Cloning of GmDFR gene from Soybean (Glycine max) and Identification of Its Function on Resistance to Iron Deficiency
SHI Zhuo1, LI Hong1, GAO Ming1, GUO Chang-Hong1, GUO Dong-Lin1,*, BI Ying-Dong2,*
1 College of Life Science and Technology/Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province, Harbin Normal University, Harbin 150025, China; 2 Tillage and Cultivation Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
Abstract：Dihydroflavonol-4-reductase (DFR) plays a key catalytic role in anthocyanin synthesis. Anthocyanin can protect tissues from oxidative damage induced by abiotic stress, and can also be used as chelating agents to promote plant iron absorption. In this study, GmDFR gene (GenBank No. MN547961) and its promoter pGmDFR fragment (GenBank No. MW455109) were cloned from soybean (Glycine max). The CDS length of GmDFR gene was 1 020 bp and encoded 339 amino acids. GmDFR had typical PLN02650 and WcaG superfamily domains, belongs to dihydroflavonol 4-reductase and nucleoside diphosphate sugar isomerase family, GmDFR protein was not a transmembrane protein. Phylogenetic analysis showed that soybean GmDFR was most closely related to wild soybean (G. soja) GsDFR, with a similarity of 99.12%. Semi-quantitative RT-PCR detection showed that GmDFR was expressed in soybean tissues and the highest expression level was in roots. The pGmDFR promoter fragment was obtained by cloning, which contained multiple hormone and stress response elements. Under iron deficiency, the anthocyanin content and chlorophyll content of GmDFR transgenic tobacco (Nicotiana tabacum) were significantly higher than that of WT (P<0.05); the contents of malondialdehyde, hydrogen peroxide and superoxide anion were significantly lower than that of WT (P<0.05); and the activities of antioxidant enzymes (SOD, POD and CAT) were significantly higher than that of WT (P<0.05). These results indicated that transgenic GmDFR tobacco had certain ability to resist iron deficiency. This study provides scientific basis for the application of GmDFR gene in soybean resistance to iron deficiency, and also provides new gene resources for soybean molecular breeding.
石卓, 李洪, 高铭, 郭长虹, 郭东林, 毕影东. 大豆GmDFR基因克隆及其抵御缺铁胁迫功能的鉴定[J]. 农业生物技术学报, 2023, 31(2): 259-272.
SHI Zhuo, LI Hong, GAO Ming, GUO Chang-Hong, GUO Dong-Lin, BI Ying-Dong. Cloning of GmDFR gene from Soybean (Glycine max) and Identification of Its Function on Resistance to Iron Deficiency. 农业生物技术学报, 2023, 31(2): 259-272.
[1] 董洁, 王学敏, 王赞, 等. 2012. 紫花苜蓿二氢黄酮醇还原酶基因(MsDFR)的克隆与分析[J]. 草业学报, 21(2): 123-132. (Dong J, Wang X M, Wang Z, et al.2012. Cloning and analysis of dihydroflavonol reductase (DFR) gene from Medicago sativa[J]. Acta Agriculturae Sinica, 21(2): 123-132.) [2] 董志强, 张丽华, 裴翠娟, 等. 2011. 石灰性土壤大豆植株铁营养障碍解除技术研究[J]. 华北农学报, 26(S2): 122-124. (Dong Z Q, Zhang L H, Pei C J, ZHAO S J. Jia X L, Zhang M C.2011. Study on technique of relieving lack iron nutrition for soybean plants in calcareous soil[J]. Acta Agriculturae Boreali-Sinica, 26(S2): 122-124.) [3] 高铭, 周思莹, 李钧儒, 等. 2021. 紫花苜蓿MsMATE1基因克隆及其抵御铁胁迫功能的鉴定[J]. 植物遗传资源学报, 22(2): 512-520. (Gao M, Zhou S Y, Li J R, et al.2021. Cloning of MsMATE1 gene from Alfalfa and identification of its resistance to iron stress[J]. Journal of Plant Genetic Resources, 22(2): 512-520.) [4] 郭凤丹, 夏晗, 袁美, 等. 2011. 花生二氢黄酮醇还原酶基因(DFR)的克隆及表达分析[J]. 农业生物技术学报, 19(5): 816-822. (Guo F D, Xia H, Yuan M, et al.2011.Cloning and expression analysis of dihydroflavonol reductase gene (DFR) in peanut[J]. Chinese Journal of Agricultural Technology, 19(5): 816-822.) [5] 贺晓岚, 王建伟, 李文旭, 等. 2016. 