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Construction and Analysis of Roots Suppression Subtraction Hybridization cDNA Library After Tobacco Topping

QI Yuan-cheng; MA Lei; WANG Fei-fei; LIU Wei-qun;   

  1. 1、College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002；
    2、College of Life Science, Henan Agricultural University, Zhengzhou 450002
  • Received:2010-07-18 Online:2011-04-02 Published:2010-10-20

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Abstract: 【Objective】 This study was carried out to construct suppression subtractive hybridization(SSH) library of tobacco roots after topping and to search some candidate genes involved in regulation of nicotine biosynthesis. 【Method】 An sSSH library was constructed using cDNA from control tobacco plants as driver and those from topped tobacco plants as tester. The positive clones were selected and further screened by reverse-northern blotting, and significantly differently expressed clones were sequenced and analyzed by bioinformatics. 【Result】 The SSH library was constructed successfully. The insert size of positive clones was 200-1 000 bp confirmed by PCR. After reverse-northern blotting further screening, 560 significantly differently expressed clones among 850 positive clones were acquired, sequenced and 273 high quality expressed sequence tags (ESTs) were acquired. The results of nucleotide blast homological analysis indicated that these ESTs mainly involved in alkaloid biosynthesis (4%), plant hormone metabolism (3%), signaling/transcription (18%), stress/defense (32%), protein metabolism (9%), carbon metabolism (6%), other metabolism (15%) and function unknown (13%). The RT-PCR result of NTAT84 and NTAT71 indicated that their transcription amount increased after tobacco topping. 【Conclusion】 The SSH library of tobacco roots after topping were well constructed, and thus a basis for screening candidate genes involved in regulation of nicotine biosynthesis has been established by the information generated in this study.

Key words: tobacco , nicotine , suppression subtraction hybridization , reverse-Northern blotting , sequence analysis , reverse- transcription PCR

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