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

RichHTML

1

PDF

645

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

[1]    Chase M W. An update of the angiosperm phylogeny group classication for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society, 2009(161): 105-122.
[2]    刘学铭, 肖更生, 陈卫东. 桑椹的研究与开发进展. 中草药, 2001, 32(6): 569-571.
Liu X M, Xiao G S, Chen W D. Advances in research and development of mulberry. Chinese Traditional and Herbal Drugs, 2001, 32(6): 569-571. (in Chinese)
[3]    Konczak I, Zhang W. Anthocyanins-more than nature's colours. Journal of Biomedicine and Biotechnology, 2004, 5: 239-240.
[4]    Castañeda-Ovando A, Pacheco-Hernández M L, Páez-Hernández M E, Rodríguez J A, Galán-Vidal C A. Chemical studies of anthocyanins: A review. Food Chemistry, 2009, 113(4): 859-871.
[5]    Kose R, Verweij W, Quattrocchio F. Flavonoids: A colorful model for the regulation and evolution of biochemical pathways. Trends in Plant Science, 2005, 10(5): 236-242.
[6]    Clifford M N. Anthocyanins-nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture, 2000, 80(7): 1063-1072.
[7]    Jaakola L. New insights into the regulation of anthocyanin biosynthesis in fruits. Trends in Plant Science, 2013, 18(9): 477-483.
[8]    Liu X, Xiao G, Chen W, Xu Y, Wu J. Quantification and purification of mulberry anthocyanins with macroporous resins.  Journal of Biomedicine and Biotechnology, 2004(5): 326-331.
[9]   Wu X, Beecher G R, Holden J M, Haytowitz D B, Gebhardt S E, Prior R L. Concentrations of anthocyanins in common foods in the united states and estimation of normal consumption . Journal of Agricultural and Food Chemistry, 2006, 54(11): 4069-4075.
[10]   Zou T B, Wang M, Gan R Y, Ling W H. Optimization of ultrasound-assisted extraction of anthocyanins from mulberry, using response surface methodology. International Journal of Molecular Sciences, 2011, 12(5): 3006-3017.
[11]   Kang T H, Hur J Y, Kim H B, Ryu J H, Kim S Y. Neuroprotective effects of the cyanidin-3-O-beta-d-glucopyranoside isolated from mulberry fruit against cerebral ischemia. Neuroscience Letters, 2006, 391(3): 122-126.
[12]   Liu L K, Lee H J, Shih Y W, Chyau C C, Wang C J. Mulberry anthocyanin extracts inhibit LDL oxidation and macrophage-derived foam cell formation induced by oxidative LDL. Journal of Food Science, 2008, 73(6): 113-121.
[13]   Jeong J C, Jang S W, Kim T H, Kwon C H, Kim Y K. Mulberry fruit (Moris fructus) extracts induce human glioma cell death in vitro through ROS-dependent mitochondrial pathway and inhibits glioma tumor growth in vivo. Nutrition and Cancer, 2010, 62(3): 402-412.
[14]   Huang Y, Gou J, Jia Z, Yang L, Sun Y, Xiao X, Song F, Luo K. Molecular cloning and characterization of two genes encoding dihydroflavonol-4-reductase from Populus trichocarpa. PLoS One. 2012, 7(2): e30364.
[15]   Kumar V, Nadda G, Kumar S, Yadav S K. Transgenic tobacco overexpressing tea cDNA encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase induces early flowering and provides biotic stress tolerance. PLoS One. 2013, 8(6): e65535.
[16]   Aljanabi S M, Martinez I. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research, 1997, 25: 4692-4693. 
[17]   Bombarely A, Rosli H G, Vrebalov J, Moffett P, Mueller L A, Martin G B. A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research. Molecular Plant Microbe Interactions, 2012, 25: 1523-1530.
