Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (22): 4524-4532.doi: 10.3864/j.issn.0578-1752.2014.22.018

• RESEARCH NOTES • Previous Articles     Next Articles

Cloning and Function Analysis of a MaDFR Gene from Mulberry

LI Jun1, 2, ZHAO Ai-chun1, UMUHOZA Diane1, WANG Xi-ling1, LIU Chang-ying1, LU Cheng1, YU Mao-de1   

  1. 1 College of Biotechnology of Southwest University/State Key Laboratory of Silkworm Genome Biology, Chongqing 400715
    2 Guiyang College of Traditional Chinese Medicine, Guiyang 550002
  • Received:2014-05-12 Revised:2014-07-24 Online:2014-11-16 Published:2014-11-16

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Abstract: 【Objective】In this study, MaDFR was isolated from mulberry (Morus alba L.) and transformed into tobacco for function analysis of flavonoids biosynthesis and providing application basement. 【Method】Fragment of MaDFR was obtained by RT-PCR and transformed into tobacco by using Agrobacterium tumerficians-mediated transformation approach. Plants with kanamycin resistance were obtained for identification by using genome PCR and insertion sites analysis by reverse PCR. The expression levels were determined by qRT-PCR. The concentration of total flavonoids was analyzed by AlCl3 and the antioxidant activity of acid hydrolysis and acid hydrolysis was showed by using DPPH scavenging ability. HPLC was used for identification of anthocyanins in tobacco. 【Result】The full-length CDS sequence of MaDFR obtabined from Zhongsang 5801 is 1026 bp in length encoding a putative protein about 341 amino acids with a typical NADPH binding site. A transgenic vector based on pBI121 was constructed and transformed into tobacco by Agrobacterium tumerficians-mediated transformation approach. Seven plants with kanamycin resistance were obtained. Four positive plants were identified by genome PCR and reverse PCR. In transgenic plants MaDFR showed different expression levels. There were significant increases of total flavonoids in transgenic plants, DFR-2(19.8%), DFR-4(30.4%), DFR-5(14.2%), and DFR-7 (27.6%), respectively. There was a large difference in the antioxidant activity between control and transgenic plants, the antioxidant activity of total flavonoids in DFR-2 and DFR-4 showed significant increase by 5 times, while the antioxidant activity of DFR-5 and DFR-7 showed no significant difference compared to control. The antioxidant activity of total flavonoids before acid hydrolysis showed a well correlation with the expression levels of MaDFR in tobacco. The antioxidant activity of acid hydrolysis of total flavonoids in transgenic plants showed a well correlation with the contents of total flavonoids. The overexpression of MaDFR improved the content of anthocyanins in transgenic tobacco corolla, DFR-4(15%), DFR-5(100%) and DFR-7 (100%), respectively, but could not change the components of anthocyanins.【Conclusion】The overexpression of MaDFR in tobacco increased the content and antioxidant activity of total flavonoids. MaDFR also deepened the color of tobacco corolla by improving the content of anthocyanins, and thus playing an important role in biosynthesis of anthocyanins, especially in cyanidin.

Key words: mulberry, DFR, tobacco, overexpression, total flavonoids, antioxidant

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