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作物学报 ›› 2009, Vol. 35 ›› Issue (4): 602-607.doi: 10.3724/SP.J.1006.2009.00602

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

中棉所36均一化全长cDNA文库的构建与鉴定

吴东，刘俊杰，喻树迅，范术丽，宋美珍   

  1. 中国农业科学院棉花研究所/农业部棉花遗传改良重点实验室，河南安阳455000
  • 收稿日期:2008-09-01 修回日期:2008-12-13 出版日期:2009-04-12 网络出版日期:2009-01-16
  • 通讯作者: 喻树迅 E-mail:yu@cricaas.com.cn Tel:0372-2562201
  • 基金资助:

    本研究由国家高技术研究发展计划（863计划）项目（2006AA10A109）资助。

Establishment and Identification of a Normalized Full-length cDNA Library of CCRI36

WU Dong,LIU Jun-Jie**,YU Shu-Xun*,FAN Shu-Li,SONG Mei-Zhen   

  1. Cotton Research Institute of Chinese Academy of Agricultural Sciences/Key Laboratory of Cotton Genetic Improvement, Ministry of Agriculture,Anyang 455000,China
  • Received:2008-09-01 Revised:2008-12-13 Published:2009-04-12 Published online:2009-01-16
  • Contact: YU Shu-Xun E-mail:yu@cricaas.com.cn Tel:0372-2562201

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2016

被引次数

29

摘要：

以短季棉中棉所36(CCRI36)顶端分生组织和花蕾为材料, 以DSN(duplex-specific nuclease)均一化技术与SMART(switching mechanism at 5¢ end of RNA transcript)建库技术相结合, 构建CCRI 36花发育期均一化全长cDNA文库。经检测原始文库滴度为1.7×106 cfu mL-1。随机挑取100个克隆, 利用PCR方法测得文库重组率达100%, 插入片段平均长度为1.2 kb。以两个高丰度表达基因Histon3和UBQ7为探针, 进行虚拟Northern blot检测显示, 其丰度在均一化cDNA中均明显降低, 说明均一化效果显著, 为节约筛库成本和EST有效测序奠定了基础；同时, 利用常规PCR扩增技术从cDNA文库中筛选阳性信号, 获得了花发育相关蛋白基因。初步证实CCRI36花发育期均一化全长cDNA文库构建成功, 为深入研究棉花花发育机理及发掘与早熟相关的功能基因奠定了基础。

关键词: 花发育, 均一化, cDNA文库

Abstract:

Short season cotton (SSC) breeding plays an important role in solving the competition for more growing area between cotton and food crops. To explore the premature mechanism of SSC, to clone genes that have a close relation with premature flowering, and to speed process of premature cultivar breeding, we established a normalized full-length cDNA library using the flower and bud of CCRI 36 by DSN (duplex-specific nuclease)-normalization method combined with SMART (switching mechanism at 5' end of RNA transcript) technique. The titer of un-amplified cDNA library was about 1.7×106 cfu mL-1. The average size of cDNA inserts was 1 200 bp with a recombination rate of 100%. The abundance of transcripts Histon3 and UBQ7 decreased obviously in normalized cDNA library comparing with that in non-normalized samples detected by Virtual Northern Blot. Meanwhile, protein genes associated with flower development were obtained on the basis of the positive signal of cDNA library by PCR. These results indicated that the normalized full-length cDNA library has been established successfully, which is convenient for further study on the molecular mechanism and gene cloning of flower development.

Key words: Flower development, Normalization, cDNA Library

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