大赖草6-SFT基因的克隆及其转基因烟草抗旱和抗寒性分析[J]. 作物学报, 42(3): 389-398. (He X L, Wang J W, Li W X, et al.2016. Cloning of Leymus herba 6-SFT gene and analysis of drought and cold resistance of transgenic tobacco[J]. Acta Agronomica Sinica, 42(3): 389-398.) [6] 焦淑珍, 刘雅莉, 娄倩, 等. 2014. 葡萄风信子二氢黄酮醇4-还原酶基因(DFR)的克隆与表达分析[J]. 农业生物技术学报, 22(05): 529-540. (Jiao S Z, Liu Y L, Lou Q, et al.2014. Cloning and expression analysis of dihydroflavanol 4-reductase gene from grape hyacinth[J]. Journal of Agricultural Biotechnology, 22(05): 529-540.) [7] 李傲辰. 2020. 大豆的主要营养成分及营养价值研究进展[J]. 现代农业科技, 23(3): 213-214. (Li A C.2020. Main nutrientional components and values of soybean[J]. Modern Agricultural Science and Technology, 23(3): 213-314.) [8] 李利敏, 吴良欢, 马国瑞. 2010. 植物吸收铁机理及其相关基因研究进展[J]. 土壤通报, 41(04): 994-999. (Li L M, Wu L H, Ma G R.2010. Research progress on the mechanism of iron uptake by plants and related genes[J]. Chinese Journal of Soil Science, 41(04): 994-999.) [9] 刘恺媛, 王茂良, 辛海波, 等. 2021. 植物花青素合成与调控研究进展. 中国农学通报, 37(14): 41-51. (Liu K Y, Wang M L, Xin H B, et al.2021. Advances in synthesis and regulation of anthocyanins in plants[J]. Chinese Agricultural Science Bulletin, 37(14): 41-51.) [10] 汤章城. 1999. 现代植物生理学实验指南[M]. 科学出版社, 北京. pp. 127-170. (Tang Z C.1999. Experimental Guide to Modern Plant Physiology[M]. Science Press, Beijing. pp. 127-170.) [11] 姚红, 周平, 范雨昕, 等. 2019. 中国水仙DFR基因启动子的克隆及功能[J]. 应用与环境生物学报, 25(4): 993-998. (Yao H, Zhou P, Fan Y X, et al.2019. Molecular cloning and functional characterization of the DFR gene promoter in Chinese daffodils[J]. Chinese Journal of Applied & Environmental Biology, 25(4): 993-998.) [12] 姚攀锋. 2016. 苦荞WD40转录因子的基因克隆及其对花青素合成的影响[D]. 硕士学位论文, 四川农业大学, 导师: 吴琦, pp. 1. (Yao P F.2016. Characterzation of tartary buckwheat WD40 transcription factor and its regulation of anthocyanin biosynthesis[D]. Thesis for M.S., Sichuan Agricultural University, Supervisor: Wu Q, pp. 1.) [13] Aebi H.1984. Catalase in vitro[J]. Methods in Enzymology, 105(2): 121-126. [14] Buchweitz M, Gudi G, Carle R, et al.2012. Systematic investigations of anthocyanin-metal interactions by raman spectroscopy[J]. Journal of Raman Spectroscopy, 43(12): 2001-2007. [15] Dai L P, Dong X J, Ma H H.2012. Antioxidative and chelating properties of anthocyanins in Azolla imbricata induced by cadmium[J]. Polish Journal of Environmental Studies, 21(4): 837-844. [16] Dhindsa R S, Wandekayi M.1981. Drought tolerance in two mosses: Correlated with enzymatic defence against lipid peroxidation[J]. Journal of Experimental Botany, 32(126): 79-91. [17] Esmaeilzadeh-Salestani K, Riahi-Madvar A, Maziyar M A.2014. Antioxidant enzymes activity and anthocyanin content in Fe2+-treated lemon balm seedlings[J]. International Journal of Farming and Allied Sciences, 3(5): 562-565. [18] Esparza I, Salinas I, Caballero I, et al.2004. Evolution of metal and polyphenol content over a 1-year period of vinification: Sample fractionation and correlation between metals and anthocyanins[J]. Analytica Chimica Acta, 524(12): 215-224. [19] Feng Z, Ding C Q, Li W H, et al.2020. Applications of metabolomics in the research of soybean plant under abiotic stress[J]. Food Chemistry, 310(25): 125914. [20] Fu D W, Zhang J C, Wang D C, et al.2020. Molecular cloning, functional characterization and transcriptional regulation of the promoter of dihydroflavonal-4-reductase gene from purple-fleshed sweet potato[J]. South African Journal of Botany, 131: 206-213. [21] Johnson E T, Ryu S, Yi H, et al.2001. Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase[J]. The Plant Journal, 25(3): 325-333. [22] Jolley V D, Brown J C, Davis T D.1986.Increased Fe-efficiency in soybeans through plant breeding related to increased response to Fe-deficiency stress. Ⅱ. Mineral nutrition[J]. Journal of Plant Nutrition, 9(3): 387-396. [23] Katsu K, Suzuki R, Tsuchiya W, et al.2017. A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity[J]. BMC Plant Biology, 17(1): 239-253. [24] Kohno Y, Kato Y, Shibata M, et al.2015. Enhanced stability of natural anthocyanin incorporated in Fe-containing mesoporous silica[J]. Microporous Mesoporous Materials, 203: 232-237. [25] Kumar V, Nadda G, Kumar S, et al.2013. Transgenic tobacco overexpressing tea cdna encoding dihydroflavonol 4-reductase and anthocyanidin reductase induces early flowering and provides biotic stress tolerance[J]. PLOS ONE, 8(6): e65535. [26] Li G, Zhao J, Qin B Y, et al.2019. ABA mediates development-dependent anthocyanin biosynthesis and fruit coloration in Lycium plants[J]. BMC Plant Biology, 19(1): 317. [27] Li H, Qiu J, Chen F, et al.2012. Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa[J]. Molecular Biology Reports, 39(3): 2991-2999. [28] Long T A, Tsukagoshi H, Busch W, et al.2010. The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots[J]. Plant Cell, 22(7): 2219-2236. [29] O'Reilly C, Shepherd N S, Pereira A.1985. Molecular cloning of the a1 locus of Zea mays using the transposable elements En and Mu1[J]. Embo Journal, 4(4): 877-882. [30] Posmyk M M, Kontek R, Janas K M.2009. Antioxidant enzymes activity and phenolic compounds content in red cabbage seedlings exposed to copper stress[J]. Ecotoxicology and Environmental Safety, 72(2): 596-602. [31] Shen X, Zhao K, Liu L, et al.2014. A role for PacMYBA in ABA-regulated anthocyanin biosynthesis in Red-Colored Sweet Cherry cv. Hong Deng (Prunus avium L)[J]. Plant and Cell Physiology, 55(5): 862-880. [32] Shi J, Fu X Z, Peng T, et al.2010. Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response[J]. Tree Physiology, 30(7): 914-922. [33] Tang L K, Chu H, Yip W K, et al.2010. An anther-specific dihydroflavonol 4-reductase-like gene is essential for male fertility in Arabidopsis[J]. New Phytologist, 181(3): 576-587. [34] Varotto C, Maiwald D, Pesaresi P, et al.2002. The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana[J]. Plant Physiology, 129(1): 85-94. [35] Veljovic-Jovanovic S, Noctor G, Foyer C H.2002. Are leaf hydrogen peroxide concentrations commonly overestimated the potential influence of artefactual interference by tissue phenolics and ascorbate[J]. Plant Physiology Biochemistry, 40(6-8): 501-507. [36] Waters B M, Blevins D G, Eide D J.2002. Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition[J]. Plant Physiology, 129(1): 85-94. [37] Winkel-Shirley B.2002. Biosynthesis of flavonoids and effects of stress[J]. Current Opinion in Plant Biology[J]. 5(3): 218-223. [38] Xu, Z, Mahmood, K, Rothstein S J.2017. Ros induces anthocyanin production via late biosynthetic genes and anthocyanin deficiency confers the hypersensitivity to ROS-generating stresses in Arabidopsis[J]. Plant Cell Physiology, 58(8): 1364-1377. [39] Zhang F, Wan X Q, Zheng Y X, et al.2014. Physiological and related anthocyanin biosynthesis genes responses induced by cadmium stress in a new colored-leaf plant Quanhong poplar[J]. Agroforestry Systems, 88(2): 343-355. [40] Zhu Z, Wang H, Wang Y, et al.2015. Characterization of the cis elements in the proximal promoter regions of the anthocyanin pathway genes reveals a common regulatory logic that governs pathway regulation[J]. Journal of Experimental Botany, 66(13): 3775-3789.
