Testis Following Exposure To Rana Chensinensis Octyl Phenol Subtractive Cdna Library Construction, And Analysis

In order to clarify the interference mechanism of alkylphenol on amphibian testis function and explore the effects of alkylphenols on gene expression, Rana chensinensis were exposured to4-tert-Octylphenol (OP) of10-6mol/L, and their testis were as research materials after being exposured30days. In order to explore OP interfered with reproductive endocrinology and the molecular mechanisms of growth and development of R. chensinensis, we constructed a subrative cDNA library using suppression subtractive hybridization (SSH) and selected the genes of R. chensinensis to effect gonadal development.1) Subtractive cDNA library in testis of R. chensinensis exposured to OP of10-6mol/L was successfully constructed using SSH, and180expressed the sequence tags (ESTs) were obtained. These differentially expressed genes were binding protein, ATP synthase, calcium-binding protein, actin, elongation factor, mitochondrion and kinase. It was worth noting that pregnancy specific beta-1-glycoprotein9(PSG9) and pregnancy-associated plasma protein A (PAPP-A) specifically expressed in the female viviparous animal, and individual oncogenes appeared. The Gene Ontology (GO) classification showed there were87biological processes,106cellular components,68molecular function (including pleiotropic effects). In biological processes, metabolic processes, cellular processes, female pregnancy, transcription regulation, response to stimulus, inflammatory response, oxidation-reduction process, gene expression accounted for45.98%,24.14%,13.79%,6.90%,3.45%,3.45%,1.15%,1.15%, respectively. In cellular components, cell part accounted for45.28%, and organelle part accounted for29.25%, and cell membrane accounted for14.15%, and cytoskeleton accounted for11.32%. In Molecular function, the elongation factor activity accounted for29.41%, and the structural molecule activity accounted for23.53%, and the translation regulator activity accounted for22.06%, and binding activity accounted for14.71%, and catalytic activity accounted for7.35%, and transporter activity accounted for2.94%. The dates showed that in biological processes metabolic process was occupied the highest proportion, and in the cellular components cell part was occupied the highest proportion, and in the extension factor activity molecular function was occupied the highest proportion. These results suggest that OP has an important impact on the growth and development of R. chensinensis testis, which mainly affected the metabolic process, cytoplasmic constituents, the elongation factor activity and feminizing effects.2) In subtractive cDNA library PSG9and PAPP-A were detected by Semi-quantitative RT-PCR. Normally, these two genes were only expressed in the mammalian placenta, but not expressed in the testis, ovary, liver and brain tissues. In testis of R. chensinensis exposured to OP of10-6mol/L after30days, the PSG9and PAPP-A were expressed. Strip brightness of PSG9and PAPP-A in the electrophoresis were significantly higher than that of the housekeeping gene β-actin, and slightly wider, and showed that the expression level was higher than β-actin. The control group PSG9and PAPP-A were not expressed. So we imagine that OP induced the expression of PSG9and PAPP-A in the testis of R. chensinensi, which may interfere with the development of the testis.