Screening of differentially expressed genes in heat-stressed mouse testis by construction of a subtractive cDNA library

In order to screen out the most sensitive genes to heat stress,we constructed a subtractive cDNA library using suppression subtractive hybridization(SSH).The total RNA was extracted from the testicle of heat-stressed and control mice,then they were reversed and transcribed into cDNA.cDNA from the testicle tissue of control mice and designated as the experimental group(the tester)and cDNA from the testicle tissue of heat-stressed mice as the control group(the driver).Only differentially expressed sequences were exponentially amplified using suppression PCR.Background was reduced and differentially expressed sequences were further enriched.The PCR products were easily ligated to T-Vector pGEM T.The positive clone was obtained and a subtractive cDNA library was constructed.Semi-quantitative RT-PCR analysis indicated that the genes revealed by subtractive hybridization were differentially expressed in the control and heat-stressed testis.932 clones were randomly selected for DNA sequencing.Among the 932 clones,565 were successfully sequenced and were analyzed homologically with the sequence published in Genbank.Homology analysis showed that homeotic genes were found for the great majority of these genes.The result showed 13 genes that included cPGES/p23 were up-regulated after heat-stressed,while 120 genes that included septin2 were down-regulated.cPGES/p23 was the gene discovered sensitive to heat stress during spermatogenesis of scrotal mammals in this study.