南方红豆杉细胞悬浮培养体系优化及动力学研究
|
摘要
[目的]南方红豆杉细胞悬浮培养体系优化及动力学模型构建。[方法]以南方红豆杉1年生枝条诱导产生的愈伤组织为材料,在优化愈伤组织接种量、培养基初始pH、植物激素类型及配比的基础上,分析南方红豆杉悬浮细胞生长和紫杉醇积累的动力学关系及主要营养成分的变化。[结果]适合南方红豆杉悬浮培养的培养基为:B5+0.4 mg·L~(-1) 2,4-D+0.3 mg·L~(-1) NAA+1.2 mg·L~(-1) 6-KT+30 g·L~(-1)蔗糖,培养基最佳初始pH为5.8,最佳接种量为0.09 g·mL~(-1)。在1个培养周期内,培养液中的糖、磷元素,铵态氮、硝态氮在细胞培养的第18天基本被消耗完全或保持不变;动力学拟合表明,南方红豆杉细胞悬浮培养生长符合Logistic生长模型,最大比生长速率为0.143 65 d~(-1),底物消耗和紫杉醇合成可用Luedeking-Piret模型描述,悬浮细胞生长与紫杉醇积累属于部分生长偶联型。[结论]南方红豆杉细胞悬浮培养体系优化后,通过模型的回归拟合,获得了反映南方红豆杉细胞培养的动力学参数,试验所构建的发酵动力学模型在一定程度上揭示了南方红豆杉细胞产紫杉醇代谢过程的特征。
[Objective] To optimizing the cell suspension culture system and building the kinetics model of Taxus chinensis var. mairer. [Method] The effects of basic callus inoculum size, initial pH value, phytohormone types and concentration on T. chinensis var. mairer cell growth was investigated using the callus induced from annual branches of T. chinensis var. mairer. Based on this, the kinetic relationship between T. chinensis var. mairer cell growth and paclitaxel production and the nutrient consumption were also analyzed. [Result] The optimal media for T. chinensis var. mairer cell suspension culture is B5 medium supplemented with 0.4 mg·L~(-1) 2,4-D, 0.3 mg·L~(-1) NAA, 1.2 mg·L~(-1) 6-KT and sucrose 30 g·L~(-1). The optimum callus inoculum size is 0.09 g·mL~(-1) and the optimum initial pH of medium is 5.8. During the process, the sugar, phosphor-N and nitrate-N in the culture medium are nearly used up or remain unchanged on the 18 th day. The growth law of T. chinensis var. mairer is consistent with Logistic growth model and the maximum specific growth rate is 0.143 65 d~(-1). Substrate consumption and paclitaxel production by Luedeking-Piret model are described. The growth of suspension cell and the accumulation of paclitaxel belong to partial growth coupling type. [Conclusion] The cell suspension culture system is optimized. Through regression model, the kinetic parameters are obtained to reflect the cell culture of T. chinensis var. mairer. The fermentation kinetics model constructed in this experiment reveals the characteristics of paclitaxel production in Taxus chinensis cells to a certain extent.
[Objective] To optimizing the cell suspension culture system and building the kinetics model of Taxus chinensis var. mairer. [Method] The effects of basic callus inoculum size, initial pH value, phytohormone types and concentration on T. chinensis var. mairer cell growth was investigated using the callus induced from annual branches of T. chinensis var. mairer. Based on this, the kinetic relationship between T. chinensis var. mairer cell growth and paclitaxel production and the nutrient consumption were also analyzed. [Result] The optimal media for T. chinensis var. mairer cell suspension culture is B5 medium supplemented with 0.4 mg·L~(-1) 2,4-D, 0.3 mg·L~(-1) NAA, 1.2 mg·L~(-1) 6-KT and sucrose 30 g·L~(-1). The optimum callus inoculum size is 0.09 g·mL~(-1) and the optimum initial pH of medium is 5.8. During the process, the sugar, phosphor-N and nitrate-N in the culture medium are nearly used up or remain unchanged on the 18 th day. The growth law of T. chinensis var. mairer is consistent with Logistic growth model and the maximum specific growth rate is 0.143 65 d~(-1). Substrate consumption and paclitaxel production by Luedeking-Piret model are described. The growth of suspension cell and the accumulation of paclitaxel belong to partial growth coupling type. [Conclusion] The cell suspension culture system is optimized. Through regression model, the kinetic parameters are obtained to reflect the cell culture of T. chinensis var. mairer. The fermentation kinetics model constructed in this experiment reveals the characteristics of paclitaxel production in Taxus chinensis cells to a certain extent.
