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园艺学报 ›› 2011, Vol. 38 ›› Issue (09): 1667-1674.

• 果树 • 上一篇    下一篇

香蕉根系均一化全长cDNA文库的构建和鉴定

王 卓1,4,殷晓敏1,王家保3,徐碧玉2,金志强1,2,*   

  1. 1中国热带农业科学院海口实验站,海口570102;2中国热带农业科学院热带生物技术研究所,农业部热带生物技术重点开放实验室,海口571101;3中国热带农业科学院环境与植物保护研究所,海南儋州571737;4海南大学,海口570228
  • 收稿日期:2011-04-13 修回日期:2011-09-02 出版日期:2011-09-25 发布日期:2011-09-25
  • 通讯作者: 金志强

Construction and Characterization of Normalized Full-length cDNA Library of Banana Roots

WANG Zhuo1,4,YIN Xiao-min1,WANG Jia-bao3,XU Bi-yu2,and JIN Zhi-qiang1,2,*   

  1. 1Haikou Experimental Station,Chinese Academy of Tropical Agricultural Sciences,Haikou 570102,China;2Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China;3Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences,Danzhou,Hainan 571737,China;4Department of Agriculture,Hainan University,Haikou 570228,China
  • Received:2011-04-13 Revised:2011-09-02 Online:2011-09-25 Published:2011-09-25
  • Contact: JIN Zhi-qiang

摘要: 以香蕉幼苗根系为材料,利用DSN(duplex-specific nuclease)均一化技术与SMART(switching mechanismat 5′ end of RNA transcript)技术相结合,构建香蕉根系均一化全长cDNA文库。经检测,初级文库滴度1.1 × 106 cfu · mL-1,库容5 × 106个独立克隆,重组率大于95%,插入片段平均长度大于1 kb,表明文库质量较好。随机挑取192个克隆进行EST(expressed sequence tags)测序,成功获得179个高质量EST,含5个conting和174个singlet,冗余度仅为2.35%。小规模测序拼接后获得145个unigenes,对unigenes分析结果表明文库的可靠性好。从序列比对结果中挑选3个与其它植物同源的抗逆基因,利用RT-PCR方法进行了干旱、低温及盐胁迫条件下基因表达分析,结果显示这3个基因在不同的逆境下差异表达。

关键词: 香蕉, 根系, 均一化, cDNA文库, 基因, 表达

Abstract: A normalized full-length cDNA library was constructed with the banana roots,by DSN(duplex-specific nuclease)normalization method combined with SMART(switching mechanism at 5′end of RNA transcript)technique. The titer of unamplified cDNA library was about 1.1 × 106 cfu · mL-1,the capacity was 5 × 106 clones,and the recombination ratio was more than 95%. The average insertion size was above 1 kb which suggested that the quality of the cDNA was better. Random selected 192 clones were sequenced and 179 high quality EST were obtained,which included 5 contings and 174 singlets. The redundancy was 2.35%. With small scale sequencing,145 unigenes were obtained. The analysis result indicated the library was efficient and reliable. Selecting 3 genes which is homological with other plants’ anti-abiotic stress genes from above sequences,they are expressed under drought,low temperature and salt tress using RT-PCR method. The result suggested that the 3 genes had different expression under different stresses.

Key words: banana, roots, normalized, cDNA library, gene, expression

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