[18]   Chang C C, Yang M H, Wen H M, Chern J C. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 2002, 10: 178-182.
[19]   Han Y, Vimolmangkang S, Soria-Guerra R E, Korban S S. Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin. Journal of Experimental Botany, 2012, 63(7): 2437-2447.
[20]   Zou T B, Wang M, Gan R Y, Ling W H. Optimization of ultrasound- assisted extraction of anthocyanins from mulberry, using response surface methodology. International Journal of Molecular Sciences, 2011, 12(5): 3006-3017.
[21]   He N, Zhang C, Qi X, Zhao S, Tao Y, Yang G, Lee T H, Wang X et al. Draft genome sequence of the mulberry tree Morus notabilis. Nature Communication, 2013, 4: 2445.
[22]   Ochman H, Gerber A S, Hartl D L. Genetic applications of an inverse polymerase chain reaction. Genetics, 1988, 120(3): 621-623.
[23]   Li W, Zhu S L, Li N, Chen X Y. Characteristics of nucleotides and amino acids in cDNA sequence of xyloglucan endotransglycosylase cloned from Anthocephalus chinensis. Forestry Studies in China, 2011, 13(1): 45-51.
[24]   Allshire R C, Javerzat J P, Redhead N J, Cranston G. Position effect variegation at fission yeast centromeres. Cell, 1994, 76(1): 157-169.
[25]   李亚丽, 刘中来, 周洁, 徐国娟, 瞿绍洪. 转 mCherry 基因水稻的遗传分析及 T-DNA 整合位点的研究. 分子植物育种, 2012, 10(2): 121-130.
Li Y L, Liu Z L, Zhou J, Xu G J, Qu S H. Genetic analysis of mCherry transgenic rice and molecular characte-rization of T-DNA integration sites in the rice genome. Molecular Plant Breeding, 2012, 10(2): 121-130. (in Chinese)
[26]   Nishihara M, Nakatsuka T, Yamamura S. Flavonoid components and flower color change in transgenic tobacco plants by suppression of chalcone isomerase gene. FEBS Letters, 2005, 579(27): 6074-6078.
[27]   Wang C K, Chen P Y, Wang H M, To K Y. Cosuppression of tobacco chalcone synthase using Petunia chalcone synthase construct results in white flowers. Botanical Studies, 2006, 47: 71-82.
[28]   Cao G, Sofic E, Prior R L. Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationships. Free Radical Biology and Medicine, 1997, 22(5): 749-760.
[29]   刘微微, 任虹, 曹学丽, 徐春明, 王巧娥. 天然产物抗氧化活性体外评价方法研究进展, 食品科学, 2010, 31(17): 415-419.
Liu W W，Ren H，Cao X L，Xu C M，Wang Q E. Progress in evaluation techniques for antioxidant activity of natural products in vitro. Food Chemistry, 2010, 31(17): 415-419. (in Chinese)
[30]   Liggins J, Bluck L J, Coward W A, Bingham S A. Extraction and quantification of daidzein and genistein in food. Analytical Biochemistry, 1998, 264(1): 1-7.
[31]   Xie D Y, Sharma S B, Dixon R A. Anthocyanidin reductase from Medicago truncatula and Arabidopsis thaliana. Archives of Biochemistry and Biophysics, 2004, 422: 91-102.
[32]   Qin C, Li Y, Niu W, Ding Y, Zhang R, Shang X. Analysis and characterisation of anthocyanins in mulberry fruit . Czech Journal of Food Sciences, 2010, 28(2): 117-126.
[1] WANG YaFei, YAN Peng, XUE JinTao, DONG XueRui, MENG FanQi, GUO LiNa, LUO Yi, ZHANG Juan, DONG ZhiQiang, LU Lin. Effects of Ethephon-Glycine Betaine-Salicylic Acid Mixture on Root System Architecture, Physiological Function and Yield of Maize Under Heat Stress [J]. Scientia Agricultura Sinica, 2026, 59(7): 1439-1455.
[2] LI SaiYa, XU YaPing, ZHENG JiaXing, ZHENG Yang, YU JiaHui, LI YongQiang, PU ShouCheng, YANG Li, GUO WeiDong. Bioactive Polyphenolic Composition and Antioxidant Activities in Leaves of 34 Vaccinium Species [J]. Scientia Agricultura Sinica, 2026, 59(4): 874-886.