引文
[1] 肖遥,楚秀丽,徐肇友,等. 南方红豆杉2年生容器苗多点试验的生长节律家系变异[J]. 林业科学研究,2016,29(2):238-244.
[2] 姚晓, 步达, 陈建伟, 等. 南方红豆杉愈伤组织培养及其紫杉烷二萜类成分的分析[J]. 中草药,2014,45(18):2696-2702.
[3] Zhang F, Wang P, Ji D, et al. Asymmetric somatic hybridization between Bupleurum scorzonerifolium Willd. and Taxus chinensis var. mairei[J]. Plant cell reports, 2011, 30(10): 1857-1864.
[4] Oh H C, Seo D W, Song T J, et al. Endoscopic ultrasonography-guided ethanol lavage with paclitaxel injection treats patients with pancreatic cysts [J]. Gastroenterology, 2011,140(1):172-179.
[5] 匡雪君, 王彩霞, 邹丽秋, 等. 紫杉醇生物合成途径及合成生物学研究进展[J]. 中国中药杂志, 2016,41(22):4144-4149.
[6] Jennewein S, Croteau R. Taxol: biosynthesis, molecular genetics, and biotechnological applications[J]. Applied Microbiology and Biotechnology, 2001,57(1-2):13-19.
[7] 白向阳, 吕建明, 周艳英, 等. 南方红豆杉细胞培养物的化学成分研究[J]. 药学学报, 2015,50(1):70-74.
[8] 徐志荣, 简方敏, 刘伯卿, 等. 南方红豆杉外植体的消毒和愈伤组织的培养[J]. 生物灾害科学, 2016,39(3):215-219.
[9] 刘华, 梅兴国. TTC法测定红豆杉细胞活力[J]. 植物生理学通讯, 2001,37(6):537-539.
[10] 林丽, 王琰, 王福星, 等. 基于硫酸-蒽酮法对不同产地藏药线叶龙胆多糖的含量测定[J]. 中国中药杂志, 2014,39(14):2774-2776.
[11] 陈帅, 朱军莉, 潘伟春. 乳糖对鱿鱼中氧化三甲胺热分解反应动力学的影响[J]. 现代食品科技, 2017,33(3):116-121.
[12] 贾洪秀, 朱云勤, 朱必学, 等. 分光光度法测定轻质碳酸钙中硝酸根含量[J]. 无机盐工业, 2010,42(5):53-55.
[13] 夏庆兵, 王军, 朱鲁生, 等. 土壤微生物对邻苯二甲酸二(2-乙基己)酯胁迫的生态响应[J]. 农业环境科学学报, 2016,35(7):1344-1350.
[14] 王晓东, 李晓灿, 翟俏丽, 等. 真菌诱导子对白桦悬浮体系中N和P的吸收利用和三萜合成的影响[J]. 中草药, 2011,42(10):2119-2124.
[15] 薛文娇, 马赛箭, 常帆, 等. 利用动力学模型探讨碳源浓度对普鲁兰分批发酵过程的影响[J]. 化学工程, 2015,43(11):5-10, 40.
[16] 朱晓媛, 胡铁, 黎继烈, 等. 基于遗传算法的纤维素酶分批发酵动力学研究[J]. 中国食品学报, 2014,14(1):47-51.
