杜洛克与梅山猪大卵泡消减文库的构建

为了鉴定杜洛克猪相对于梅山猪在大卵泡中高表达的基因,本研究应用抑制性消减杂交技术成功构建了以梅山大卵泡c DNA为driver,杜洛克大卵泡c DNA为tester的消减c DNA文库。结果显示:以G3PDH和β-actin为指标检测文库的消减效率为25,从该文库中获取了350个有效的阳性克隆,PCR检测插入片段主要分布在150-750 bp。克隆测序得到74个有效的EST序列,GO分析表明主要与细胞信号、细胞结构、代谢、细胞分化、基因/蛋白质合成、细胞组织防护等功能相关。利用q PCR技术验证了ELTD1、Grb14、SNRPE、CSDE1、ALDH18A1、e IF4E、BMPR-IB等基因在梅山和杜洛克大卵泡中的表达模式。结果发现在两猪种的大卵泡间存在显著性差异。本研究有助于揭示影响猪卵泡发育和生殖数量控制的分子基础。

Construction of Subtracted cDNA Library of Large Follicle Tissue of Duroc and Meishan Sows

By using suppression subtractive hybridization （SSH） technique,subtracted cDNA Library between large Follicles from Meishan and Duroc Pigs was constructed.A housekeeping gene G3PDH was used to estimate the efficiency of subtractive cDNA. In Duroc large follicle cDNA library,G3PDH was subtracted efficiently at appropriate 25 folds.A total of 350 positive clones were acquired from the subtracted cDNA library.PCR detection showed that most of the clones contained inserts of 150-750 bp. Seventy-four nun-redudent ESTs were identified by sequencing.GO analysis showed that the genes were related to the cell division,signal transduction, cell structure,metabolism or cell defense.To validate the results of SSH,some genes were selected for conformational ananlysis by using quantitative real-time PCR （qPCR）.All the genes were differential expression in two pigs.This study would be helpful to reveal the molecular basis of follicle development and fertility control.