半番鸭羽色相关基因均一化差减文库的构建和鉴定

doi:10.3864/j.issn.0578-1752.2012.22.015

中国农业科学 ›› 2012, Vol. 45 ›› Issue (22): 4688-4696.doi: 10.3864/j.issn.0578-1752.2012.22.015

半番鸭羽色相关基因均一化差减文库的构建和鉴定

郑嫩珠, 陈晓燕, 卢立志, 朱志明, 缪中纬, 辛清武, 陈晖, 肖天放

1.福建省农业科学院畜牧兽医研究所, 福州 350013
2.浙江省农业科学院畜牧兽医研究所，杭州 310021
3.福建农林大学动物科学学院，福州 350002

收稿日期:2012-04-19 出版日期:2012-11-15 发布日期:2012-08-27
通讯作者: 通信作者陈晖，Tel：0591-83815170；E-mail：chh5555@163.com。通信作者肖天放，Tel：0591-83789172；E-mail：xiaotf@163.com
作者简介:郑嫩珠，Tel：0591-83815170；E-mail：zhengnz@163.com
基金资助:
福建省自然基金项目（2010J01107）、福建省种业创新与产业化工程项目（2011FJZY-9-02）、现代农业产业技术体系建设专项（nycytx- 45-06）

Construction and Identification of Normalized Subtractive cDNA Library of Plumage Color-Related Genes in Mule Duck

ZHENG Nen-Zhu, CHEN Xiao-Yan, LU Li-Zhi, ZHU Zhi-Ming, MIAO Zhong-Wei, XIN Qing-Wu, CHEN Hui, XIAO Tian-Fang

1.Institute of Animal Science and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013
2.Institute of Animal Science and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
3.Institute of Animal Science, Fujian Agricultural and Forestry University, Fuzhou 350002

Received:2012-04-19 Online:2012-11-15 Published:2012-08-27

摘要/Abstract

摘要： 【目的】分离半番鸭羽色相关基因，探索半番鸭羽色基因表达调控的分子机制。【方法】以半番鸭白羽皮肤和黑羽皮肤分别作为试验组（tester）和驱动组（driver），利用双链特异性核酸酶（duplexspecific nuclease，DSN）介导的均一化消减杂交方法，构建半番鸭羽色相关基因消减cDNA文库，同时采用实时荧光定量PCR方法对文库质量进行验证。【结果】①从文库中随机选取144个阳性克隆进行测序分析，最终共获得64条有效序列，平均长度为1 031 bp；经BLAST比对发现，21条具有同源序列，且同源性平均为92.8%；43条未找到匹配序列，推测可能为羽色相关新基因；②Geneontology功能分析表明，已知基因参与信号转导、细胞结构、物质转运、细胞凋亡、细胞与机体防御、转录与表达调控等诸多生物学过程，并且与色素形成和转运存在不同程度的相关性。【结论】经实时荧光定量PCR鉴定后，所建文库质量良好，能够有效地富集白羽皮肤特异表达基因。

关键词: 半番鸭 , 羽色基因 , 双链特异性核酸酶（DSN） , 消减杂交 , 荧光定量PCR

Abstract: 【Objective】 The aim of this study was to isolate plumage color related-genes in mule duck and explore its molecular mechanism. 【Method】 A normalized subtractive library of plumage color related-genes of mule duck was constructed. White plumage skin and black plumage skin were adopted respectively as the tester and driver, and a duplex-specific nuclease (DSN) normalized subtractive hybridization method was used to construct the library, and real-time PCR was employed to identify the quality of the library.【Result】Results showed that 64 genes with the average size of 1 031 bp were obtained by selecting and sequencing 144 positive clones randomly. Nucleotide BLAST homological analysis incidated that 21 genes had similarities to known genes which enjoy 92.8% homology, and 43 genes were not matched which presumably may be new genes related to plumage color. Geneontology displayed that these known genes were involved in many biological processes such as signal transduction, cell structure, material transport, apoptosis, cell and organism defense, transcription and expression regulation，and had different correlations with formation and transshipment of pigment. 【Conclusion】 It was determined that the library had good quality and could enrich the genes of the white plumage via real-time PCR.

Key words: mule duck , plumage color gene , duplex-specific nuclease (DSN) , subtractive hybridization , real-time PCR

郑嫩珠, 陈晓燕, 卢立志, 朱志明, 缪中纬, 辛清武, 陈晖, 肖天放. 半番鸭羽色相关基因均一化差减文库的构建和鉴定[J]. 中国农业科学, 2012, 45(22): 4688-4696.

ZHENG Nen-Zhu, CHEN Xiao-Yan, LU Li-Zhi, ZHU Zhi-Ming, MIAO Zhong-Wei, XIN Qing-Wu, CHEN Hui, XIAO Tian-Fang. Construction and Identification of Normalized Subtractive cDNA Library of Plumage Color-Related Genes in Mule Duck[J]. Scientia Agricultura Sinica, 2012, 45(22): 4688-4696.

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半番鸭羽色相关基因均一化差减文库的构建和鉴定

Construction and Identification of Normalized Subtractive cDNA Library of Plumage Color-Related Genes in Mule Duck

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