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首页> 中文期刊> 《中国农业科学》 >福美双诱发肉鸡胫骨软骨发育不良早期生长板消减cDNA文库的构建

福美双诱发肉鸡胫骨软骨发育不良早期生长板消减cDNA文库的构建

         
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摘要

[Objective] An experiment was conducted to construct subtracted cDNA library for selecting time series genes differentially expressed in the TD growth plate of broiler chickens at the early stage with cDNA microarray. [Method] AVIAN (AV) broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 48h to induce TD (thiram diet-fed). Forward and reverse-subtracted eDNA libraries were generated by suppression subtractive hybridization technology (SSH). Identification of the inserted cDNA fragments in subtractive library was done using PCR. One hundred clones were randomly selected for further DNA sequencing, blast homology analysis and function prediction. [Result] A total of 2 227 positive clones were obtained and the size of inserts was between 200 bp and 1 000 bp. There were 97 homologous gene sequences shared more than 99％ identity with genes known in chicken (Gallus gallus). Non-redundancy of sequenced genes was 68.7％. Meanwhile, 3 clones were found to be novel EST as no functional clues were associated with them by bioinformatic analysis. Most of these genes are involved in matrix formation,endochondral ossification and remodelling, developmental regulation, signal transduction, electron transport in mitochondrial respiratory chain and vascularization. [ Conclusion ] Successfully produced cDNA library would make a good foundation for further printing cDNA microarray, screening differential expression genes of TD growth plates at different stages, and also may provide new insights into understanding the pathogenesis of TD.%[目的]为应用cDNA芯片技术筛选肉鸡胫骨软骨发育不良(TD)时序性差异表达基因,本研究构建了早期生长板消减cDNA文库.[方法]将7日龄AV肉鸡随机分为两组,对照组(control,C)饲以基础日粮,试验组(thiram diet-fed,T)饲以基础日粮添加福美双100 mg·kg(-1),2 d后继续饲以墓础日粮,诱发肉鸡TD.应用抑制消减杂交技术(SSH)构建正反向消减cDNA文库,用PCR验证两个文库中克隆的插入片段,随机抽取100个阳性克隆测序,对所测序列同源性分析和功能预测.[结果]从两个文库共获得2 227个有效阳性克隆,插入片段大小主要分布在200--1 000bp之间;所测序列中有97条ESTs与GenBank中的鸡基因序列同源性达到99%,且非冗余度达到68.7%;此外,有3条ESTs未找到同源序列.这些基因具有构成细胞外基质,参与软骨内骨化和骨的重构,调节骨发育,行使信号转导、血管发生,参与电子传递及能量代谢等功能.[结论]成功构建了消减cDNA文库,将为进一步点制cDNA芯片,筛选不同阶段的差异表达基因奠定了基础,也为肉鸡TD机理研究提供新线索.

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  • 来源
    《中国农业科学》 |2011年第4期|829-834|共6页
  • 作者

    宁官保; 郭定宗; 田文霞; 王瑞; 覃平; 乔建钢; 李宏全; 李家奎; 毕丁仁; 潘思轶;

  • 作者单位

    山西农业大学动物科技学院;

    山西;

    太谷;

    030801;

    华中农业大学动物医学院;

    武汉;

    430070;

    山西农业大学动物科技学院;

    山西;

    太谷;

    030801;

    华中农业大学动物医学院;

    武汉;

    430070;

    华中农业大学食品科技学院;

    武汉;

    430070;

    华中农业大学动物医学院;

    武汉;

    430070;

    华中农业大学动物医学院;

    武汉;

    430070;

    山西农业大学动物科技学院;

    山西;

    太谷;

    030801;

    山西农业大学动物科技学院;

    山西;

    太谷;

    030801;

    华中农业大学动物医学院;

    武汉;

    430070;

    华中农业大学动物医学院农业微生物学国家重点实验室;

    武汉;

    430070;

    华中农业大学食品科技学院;

    武汉;

    430070;

  • 原文格式 PDF
  • 正文语种 chi
  • 中图分类
  • 关键词

    消减cDNA文库; 胫骨软骨发育不良; 福美双; 抑制消减杂交(SSH); 差异表达基因;

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