| Hyperthermia is a harmful condition being increasingly recognized in military area. Many studies had been intensely focused on to what extent and by what mechanism an individual had adapted himself to hyperthermia stress. Although many study to learn the pathophysiology of hyperthermia stress, hyperthermia injury and hyperthermia adaptation through traditional technology had achieved some progress in the past, molecular biology and biochemisty mechanisms underlining the pathophysiology condition of hyperthermia stress were still left largely unknown. Under hyperthermia stress, gene-ragulated adaptation will play pivotal roles in maintaining relatively stablility of internal environment by expressing regulated protein, including some hormones and enzymes. Fully understanding of this differently expression phenomena will help to disclose the molecular mechanism of hyperthermia stress. But by present, little work had been reported in this field.Objective To study the differently expressed genes of monocyte cells in the peripheral blood of heat-adaptation rabbits, a PCR based different expression technology with highly discriminating ability, the Suppression SubtractiveHybridization (SSH), was introduced to find out differently expressed genes and to further reveal the possible gene-marker during the process of heat adaptation.Method Heat adaptation rabbit model was constructed by standard procedure. Peripheral blood was extracted from both normal rabbits and heat-adaptation ones, then cDNA was synthesized by RT-PCR and digested with Rsa I . After ligating the digested cDNA with two adaptors, two steps of hybridization and two steps of PCR were followed to normalize and enrich differently expressed genes and construct the subtractive cDNA library. A PCR-select differential screening procedure was employed to reduce the background, Clones containing the differently expressed genes was selected for DNA sequencing and analysised with BLAST program in GenBank wired NCBI website.Results Sufficient heat adaptation was achieved. Total RNA was exacted and purified in good quality and quantity. Double strand cDNA was reverse transcripted integrately,and then cut with Rsa I into even length short segments. Ligation was effective. After two stage of subtractive hybridization, ascending and descending expressed heat adaptation related cDNA subtractive libraries were constructed.Nested-PCR wsa specially,which enlarged differential expressed gene exponentialy. Procucts were ligated with T vector and then transformed into Escherichia coli DH5acompetent cells successfully, which dispersed differentialy expressed genes of the libraries into single copy. 96 target gene segments were isolated primarily from it.After differential screening and virtual Northern Blot hybridization, 7 clones contain differently express genes was identified. Sequences was submitted to GeneBank for homogenous contrast to make clear their propert. DNA sequencing and analysis reveal 1 known sequences and 6 known sequences.Conclusion SSH PCR had been applied successfully in our study to construct heat adaptation related subtractive cDNA library containing differently expressed ' up-regulated and down-regulated genes from heat-adaptation rabbits. Some up-regulated genes were identified and were believed to play important roles in metabolic adjust and tisssue repairing during the heat-adaptation. Some genes identified as differently expressed are from complete mitochondrial genome, but its functions remain unknown. Several sequence genes cloned in our study was foundto have little homology with sequences registered in GeneBank after homogenous contrast. Further study is required to determine their gene structure and biological functions. Our study also demonstrated that PCR-based SSH techenology (including Differently Screening and DIG DNA labeling and detection system) is a powerful tool to identified differently expressed genes in heat-adaptation tissues.
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