蓝光对龙眼细胞培养及类黄酮代谢的影响
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摘要
采用搅拌式生物反应器放大培养龙眼悬浮细胞,探讨蓝光对龙眼细胞生长及类黄酮积累的影响。基于已建立并优化的龙眼细胞悬浮培养体系,首先研究龙眼细胞在黑暗和蓝光的培养过程中,细胞生长量、类黄酮含量、细胞活力、培养液的底物消耗量等的变化情况。结果发现:龙眼细胞培养9 d后,蓝光的细胞干重比黑暗增长了0.28 g/L,类黄酮含量增长了0.77 mg/g。细胞培养前期蓝光的培养液蔗糖消耗速度慢于黑暗培养,此后蔗糖含量均稳定在2 g/L。培养过程中,蓝光培养的还原糖含量均高于黑暗培养,蓝光的磷酸盐的消耗量基本大于黑暗培养。其次,通过qPCR技术分析光信号转录因子DlHY5、调控基因DlPAP1及类黄酮途径合成基因DlCHS的表达差异。结果表明蓝光可能通过光信号转录因子DlHY5调控基因DlPAP1的表达,进而调控龙眼类黄酮代谢途径合成基因DlCHS的表达,从而导致类黄酮的积累。
The effects of blue light on the growth of longan cells and flavonoid accumulation were investigated using a stirred bioreactor with enlarged culture of longan cells in this study. Based on the established and optimized suspension culture system of longan cells, the changes of the growth of cells, flavonoid contents, cell vitality, substrate consumption and so on were firstly studied in the dark and blue light treatments. Results found that the dry weight of blue light increased by 0.28 g/L and the flavonoid contents increased by 0.77 mg/g after 9 days culture. The sucrose consumption under blue light was slower than that under the darkness in the early stages of culture, and then the sucrose contents were stable at 2 g/L. In the process of culture, the contents of reducing sugar under blue light was higher than that under darkness, and the phosphate consumption under blue light was more than that under darkness. Then, the expression of light signal transcription factor DlHY5, regulatory gene DlPAP1 and flavonoid biosynthesis pathway structural gene DlCHS were analyzed by qPCR. Results indicated that the regulation of flavonoid accumulation in longan by blue light is probably through the light signal transcription factor DlHY5, and then regulated the expression of flavonoid biosynthesis pathway regulatory gene DlPAP1 and structural genes DlCHS.
The effects of blue light on the growth of longan cells and flavonoid accumulation were investigated using a stirred bioreactor with enlarged culture of longan cells in this study. Based on the established and optimized suspension culture system of longan cells, the changes of the growth of cells, flavonoid contents, cell vitality, substrate consumption and so on were firstly studied in the dark and blue light treatments. Results found that the dry weight of blue light increased by 0.28 g/L and the flavonoid contents increased by 0.77 mg/g after 9 days culture. The sucrose consumption under blue light was slower than that under the darkness in the early stages of culture, and then the sucrose contents were stable at 2 g/L. In the process of culture, the contents of reducing sugar under blue light was higher than that under darkness, and the phosphate consumption under blue light was more than that under darkness. Then, the expression of light signal transcription factor DlHY5, regulatory gene DlPAP1 and flavonoid biosynthesis pathway structural gene DlCHS were analyzed by qPCR. Results indicated that the regulation of flavonoid accumulation in longan by blue light is probably through the light signal transcription factor DlHY5, and then regulated the expression of flavonoid biosynthesis pathway regulatory gene DlPAP1 and structural genes DlCHS.
引文
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[8]钟春水,赖瑞联,刘生财,等.光源或培养基成分对金花茶愈伤组织中DFR、LAR与PPO基因表达及总儿茶素含量的影响[J].广西植物,2016,36(12):1 410-1 415.
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[12]赖钟雄,陈振光.龙眼胚性细胞悬浮培养再生植株[J].应用与环境生物学报,2002,8(3):485-491.
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[14]李琨,张学杰,张德纯,等.不同芹菜品种叶与叶柄黄酮含量及其与抗氧化能力的关系[J].园艺学报,2011,38(1):69-76.
