马铃薯块茎低温反应抑制差减文库的构建及鉴定

doi:10.3969/j.issn.1008-0864.2010.06.12

›› 2010, Vol. 12 ›› Issue (6): 68-74.DOI: 10.3969/j.issn.1008-0864.2010.06.12

马铃薯块茎低温反应抑制差减文库的构建及鉴定

陈霞,宋波涛,杨建文,谢从华,柳俊

(华中农业大学国家蔬菜改良中心华中分中心, 教育部园艺植物生物学重点实验室, 湖北省马铃薯工程技术研究中心, 武汉 430070)

收稿日期:2010-07-06 修回日期:2010-07-29 出版日期:2010-12-15 发布日期:2010-11-09
通讯作者: 柳俊,教授,博士生导师,主要从事马铃薯细胞与分子生物学研究。E-mail:liujun@mail.hzau.edu.cn
作者简介:陈霞,博士研究生,研究方向为马铃薯分子生物学。E-mail:silly80@webmail.hzau.edu.cn。
基金资助:
国家自然科学基金(30571181);国家863计划项目(2006AA100107)资助。

Construction and Identification of Potato Tuber SSH Library Induced by Low Temperature

CHEN Xia, SONG Bo-tao, YANG Jian-wen, XIE Cong-hua, LIU Jun

(National Centre for Vegetable Improvement (Central China), Huazhong Agriculture University, Key Laboratory of
Horticultural Plant Biology, Ministry of Education, Potato Engineering and Technology Research Center of
Hubei Province, Wuhan 430070, China)

Received:2010-07-06 Revised:2010-07-29 Online:2010-12-15 Published:2010-11-09

摘要/Abstract

摘要：

本研究以4℃(Tester)和20℃(Driver)处理的马铃薯野生种Solanum berthaultii的无性系CW2-1块茎为材料,构建马铃薯块茎低温反应抑制差减文库以进一步筛选差异表达基因。通过PCR鉴定后,获得了含有2 112个阳性克隆的差减杂交文库。采用反向Northern方法对差减文库进行筛选鉴定,得到274个含有显著表达差异基因（P/N>3）的阳性克隆。从274个克隆中挑选了53个克隆进行测序和同源比对分析,合并后获得29个代表不同基因的非重复序列片段,表明文库具有较高质量。并随机选取了其中4个EST进行基因表达分析,结果显示,在耐低温糖化的S. berthaultii块茎中,4℃下贮藏的块茎基因表达强度高于20℃贮藏的块茎；同在4℃贮藏条件下,S. berthaultii和易低温糖化栽培种E1的块茎中基因的表达量亦存在差异,证明所构建的差减文库富集了马铃薯块茎对低温响应的基因。

关键词: 马铃薯;差减杂交库;低温

Abstract:

A SSH (suppression subtractive hybridization) library was constructed to identify the genes of potato tubers response to low temperature, with wild species S. berthaultii CW2-1 tubers as material, which was treated at 4℃ (tester) and 20℃ (driver), respectively. A library including 2 112 positive clones was constructed by PCR identification. 274 differentially expressed fragments (P/N>3) were obtained through Northern blotting. Then 53 fragments were selected randomly for sequencing and homologous analysis. And 29 non-redundant gene fragments were acquired, which showed high quality of the library. Among them 4 ESTs (expression sequence tags) were selected randomly for further analysis, The results showed that the expression levels in the tubers of CW2-1 were higher at 4 ℃ than at 20℃ by semi-quantitative reverse transcription PCR; while stored at 4℃, their expression levels were also different between CW2-1 and E1 (E-potato 1, a potato cultivar sensitive to cold sweetening). This indicated that some cold-responsive genes in potato tuber were enriched by the SSH library constructed.

Key words: potato, SSH library, low temperature induction

中图分类号:

Q786

陈霞,宋波涛,杨建文,谢从华,柳俊. 马铃薯块茎低温反应抑制差减文库的构建及鉴定[J]. , 2010, 12(6): 68-74.

CHEN Xia, SONG Bo-tao, YANG Jian-wen, XIE Cong-hua, LIU Jun. Construction and Identification of Potato Tuber SSH Library Induced by Low Temperature[J]. , 2010, 12(6): 68-74.

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马铃薯块茎低温反应抑制差减文库的构建及鉴定

Construction and Identification of Potato Tuber SSH Library Induced by Low Temperature

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