中国农业科学 ›› 2009, Vol. 42 ›› Issue (10): 3749-3754 .doi: 10.3864/j.issn.0578-1752.2009.10.0047

• 研究简报 • 上一篇    

绒山羊生长期次级毛囊cDNA消减文库的构建及其序列分析

席海燕,周欢敏,郑琰

  

  1. (内蒙古农业大学生物工程学院)
  • 收稿日期:2009-03-16 修回日期:2009-06-04 出版日期:2009-10-10 发布日期:2009-10-10
  • 通讯作者: 周欢敏

Construction and Analysis of Subtractive cDNA Library of Secondary Follicle in Anagen in Cashmere Goat

XI Hai-yan, ZHOU Huan-min, ZHENG Yan   

  1. (内蒙古农业大学生物工程学院)
  • Received:2009-03-16 Revised:2009-06-04 Online:2009-10-10 Published:2009-10-10
  • Contact: ZHOU Huan-min

摘要:

【目的】构建绒山羊生长期次级毛囊的消减文库,寻找绒毛生长的相关候选基因。【方法】以生长期次级毛囊cDNA为tester,休止期次级毛囊cDNA为driver,利用抑制性消减杂交技术构建生长期次级毛囊消减文库并进行生物信息学分析。【结果】构建了具有高消减效率的cDNA文库。随机挑取20个阳性克隆进行PCR扩增,插入片段长度主要分布在250~1 000 bp之间。挑取750个克隆进行测序,得到342条有效序列,平均长度596 bp。进行同源性分析,有298个与nucleotide数据库山羊及其它物种的已知序列同源。在EST数据库中找到38个相似EST序列和6个无同源性序列。对298个已知功能基因的ESTs进行分类,细胞分裂类为13个、细胞信号类为49个、细胞结构蛋白52个、细胞防御类21个、代谢类46个、基因/蛋白表达类37个、未分类的80个。【结论】应用SSH技术,构建了绒山羊生长期和休止期次级毛囊差异表达基因的cDNA文库,为进一步筛选绒毛生长相关的候选基因奠定了基础。

关键词: 绒山羊, 次级毛囊, 生长期, 休止期, 抑制性消减杂交, 序列分析

Abstract:

【Objective】 This study was carried out to construct suppression subtractive hybridization(SSH) library of secondary follicle in anagen and to search some candidate genes involved in cashmere development in Inner Mongolia cashmere goat. 【Method】 cDNA subtracted library was performed with cDNA from secondary follicle in anagen as the “tester” and cDNA from secondary follicle in telogen as the “driver”. The gene fragments were sequenced and analyzed by bioinformatics. 【Result】 The cDNA library was constructed successfully. Twenty positive clones were amplified by using nested PCR primer 1 and 2R, the size of inserts was 250-1 000 bp. Three hundred and fourty-two genes were obtained by DNA sequencing from 750 positive clones picked randomly. Their average length is 596 bp. After nucleotide blast homological analysis, 298 matched to known genes, 38 matched only to other ESTs in dbEST, and the remaining six showed no match to any ESTs or known genes. A total of 298 unique known genes were used to analyze the gene expression patterns. These were categorized into seven categories on the basis of gene function. They were divided into cell division (13), cell signaling/communication (49), cell structure/motility (52), cell/organism defense (21), metabolism (46), gene/protein expression (37), and unclassified (80). 【Conclusion】 The subtractive cDNA library of secondary follicles in anagen and telogen were obtained by SSH, and the information generated in this study had established a basis for screening candidate genes involved in cashmere development.

Key words: cashmere goat, secondary follicle, anagen, telogen, suppression subtractive hybridization, sequence analysis