[15]张建文,刘禹,夏建业,等.机械搅拌生物反应器的CFD模拟及剪切力对红花细胞悬浮培养的影响[J].华东理工大学学报(自然科学版),2016,42(3):492-498.
[16]单会娇,王冰.龙葵药材中可溶性糖的动态积累[J].中国实验方剂学杂志,2010,16(9):100-102.
[17]赵凯,许鹏举,谷广烨.3,5-二硝基水杨酸比色法测定还原糖含量的研究[J].食品科学,2008,29(3):534-536.
[18]李永远,杨坤朋.水中总磷酸盐(Phosphate salt)的测定方法[J].中国化工贸易,2013(3):212.
[19]Lin Y L,Lai Z X.Reference gene selection for q PCR analysis during somatic embryogenesis in longan tree[J].Plant Science,2010,178(2):359-365.
[20]Meijer J J,Hoopen H J G T,Luyben K C A M,et al.Effects of hydrodynamic stress on cultured plant cells:A literature survey[J].Enzyme&Microbial Technology,1993,15(3):234-238.
[21]侯学文,郭勇.不同碳源对悬浮培养玫瑰茄细胞呼吸强度及产生辅酶Q的影响[J].广西植物,1999,19(1):150-153.
[22]Pratt J,Boisson A M,Gout E,et al.Phosphate(Pi)starvation effect on the cytosolic Pi concentration and Pi exchanges across the tonoplast in plant cells:an in vivo 31P-nuclear magnetic resonance study using methylphosphonate as a Pi analog[J].Plant Physiology,2009,151(3):1 646-1 657.
[23]李雅丽,孟婷婷,王毛毛,等.甘草细胞在搅拌式生物反应器中的放大培养[J].植物生理学报,2015(3):302-306.
[24]Yu X,Liu H,Klejnot J,et al.The CRY blue light receptors[J].Arabidopsis Book,2010,8:e0135.
[25]于丹,朴炫春,李阳,等.利用生物反应器大量生产东北刺人参不定根的研究[J].中国农学通报,2014,30(16):252-255.
[26]高慧君.利用柑橘愈伤组织研究植物类胡萝卜素积累的调控机理[D].武汉:华中农业大学,2013.
[27]王娟,高文远,黄滔,等.培养条件对西洋参悬浮细胞生物量和活性成分的影响[J].中国中药杂志,2009,34(4):375-378.
[28]Dan Q U,Wang H M,Ren J.Effects of sugar content on cell growth,accumulation of rosemary acid,and antioxidative enzyme activity in suspension cells of rosemary[J].Bulletin of Botanical Research,2015.
[29]Hummel M,Rahmani F,Smeekens S,et al.Sucrose-mediated translational control[J].Annals of Botany,2009,104(1):1-7.
[30]Zheng Y,Li T,Liu H,et al.Sugars induce anthocyanin accumulation and flavanone 3-hydroxylase expression in grape berries[J].Plant Growth Regulation,2009,58(3):251-260.
[31]Price J,Laxmi A,St Martin SK,et al.Global transcription profiling reveals multiple sugar signal transduction mechanisms in Arabidopsis[J].Plant Cell,2004,16(8):2 128-2 150.
[32]Solfanelli C,Perata P.Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis[J].Plant Physiology,2006,140(2):637.
[33]刘忠娟.PIFs在糖调节植物生长代谢中的作用机理研究[D].兰州:兰州大学,2011.
[34]程华,余龙江.不同因素对岩黄连悬浮细胞生长的影响[J].生物技术,2007,17(3):56-59.
[35]王静,王渭玲,徐福利,等.氮磷钾对桔梗生长及次生代谢产物的影响[J].草业科学,2012,29(4):586-591.
[36]Jiao Y,Lau O S,Deng XW.Light-regulated transcriptional networks in higher plants[J].Nature Reviews Genetics,2007,8(3):217-230.
[37]Lee J,He K,Stolc V,et al.Analysis of transcriptional factor HY5genomic binding sites revealed its hierarchical role in light regulation of development[J].Plant Cell,2007,19(4):731-749.