[3] YANG KeXin, ZHANG Yong, LI YanXiu, XIE SiYao, XUE Bo, YANG ShaoJie, SONG DeWei, MA Qiang, ZOU Ping, LI Yang, MA SiQi, JING ChangLiang. Effects of Alginate Oligosaccharides on Alleviating Atrazine Phytotoxicity in Tobacco [J]. Scientia Agricultura Sinica, 2026, 59(1): 101-113.
[4] CHEN GuiPing, LI Pan, SHAO GuanGui, WU XiaYu, YIN Wen, ZHAO LianHao, FAN ZhiLong, HU FaLong. The Regulatory Effect of Reduced Irrigation and Combined Organic- Inorganic Fertilizer Application on Stay-Green Characteristics in Silage Maize Leaves After Tasseling Stage [J]. Scientia Agricultura Sinica, 2025, 58(7): 1381-1396.
[5] ZHENG Yu, CHEN Yi, TI JinSong, SHI LongFei, XU XiaoBo, LI YuLin, GUO Rui. Evaluation of Carbon Footprint and Economic Benefit of Different Tobacco Rotation Patterns [J]. Scientia Agricultura Sinica, 2025, 58(4): 733-747.
[6] GUO AoLin, LIN JunXuan, LAI GongTi, HE LiYuan, CHE JianMei, PAN Ruo, YANG FangXue, HUANG YuJi, CHEN GuiXin, LAI ChengChun. Effect of VdF3′5′H2 Overexpression on the Accumulation of Anthocyanin Composition in Spine Grape Cells [J]. Scientia Agricultura Sinica, 2025, 58(4): 802-818.
[7] LÜ HuanHuan, LI RuYue, LIU QingSong, XU Lei, XU YanRan, YU HaoJie, GUO ChangHong, LONG RuiCai. Cloning and Salt Tolerance Function Analysis of MsKTI3 Gene in Alfalfa [J]. Scientia Agricultura Sinica, 2025, 58(21): 4497-4511.
[8] WU DongMing, XU Jing, YUAN JiaMei, WANG KeXuan, LI YuYi, HE Ping, ZHANG JianFeng, SONG DaLi, GAO Miao, ZHOU Wei. Isolation and Identification of Rhizosphere Growth-Promoting Bacteria of Myroides odoratimimus PJ-3 and Their Salt/ Alkali-Tolerance and Growth-Promoting Effects on Maize [J]. Scientia Agricultura Sinica, 2025, 58(20): 4131-4143.
[9] LUO ZiShu, ZHANG YiJia, ZHOU Rong, ZHANG YanXin, ZHOU Ting, YOU Jun, WANG LinHai. Characterization of Antioxidant Activities in Sesame Leaves and Screening of High Antioxidant Germplasm [J]. Scientia Agricultura Sinica, 2025, 58(19): 3814-3824.
[10] ZHANG Jie, HU ChenXi, QI JianBo, ZHANG YongTai, CHEN YiBo, ZHANG YongJi. Effects of Exogenous Zeatin on Photosynthetic Parameter, Antioxidant System and Expression of Genes Related to Zeatin Synthesis in Pepper Under Low-Temperature Combined with Low-Light Stress [J]. Scientia Agricultura Sinica, 2025, 58(19): 3959-3969.
[11] LI RuoHong, MA Wen, ZHAO ShiQiang, MAO PeiSheng. Antioxidant Physiology and Hormonal Response Pattern of Embryonic Root Mitochondria During Imbibition of Aging Seeds of Alfalfa [J]. Scientia Agricultura Sinica, 2025, 58(19): 4014-4025.
[12] CHEN JuanNi, CHEN PinLu, LI Yu, XIE MengXiao, LI XinBei, DING Wei. Mechanism of Tobacco Resistance to Bacterial Wilt Induced by Magnesium Oxide Nanoparticles [J]. Scientia Agricultura Sinica, 2025, 58(16): 3327-3344.
[13] YANG WenJuan, GAO JiaCheng, WANG YanTing, LI Yan, GUO Ming, WANG JunCheng, MENG YaXiong, WANG HuaJun, SI ErJing. Function of Effector Pg00778 Regulation on the Pathogenicity of Pyrenophora graminea to Barley [J]. Scientia Agricultura Sinica, 2025, 58(15): 3020-3035.
[14] ZHAO LinLin, HE YuXi, PENG JieLi, WANG Xu, MA Jia, ZHANG XiuMin, HU Dong. Streptomyces TOR3209 and Its Volatile Organic Compounds Enhance Tobacco Resistance to Fusarium equiseti [J]. Scientia Agricultura Sinica, 2025, 58(11): 2162-2175.
[15] PANG QiangQiang, SUN XiaoDong, CHEN YiSong, ZHOU Man, WANG YaQiang, SHI GuoBin. Heat Resistance Analysis of Meiosis Related Gene BrASY2 in Chinese Flowering Cabbage [J]. Scientia Agricultura Sinica, 2025, 58(11): 2239-2252.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd