应用抑制消减杂交分离雌、雄鸡胚性分化早期的差异表达基因  

Isolation of sexual differentially expressed genes in the chicken embryonic gonads during early sexual-differentiation period by using suppression subtractive hybridization

作　　者：俸艳萍[1] 彭秀丽[1] 李世军[1] 张淑君[1] 袁金锋[1] 杨永平[1] 詹慧琴[1] 龚炎长[1] 

机构地区：[1]华中农业大学动物科技学院农业动物遗传育种与繁殖教育部重点实验室,武汉430070

出　　处：《动物学报》2007年第2期315-324,共10页ACTA ZOOLOGICA SINICA

基　　金：湖北省杰出青年基金及国家自然科学基金(30671494)资助项目~~

摘　　要：分离不同性别的鸡胚性分化早期差异表达基因,可为禽类性别决定和性分化机制研究提供基本信息。本研究分别以孵化3.5-6d的雌、雄鸡胚性腺为材料,利用抑制性消减杂交技术成功构建了雌-雄鸡胚间正、反向消减cDNA文库,并利用斑点印迹杂交从中筛选出了39个性别差异表达的阳性cDNA克隆。以持家基因GAPDH为参照指标检测消减文库的消减效率,结果发现两个文库的消减效率均高达25倍。插入片段PCR鉴定结果显示,消减文库中cDNA插入片段的长度主要分布于250-750bp之间。分别对雌、雄鸡胚消减文库中的252和168个cDNA克隆进行斑点杂交筛选,再随机从两个消减文库中共抽取39个阳性差异表达克隆进行序列测定及序列比对分析。结果表明:这39个cDNA克隆分别代表了定位于鸡不同染色体上的18个已知功能的基因和11个假定基因;参照哺乳动物同源基因的功能,所得的18个已知差异基因可能参与多种生物反应过程。用半定量RT-PCR方法对雌、雄鸡胚消减文库中各5个基因的表达情况进行进一步验证,发现除雌性库中的一个基因外其它9个基因均有较明显的性别差异表达。Isolation of sexual differentially expressed genes in chicken embryonic gonads during early sexual-differentiation period could provide basic information for studies on the genetic mechanism of avian sex determination and sex differentiation. In present study, total RNAs from female and male chicken embryonic gonads at days 3.5 - 6 post incubation were used to construct, respectively, forward and reverse subtracted cDNA libraries by using the suppression subtractive hybridization, and 39 positive differentially expressed cDNA clones were isolated from the libraries by dot blotting. The subtractive efficiency of 2^5 folds was obtained in the two libraries by using the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase （GAPDH） as the reference. PGR analysis showed that most of the differentially expressed clones contained inserts of 250 - 750 bp. Dot blot analysis of 252 and 168 eDNA clones respectively from female and male cDNA libraries identified 39 differentially expressed clones. The sequence analysis of these 39 clones revealed that they represented 18 known genes and 11 putative genes respectively localized on different chromosomes. Based on the function of the homologous genes in mammals, 18 known genes identified here were predicted to involve in multiple biological processes. The expression of 10 genes, respectively, five from female subtracted library and five from male＇s, was further analyzed by semi-quantitative RT-PCR and results showed that nine of ten genes had the significant different expression between females and males with the exception of one gene from female cDNA library. The obtained sexual differentially expressed genes would provide the bases for further research on the regulation of gene expression during chicken gonadal development .

关 键 词：抑制消减杂交 斑点杂交 性别差异表达 鸡胚 

分 类 号：Q344[生物学—遗传学]