利用抑制消减杂交技术筛选猪蛔虫性别差异表达基因.pdfVIP

中国农业科学 2005,38(5):1040-1045 Scientia Agricultura Sinica 利用抑制消减杂交技术筛选猪蛔虫性别差异表达基因 吴绍强，邹丰才，翁亚彪，宋慧群，林瑞庆，朱兴全 （华南农业大学兽医学院，广州 510642 ） 摘要：为了筛选线虫的性别特异性基因，为线虫乃至寄生虫的性别控制提供新的工具，本研究选择猪蛔虫 （ TM Ascaris suum ）为模型，分别提取雌性和雄性成虫的 mRNA 后，采用 Clontech 公司的 PCR-select 试剂盒进行 反转录合成 cDNA 并进行抑制消减杂交（SSH），构建猪蛔虫雌、雄虫性别差异表达的消减 cDNA 文库，并采用地高 辛（DIG）标记的 cDNA 探针进行 Southern 杂交检验所构建文库的消减效率。结果表明，雌、雄虫性别差异表达 的消减 cDNA 文库均具有很强的性别特异性。随机从各库中各抽取 25 个克隆进行测序及在线 BLAST 分析，发现在 25 个雄虫 ESTs 中有 20 个已知 ESTs，5 个新的 ESTs；25 个雌虫 ESTs 中有 11 个已知 ESTs，还有 8 个可能为新基 因。猪蛔虫性别差异表达的消减cDNA文库的成功构建，为进一步研究性别特异基因的功能奠定了基础。 关键词：猪蛔虫；抑制消减杂交；性别特异性基因；cDNA文库；Southern blot Profiling of Gender-Specific Gene Expression for Ascaris suum by Suppression Subtractive Hybridization WU Shao-qiang, ZOU Feng-cai, WENG Ya-biao, SONG Hui-qun, LIN Rui-qing, ZHU Xing-quan (College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642) Abstract: Attempting a new method of gender-controlling nematode infection in animal production, differentially expressed male and female cDNA libraries of the adult Ascaris suum were constructed using the technique of suppression subtractive hybridization (SSH). The gender specificity of the constructed archives was determined using Southern blots with Digoxigenin(DIG)- labeled male and female cDNA probes. Twenty-five clones from each archive were selected ad libitum for sequence determination and homology analysis by BLAST online. Twenty of the 25 ESTs from the male cDNA library have a homology to the genes from C. elegans and 11 of the 19 ESTs from the female cDNA library have a homology to the genes from C. elegans