[38]Stracke R,Favory J J,Gruber H,et al.The Arabidopsis b ZIP transcription factor HY5 regulates expression of the PFG1/MYB12gene in response to light and ultraviolet-B radiation[J].Plant Cell&Environment,2010,33(1):88.
[39]Maier A,Schrader A,Kokkelink L,et al.Light and the E3ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis[J].Plant Journal for Cell&Molecular Biology,2013,74(4):638-651.
[40]Peng T,Saito T,Honda C,et al.Screening of UV-B-induced genes from apple peels by SSH:possible involvement of Md COP1-mediated signaling cascade genes in anthocyanin accumulation[J].Physiologia Plantarum,2013,148(3):432-444.
[41]Li J,Li G,Gao S,et al.Arabidopsis transcription factor ELONGATED HYPOCOTYL5 plays a role in the feedback regulation of phytochrome A signaling[J].Plant Cell,2010,22(11):3 634-3649.
[2]Santos-Buelga C.Flavonoids:Functions,Metabolism and Biotechnology[M].Weinheim:Wiley-VCH Verlag Gmb H&Co.KGa A,2016.
[3]Xu J,Ge X,Dolan M C.Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures[J].Biotechnology Advances,2011,29(3):278-299.
[4]李丽琴.红豆杉细胞合成紫杉醇的分子调控机理[D].武汉:华中科技大学,2009.
[5]葛锋,王剑平,王晓东,等.新疆紫草细胞悬浮培养过程研究[J].天然产物研究与开发,2010,22(3):460-465.
[6]Zoratti L,Karppinen K,Escobar AL,et al.Light-controlled flavonoid biosynthesis in fruits[J].Frontiers in Plant Science,2014,5(5):534.
[7]王璟.贯叶连翘悬浮细胞培养生产黄酮类物质的研究[M].上海:华东理工大学,2015.
[8]钟春水,赖瑞联,刘生财,等.光源或培养基成分对金花茶愈伤组织中DFR、LAR与PPO基因表达及总儿茶素含量的影响[J].广西植物,2016,36(12):1 410-1 415.
[9]Dong H S,Choi M G,Kim K,et al.HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis[J].Febs Letters,2013,587(10):1 543-1 547.
[10]Takayuki T,Mutsumi W,Rainer H,et al.Shikimate and phenylalanine biosynthesis in the green lineage[J].Frontiers in Plant Science,2013,4(1):62.
[11]董慧雪,周燕蓉,田奇琳,等.不同光质对龙眼胚性愈伤组织类黄酮含量的影响[J].热带作物学报,2014,35(12):2374-2377.
[12]赖钟雄,陈振光.龙眼胚性细胞悬浮培养再生植株[J].应用与环境生物学报,2002,8(3):485-491.
[13]赖钟雄,潘良镇,陈振光.龙眼胚性细胞系的建立与保持[J].福建农林大学学报(自然版),1997(2):160-167.
[14]李琨,张学杰,张德纯,等.不同芹菜品种叶与叶柄黄酮含量及其与抗氧化能力的关系[J].园艺学报,2011,38(1):69-76.
[15]张建文,刘禹,夏建业,等.机械搅拌生物反应器的CFD模拟及剪切力对红花细胞悬浮培养的影响[J].华东理工大学学报(自然科学版),2016,42(3):492-498.
[16]单会娇,王冰.龙葵药材中可溶性糖的动态积累[J].中国实验方剂学杂志,2010,16(9):100-102.
[17]赵凯,许鹏举,谷广烨.3,5-二硝基水杨酸比色法测定还原糖含量的研究[J].食品科学,2008,29(3):534-536.
[18]李永远,杨坤朋.水中总磷酸盐(Phosphate salt)的测定方法[J].中国化工贸易,2013(3):212.
[19]Lin Y L,Lai Z X.Reference gene selection for q PCR analysis during somatic embryogenesis in longan tree[J].Plant Science,2010,178(2):359-365.
