| Alkylphenols(APEs) are well known because it has been identified as endocrine disruptive chemicals,4-tert-Octylphenol(OP) which is derivatives of alkylphenols is lipophilic substances, it is chemically stable substance and demonstrate estrogenic effects, toxicity and carcinogenic effects in the environment. Suppression subtractive hybridization(SSH) study differential gene expression at the transcriptional level, because of its high specificity, high sensitivity and high efficiency, therefore, it can be as a powerful means to screening differentially expressed genes. In this study, we use Rana chensinensis as our experimental subjects, Rana chensinensis was exposed to OP, then the cDNA library of differentially expressed genes of Rana chensinensis liver was constructed by using suppression subtractive hybridization technique, and screening differentially expressed genes of Rana chensinensis liver after OP exposure, the classification and functional annotation of known genes which come from the cDNA library was done, all of these is helpful to understand toxicity effect mechanism of APEs on animal liver from the level of gene expression and gene interaction. The main results and conclusions which obtained in our study as follows:1. Extracted total RN A of Rana chensinensis liver tissue in the control group and treated group, then used agarose gel electrophoresis and nucleic acid spectrometer to detect concentration and purity of total RNA, we found that three bars of rRNA was clear, distribution of mRNA band showing diffusion, the ratio of RNA OD260/OD280was between1.8and2.0. So we believed that the quality of extracted total RNA to be in line with the standards of construction cDNA subtracted library. Rana chensinensis liver tissue cDNA in the treated group was used as tester, liver tissue cDNA in the control group was used as driver, cDNA library of differentially expressed genes of Rana chensinensis liver was successfully constructed by using suppression subtractive hybridization technique.2. We got131EST sequences, vector sequences would been cut from EST sequences, we used blast which provided by NCBI to search for sequence homology. We found75EST sequences of the cDNA library of differentially expressed genes of Rana chensinensis liver after OP exposure with high homology compared with the known genes,56EST sequences with low homology compared with the known genes or no significant similarity been found, the proportion of high homology and low homology(or no significant similarity) were57%and43%. These results showed that there were a large number of new genes need to be further study, or compared with genes of other species, the homology of genes which been expressed in liver tissue of Rana chensinensis was low, therefore the genes remains to be further research.3. The serum albumin gene and the fibrinogen y gene which selected from cDNA library were analysised by real-time quantitative PCR. The results showed that relative expression amount of the two genes expression in liver tissues of treated group significantly increases compare with control group. There was an increase in expression of serum albumin gene which leaded to increase in the amount of serum albumin synthesis, it is adapt to the detoxification function of serum albumin. The stress response of Rana which induced by OP brought about high expression of the fibrinogen gamma gene, causing expression of acute-phase response system related genes in liver tissue. This indicated that the cDNA library of differentially expressed genes of Rana chensinensis liver was successfully constructed by using SSH.4. The known genes in the cDNA library would been classified according to their function. The results showed that the proportion of genes involved in cell material transport and cellular signal transduction were13%, genes involved in cellular immunity, cell structure and material metabolism were10%, genes involved in cellular transcriptional regulation were7%, genes involved in cell apoptosis, cell proliferation, cell cycle, cell differentiation were3%, respectively. There are still some known genes, but its function remains unclear, this part of genes accounted for25%. The results indicated that the genes which differentially expressed in Rana liver tissue after OP exposure were mainly involved in cellular material transport, cell signal transduction, cellular immunity, cell structure and material metabolism. |
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