用差减cDNA文库筛选由热激导致的小鼠睾丸中差异表达的基因

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

采用抑制性差减杂交(Suppression Subtractive Hybridization,SSH)方法,构建正常和热激小鼠睾丸组织的差减cDNA文库,以期筛选出小鼠生精过程中对热敏感的基因。分别从正常和热激小鼠睾丸组织提取总RNA,反转录成cDNA,以正常小鼠睾丸组织cDNA作为待检组织(tester),以热激小鼠睾丸组织cDNA作为驱动组织(driver),经过2轮杂交和抑制性PCR扩增,产物与T载体连接,经蓝白斑筛选,提取质粒,经EcoRI酶切鉴定插入片段并测序,由此构建了2种组织间差异表达基因的差减cDNA文库。用半定量RT-PCR方法,进一步验证了该文库中差减出的基因基本上为差异表达的基因。对随机挑选其中的932个克隆测序,对有效测序的565个基因序列与GenBank数据库中发布的序列进行同源性比较,大部分片段都可以检索到同源序列。结果显示,cPGES/p23等13个基因热激后表达量上调;septin2等120个基因热激后表达量下调。其中cPGES/p23为本研究中首次发现的小鼠生精过程中对热敏感的基因。

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 ex- pressed 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 homologicaUy 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 serotal mammals in this study.