[20]Meijer J J,Hoopen H J G T,Luyben K C A M,et al.Effects of hydrodynamic stress on cultured plant cells:A literature survey[J].Enzyme&Microbial Technology,1993,15(3):234-238.
[21]侯学文,郭勇.不同碳源对悬浮培养玫瑰茄细胞呼吸强度及产生辅酶Q的影响[J].广西植物,1999,19(1):150-153.
[22]Pratt J,Boisson A M,Gout E,et al.Phosphate(Pi)starvation effect on the cytosolic Pi concentration and Pi exchanges across the tonoplast in plant cells:an in vivo 31P-nuclear magnetic resonance study using methylphosphonate as a Pi analog[J].Plant Physiology,2009,151(3):1 646-1 657.
[23]李雅丽,孟婷婷,王毛毛,等.甘草细胞在搅拌式生物反应器中的放大培养[J].植物生理学报,2015(3):302-306.
[24]Yu X,Liu H,Klejnot J,et al.The CRY blue light receptors[J].Arabidopsis Book,2010,8:e0135.
[25]于丹,朴炫春,李阳,等.利用生物反应器大量生产东北刺人参不定根的研究[J].中国农学通报,2014,30(16):252-255.
[26]高慧君.利用柑橘愈伤组织研究植物类胡萝卜素积累的调控机理[D].武汉:华中农业大学,2013.
[27]王娟,高文远,黄滔,等.培养条件对西洋参悬浮细胞生物量和活性成分的影响[J].中国中药杂志,2009,34(4):375-378.
[28]Dan Q U,Wang H M,Ren J.Effects of sugar content on cell growth,accumulation of rosemary acid,and antioxidative enzyme activity in suspension cells of rosemary[J].Bulletin of Botanical Research,2015.
[29]Hummel M,Rahmani F,Smeekens S,et al.Sucrose-mediated translational control[J].Annals of Botany,2009,104(1):1-7.
[30]Zheng Y,Li T,Liu H,et al.Sugars induce anthocyanin accumulation and flavanone 3-hydroxylase expression in grape berries[J].Plant Growth Regulation,2009,58(3):251-260.
[31]Price J,Laxmi A,St Martin SK,et al.Global transcription profiling reveals multiple sugar signal transduction mechanisms in Arabidopsis[J].Plant Cell,2004,16(8):2 128-2 150.
[32]Solfanelli C,Perata P.Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis[J].Plant Physiology,2006,140(2):637.
[33]刘忠娟.PIFs在糖调节植物生长代谢中的作用机理研究[D].兰州:兰州大学,2011.
[34]程华,余龙江.不同因素对岩黄连悬浮细胞生长的影响[J].生物技术,2007,17(3):56-59.
[35]王静,王渭玲,徐福利,等.氮磷钾对桔梗生长及次生代谢产物的影响[J].草业科学,2012,29(4):586-591.
[36]Jiao Y,Lau O S,Deng XW.Light-regulated transcriptional networks in higher plants[J].Nature Reviews Genetics,2007,8(3):217-230.
[37]Lee J,He K,Stolc V,et al.Analysis of transcriptional factor HY5genomic binding sites revealed its hierarchical role in light regulation of development[J].Plant Cell,2007,19(4):731-749.
[38]Stracke R,Favory J J,Gruber H,et al.The Arabidopsis b ZIP transcription factor HY5 regulates expression of the PFG1/MYB12gene in response to light and ultraviolet-B radiation[J].Plant Cell&Environment,2010,33(1):88.
[39]Maier A,Schrader A,Kokkelink L,et al.Light and the E3ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis[J].Plant Journal for Cell&Molecular Biology,2013,74(4):638-651.
[40]Peng T,Saito T,Honda C,et al.Screening of UV-B-induced genes from apple peels by SSH:possible involvement of Md COP1-mediated signaling cascade genes in anthocyanin accumulation[J].Physiologia Plantarum,2013,148(3):432-444.
[41]Li J,Li G,Gao S,et al.Arabidopsis transcription factor ELONGATED HYPOCOTYL5 plays a role in the feedback regulation of phytochrome A signaling[J].Plant Cell,2010,22(11):3 634-3649.