. Treatment group3:temperature is5℃lower than the room temperature for6hours. This experiment continued one week. We filtered out specific genes associated with chronic cold stress of broilers by the high-throughput sequencing technology and analysised gene function. Quantitative PCR technique was used to further analysis the cold stress-related gene expression of broilers under different condition of cold stress. The results show that:1) According to solexa sequencing, we obtained15779differential expression tags. There were4721differential expression tags which were biological significance with the chicken genome database comparison. It included992up-regulated genes,3088down-regulated genes,469only in the control group and172only in the experiment group.2) The analysis results of GO showed that, differential expression genes were mainly located in11cell components such as cell part, organelle, synapse and membrane-enclosed lumen. Differential expression genes were significantly enriched in12molecular functions such as binding, the catalytic activity, molecular transducer activity, transporter activity and nucleic acid binding transcription factor activity. These differential expression genes were involved in23biological processes such as cellular processes, the developmental process, biological regulation and metabolic process.3) The analysis results of pathway showed that differential expression genes were significantly enriched in39pathways such as Pathway’s in cancer, MAPK signaling pathway, oxidative phosphorylation, apoptosis and PPAR signaling pathway.4) During cold stress, the relative expression level of HSP70gene in heart and kidney were significantly higher than the other tissues in treatment group1(P<0.05). The relative expression levels of HSP70gene in the kidney was significantly higher than the other tissues in treatment group2(P<0.05). The relative expression levels of HSP70gene in the heart was significantly higher than the liver, spleen, lung and duodenum (P<0.05). The relative expression levels of HSP70gene in the duodenum was significantly higher than the other tissues in treatment group3(P<0.05). The relative expression levels of HSP70gene in the heart, liver, spleen and lung of control group broilers were significantly higher than the cold stimulation treatment group (P<0.05). The relative expression levels of HSP70gene in the duodenum of control group was significantly increased compared to the conditions of cool5℃for6hours (P<0.05). When the cooling rate was7℃and cold stimulation time respectively was6hours and3hours, the relative expression levels of HSP70gene showed an increasing trend in the heart, spleen, kidney and duodenum. But the relative expression levels of HSP70gene showed a downward trend in the liver and lung. When the cold stimulation time was same and cooling rate respectively was7℃and5℃the relative expression levels of HSP70gene showed an increasing trend in all the tissues except in heart and kidney.5) During cold stress, the relative expression level of Fas gene in the duodenum was significantly higher than the other tissues in treatment group1(P<0.05). The relative expression levels of Fas gene in the liver was significantly higher than the heart, lung and kidney (P<0.05). The relative expression level of Fas gene in the lung and duodenum were significantly higher than the other tissues in treatment group2(P<0.05). The relative expression level of Fas gene in the liver was significantly higher than the spleen (P<0.05). The relative expression level of Fas gene in the duodenum was significantly higher than the other tissues in treatment group3(P<0.05). The relative expression levels of Fas gene in the heart, liver, spleen and lung of control group broilers were significantly higher than the cold stimulation treatment group (P<0.05). When the cooling rate was7℃and cold stimulation time respectively was6hours and3hours, the relative expression levels of HSP70gene showed an increasing trend in the heart, spleen, lung and kidney. But the relative expression levels of HSP70gene showed a downward trend in the liver and duodenum. When the cold stimulation time was same and cooling rate respectively was7℃and5℃, the relative expression levels of Fas gene showed an increasing trend in all the tissues except in liver.6) During cold stress, the relative expression level of PPAR-a gene in liver was significantly higher than the other tissues in treatment group1(P<0.05). The relative expression level of PPAR-a gene in heart and kidney was significantly higher than the spleen and lung (P<0.05). The relative expression levels of PPAR-a gene in the liver and kidney were significantly higher than the other tissues in treatment group2and3(P<0.05). The relative expression level of PPAR-a gene in heart and duodenum were significantly higher than the spleen and lung (P<0.05). The relative expression levels of PPAR-a gene in the liver, lung and kidney of control group broilers were significantly higher than the cold stimulation treatment group (P<0.05). When the cooling rate was 7℃and cold stimulation time respectively was6hours and3hours, the relative expression levels of PPAR-a gene showed an increasing trend in the heart and liver. But the relative expression levels of PPAR-a gene showed a downward trend in the spleen, lung, kidney, and duodenum. When the cold stimulation time was same and cooling rate respectively was7℃and5℃, the relative expression levels of PPAR-a gene showed a downward trend in all the tissues except in liver.Through the sequencing technologies-the next generation, we filtered out genes related to chronic cold stress which were different expression in broilers. It showed that cold stress was an process of multi-gene co-regulation and these genes were enriched in some biological pathways such as energy metabolism, apoptosis, and PPAR signal pathways. We founded out the expression regular of HSP70, Fas and PPAR-α in different tissues of broilers by the quantitative PCR technique and revealed apoptosis and energy metabolism pathway played an important role in the process of cold stress. We laid the foundation for the study of the molecular mechanism of chronic cold stress in broilers. |
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