Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (2): 325-333.doi: 10.3864/j.issn.0578-1752.2013.02.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Prokaryotic Expression of Flavonoid O-methyltransferase from Camellia sinensis

 MA  Cheng-Ying, 吕Hai-Peng , LIN  Zhi, ZHANG  Yue, GUO  Li, TAN  Jun-Feng   

  1. 1.Tea Research Institute, Chinese Academy of Agricultural Sciences/Engineering Research Center for Tea Processing,      Hangzhou 310008
    2.Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2012-08-21 Online:2013-01-15 Published:2012-12-27

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Abstract: 【Objective】Cloning, sequence analysis of the CsFOMT genes from Camellia sinensis and expression of recombinant proteins in Escherichia coli were conducted to further research the enzymatic characteristics and the metabolic mechanism of regulating the flavonoid in tea plant. 【Method】 The CsFOMT cDNA fragments were amplified from Camellia sinensis by reverse transcription PCR (RT-PCR) and RACE, and then the cloned genes of CsFOMT were inserted into vector pET-28a. The recombinant plasmids pET-28a-CsFOMT were expressed in the prokaryotic expression system after its transformation into E. coli BL21. 【Result】 The whole cDNA sequence was 1 246 bp which contains an ORF of 1 077 bp and encodes 358 amino acids. The putative protein of the gene had an isoelectric point of 5.62 and a calculated molecular weight of 40.2 kD. The coding nucleotide sequence of tea showed 80% and 81% identity with that of Vitis vinifera and Populus trichocarpa, respectively. Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG. 【Conclusion】In this study, a full length cDNA of flavonoid O-methyltransferase gene was obtained from Camellia sinensis by RT-PCR and RACE，and the prokaryotic expression vector for this gene was constructed, and then successfully induced to express by IPTG in BL21.

Key words: Camellia sinensis , flavonoid O-methyhransferase , cloning , prokaryotic expression

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