Construction Of Subtractive CDNA Library Related To Heat Adaptation And Isolation,Evaluation,Sequence Analysis Of Differential Expressed Genes

The research of ability for human to endure hot environment has been paid attention all through the ages. It determined whether human being could adapt for hot environment and survive themselves effectively during the course of interaction of human being and environment. It was more important for soldiers to maintain battle effectiveness in the hot battle field environment from the angle of military special environmental medicine. So, it is essential for us to investigate heat adaptation phenomenon, seeking for more effective approaches to accelerate heat adaptation. It will help to increase battle effectiveness of soldiers and decrease non-battle sacrifice in the hot battle field environment.The effective approaches for human to gain ability of heat adaptation must be base on clarifying the molecular mechanism of heat adaptationthoroughly. It has been proved by former research that heat adaptation was a complex course controlled by many factors. However, it is still not enough to explain this phenomenon on current research data. So, thoroughly isolating biological active substance, uncovering the approaches that these substances exerting their biological activities, all are very significative for investigating molecular mechanism of heat adaptation.Aim: Investigating gene differential expression in rats liver during the course of heat adaptation using the technique of suppression subtractive hybridization(SSH), in order to clarify molecular mechanism of heat adaptation on the gene level.Method: Rats(n=6) of the same parents and sex are divided into 2 groups randomly in the experiment: heat adaptation group(n=3) and normal temperature control group(n=3). Animal model are made in the digital emulational climate room. mRNA are extracted from liver tissue of the two groups individually, and then reverse transcripted to be double strand cDNA. After universal cutting with Rsal endonuclease and adaptors ligation, cDNA from these two groups were used as "tester" and the other one as "driver" to hybridize with each other in turn. Ascending and descending subtractive libraries related to heat adaptation were constructed by SSH. Subtractive products were ligated with T vector, and then transformed into host bacteria, which dispersed differential expressed gene mixture into single ones. Target genes were achieved with PCR, and then screened with Differential Screening and reverse Northern Blot technique. Positive differential expressed genes are submitted to GeneBank for homogenous contrast.Conclusion: Sufficient heat adaptation was achieved. Total RNA and mRNA were exacted and purified in good quality and quantity. Double strand cDNA was reverse transcripted integrately, and then cut with Rsal into even length short segments. Ligation was effective. After two-some subtractive hybridization, ascending and descending expressed heat adaptation related cDNA subtractive libraries were constructed. Nested-PCR was specially, which enlarged differential expressed gene exponentially. Products wereligaled with T vector and then transformed into TOP10F' competent cell successfully, which dispersed differentially expressed genes of the libraries into single copy. 200 target gene segments were isolated primarily from it. After Differential Screening and reverse Northern Blot hybridization, 25 genes were confirmed to be differentially expressed. Sequences were submitted to GeneBank for homogenous contrast to make clear what kind of protein they are encoding.Result: (1) SSH was applied in the thermal environmental medicine research work for the first time. Molecular mechanism of heat adaptation phenomenon was investigated from the angle of gene differential expression. (2) ascending and descending expressed gene subtractive libraries related to heat adaptation were constructed ,it was the base for further systematic research work in molecular mechanism of heat adaptation. (3) HSP27, HSP86 and p53 protein encoding genes were found to upregulate in the tissue level during heat adaptation, it indicated that HSP27, H...