鸡脾脏组织抑制消减cDNA文库构建及其差异表达基因的分析

Construction of suppression subtractive cDNA library and analysis of differentially expressed genes in the spleen of the chicken

为分离与鸡抗病性密切相关的差异表达基因,并进行功能分析,利用抑制性消减杂交技术,以大骨鸡和海兰褐商品代蛋鸡20周龄时的脾脏组织为试验材料构建消减cDNA文库。文库的插入片段集中在600 bp左右,挑取760个克隆进行PCR筛选获得663个阳性克隆,经过点杂交筛选后选择了531个阳性克隆,从中随机挑取100个阳性克隆进行测序。经过同源性比对归并后得到37个差异表达基因或ESTs序列,其中,32个是已知基因,包括一般抗病性或免疫性能、特异抗病相关基因、细胞信号分子、膜蛋白质、转录因子等差异表达基因;5个为功能尚未确定的基因。并对4个可能影响鸡抗病性能的差异表达基因进行了RT-PCR半定量检测鉴定。该试验结果为进一步研究这些差异表达基因在抗病过程中的重要功能及其调控作用机理奠定了基础。

To screen the differentially expressed genes related to anti-disease activity in chicken, a suppression subtractive hybridization （SSH） was employed to construct the SSH eDNA library in the spleen by using Dagu chicken and commercial Hyland brown chicken at 20 weeks of age. The inserted fragment sizes at 600 bp and 760 clones were verified by PCR, of which 663 clones were used to perform dot blotting hybridization and 531 clones were obtained. Subsequently, 100 clones were randomly picked and sequenced. 37 differentially expressed genes were isolated by homologous BLAST search. Besides 5 unknown function genes, the other 32 genes implicated in general disease resistance-related and immunity, specific disease resistance-related, cell signaling molecules, membrane protein and transcriptional factor genes, and so on. Four differentially expressed genes probably impacting anti-disease activity of chicken were identified using semi-quantitative RT- PCR method. The Construction of SSH eDNA Library was expected to lay the foundation for isolating and cloning the novel genes from chicken spleen, which potentially involved in spleen performance, and for further investigating the functions and the regulatory mechanisms of these genes during anti-disease activity.