大鼠肝细胞热适应差异表达基因消减文库的构建与鉴定

Construction and evaluation of subtractive libraries in association with heat adaptation differentially expressed genes in the rat liver

目的 :构建大鼠肝细胞热适应差异表达基因消减文库。方法 :实验采用热适应大鼠模型、常温对照。提取肝组织mRNA ,反转录cDNA并酶切、接头连接后分别以其中一组cDNA片段为tester,另一组为driver进行抑制性消减杂交 (SSH)构建消减文库。PCR扩增文库和未消减对照样品中的G3PDH基因以对比评估文库特异性。初步分离、筛选以评估文库的可靠性。结果 :PCR证实文库中G3PDH丰度大幅低于对照组 ,说明文库特异性较高。从文库中分离获得 30 0个目的基因片段 ,其中 2 7个经鉴定为差异表达基因 ,证实文库高效可靠。结论 :通过构建优质的热适应差异表达基因消减文库 。

AIM: To construct subtractive libraries in association with heat adaptation differential expressed genes. METHODS: The experiment was carried out with heat adapted rat model and normal temperature control. mRNA was extracted from liver tissue and reverse-transcripted into cDNA. After cut and ligation, suppression subtractive hybridization was executed with each group of cDNA as tester and the other one as driver to construct subtractive libraries. Specificity of the libraries was evaluated by approach of comparing G3PDH gene PCR products with template from the library and unsubtractive sample. Reliability of the libraries was evaluated by primarily isolation and screening. RESULTS: PCR results confirmed that G3PDH concentration was greatly reduced compared with control group, which suggested that specificity of the libraries was high. 300 target segments were isolated from the library, 27 of them were verified to be differential expressed genes, which suggested that the libraries were reliable and efficient. CONCLUSION: This study founded the basis of further investigation on heat adaptation mechanism by approach of constructing subtractive libraries in association with heat adaptation differential expressed genes.