遗传 ›› 2010, Vol. 32 ›› Issue (2): 148-152.doi: 10.3724/SP.J.1005.2010.00148

• 研究报告 • 上一篇    下一篇

利用混合样本池法对鸡显性白羽基因PMEL17突变位点的检测

刘文博, 陈思睿, 郑江霞, 徐桂云, 李俊英, 曲鲁江, 杨宁   

  1. 中国农业大学动物科技学院, 北京 100193
  • 收稿日期:2009-08-13 修回日期:2009-12-10 出版日期:2010-02-20 发布日期:2010-01-15
  • 通讯作者: 杨宁 E-mail:nyang@cau.edu.cn
  • 基金资助:

    国家高技术研究发展计划(863计划)项目(编号:2006AA10A121)和国家重点基础研究发展计划(973计划)项目(编号:2006CB102102)资助

Detection of chicken dominant white gene PMEL17 mutation site by a pooling method

LIU Wen-Bo, CHEN Si-Rui, ZHENG Jiang-Xia, XU Gui-Yun, LI Jun-Ying, QU Lu-Jiang, YANG Ning   

  1. College of Animal Science & Technology, China Agricultural University, Beijing 100193, China
  • Received:2009-08-13 Revised:2009-12-10 Online:2010-02-20 Published:2010-01-15
  • Contact: YANG Ning E-mail:nyang@cau.edu.cn

摘要:

显性白羽基因座是影响鸡羽色形成的重要基因座位之一, 该基因座上的显性等位基因I 会抑制黑色素合成, 从而使携带该基因的个体全身羽毛呈现白色。目前已确认鸡显性白羽基因座编码PMEL17蛋白: 是一种黑素细胞特异性蛋白, 在黑素细胞的分化与成熟中起到重要作用, 并证明PMEL17基因的突变与显性白羽的形成有关。文章利用混合样本池建立了一种低成本、高效率, 并能在大规模群体中检测PMEL17基因突变的方法, 称为PCR产物混合样本池法。该方法的基本步骤如下: 首先, 提取个体基因组DNA, 并设计相关引物对每一个体单独进行PCR扩增; 其次, 将PCR产物等比例混合, 10个样品混在一个池中; 然后, 将PCR产物混合池样品于非变性聚丙烯酰胺凝胶上进行电泳; 最后, 待电泳结束后进行银染, 根据凝胶上所显条带判定是否存在突变体。此外, 文章还将这种方法与传统基因组DNA混合样本池法进行了比较试验, 并利用该方法对试验鸡群显性白羽基因PMEL17突变进行检测, 证实该方法具有较高准确度。

关键词: 混合样本池法, 凝胶电泳, 显性白羽, PMEL17

Abstract:

Dominant white locus is one of the major loci affecting feather color in the domestic chicken and its dominant allele I can inhibit the synthesis of the melanin. Therefore, the homozygotes (I/I) or heterozygotes (I/i) show a white phenotype. It has been confirmed that the Dominant white locus encodes PMEL17 protein which is a specific protein and plays a key role in the development of melanocytes, thus PMEL17 gene is identified as a positional candidate gene for the dominant white phenotype in chicken. In our present study, we created an economic and efficient pooling method for detecting PMEL17 mutations in large populations, known as PCR product pooling method, and the steps are as follows: firstly, PMEL17 segments containing the mutation site from individual genomic DNA samples were amplified by PCR; secondly, 10 PCR products were mixed in a pool, and then the pooled PCR samples were separated on non-denatured PAGE gels; and finally, the mutation profile of PMEL17 in certain populations were analyzed. In addition, a comparative study between the genomic DNA pooling and the PCR product pooling method was performed, and the mutation of PMEL17 was also ana-lyzed in our experimental population. In conclusion, the PCR product pooling method proved to be appropriate power to test gene mutations.

Key words: dominant white, PMEL17, polling method, electrophoresis