[17] 张阳阳, 曾柏全. 曲酸产生菌米曲霉Co-26型分批发酵动力学研究[J]. 中国食品学报, 2013,13(11):43-47.
[18] Rahpeyma S A, Moieni A, Jalali Javaran M. Paclitaxel production is enhanced in suspension-cultured hazel (Corylus avellana L.) cells by using a combination of sugar, precursor, and elicitor [J]. Engineering in Life Sciences, 2015,15(2):234-242.
[19] 陈红贤, 于笑笑, 王晨阳, 等. 国槐槐角种胚细胞悬浮培养的动力学研究[J]. 浙江农林大学学报, 2016,33(2):272-279.
[20] 张珏, 尹欢, 阮利霞, 等. 高生物产量川芎细胞悬浮培养条件优化[J]. 成都大学学报:自然科学版, 2014,33(1):10-13.
[21] 黄卫文, 黎继烈, 戴梓茹, 等. 花生根细胞悬浮培养制备白藜芦醇条件的优化[J]. 食品科学, 2011,32(7):233-236.
[22] 王娟, 高文远, 尹双双, 等. 药用植物细胞悬浮培养的研究进展[J]. 中国中药杂志, 2012,37(24):3680-3683.
[23] 盛长忠, 王淑芳, 王勇, 等. pH对红豆杉愈伤组织生长、PAL活性和紫杉醇含量的影响[J]. 中草药, 2001,32(10): 929-931.
[24] 陈继光, 上官新晨, 尹忠平, 等. 青钱柳悬浮细胞的培养及其基质消耗的规律[J]. 现代食品科技, 2014,30(1):44-49.
[25] 王沐兰, 杨生超, 郁步竹, 等. 红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展[J]. 广西植物, 2016,36(9):1137-1146.
[26] Hirasuna T J, Pestchanker L J, Srinivasan V, et al. Taxol production in suspension cultures of Taxus baccata[J]. Plant Cell, 1996(44):95-102.
[27] Liu C, Guo C, Wang Y, et al. Effect of light irradiation on hairy root growth and artemisinin biosynthesis of Artemisia annua L.[J]. Process Biochemistry, 2002,38(4):581-585.
[28] 董杰, 詹亚光, 任健. 茶条槭悬浮培养的动力学[J]. 林业科学, 2012,48(10):18-23.
[29] Omar R, Abdullah M A, Hasan M A, et al. Kinetics and modelling of cell growth and substrate uptake in Centella asiatica cell culture[J]. Biotechnology and Bioprocess Engineering, 2006,11(3):223-229.
[2] 姚晓, 步达, 陈建伟, 等. 南方红豆杉愈伤组织培养及其紫杉烷二萜类成分的分析[J]. 中草药,2014,45(18):2696-2702.
[3] Zhang F, Wang P, Ji D, et al. Asymmetric somatic hybridization between Bupleurum scorzonerifolium Willd. and Taxus chinensis var. mairei[J]. Plant cell reports, 2011, 30(10): 1857-1864.
[4] Oh H C, Seo D W, Song T J, et al. Endoscopic ultrasonography-guided ethanol lavage with paclitaxel injection treats patients with pancreatic cysts [J]. Gastroenterology, 2011,140(1):172-179.
[5] 匡雪君, 王彩霞, 邹丽秋, 等. 紫杉醇生物合成途径及合成生物学研究进展[J]. 中国中药杂志, 2016,41(22):4144-4149.
[6] Jennewein S, Croteau R. Taxol: biosynthesis, molecular genetics, and biotechnological applications[J]. Applied Microbiology and Biotechnology, 2001,57(1-2):13-19.
[7] 白向阳, 吕建明, 周艳英, 等. 南方红豆杉细胞培养物的化学成分研究[J]. 药学学报, 2015,50(1):70-74.
[8] 徐志荣, 简方敏, 刘伯卿, 等. 南方红豆杉外植体的消毒和愈伤组织的培养[J]. 生物灾害科学, 2016,39(3):215-219.
[9] 刘华, 梅兴国. TTC法测定红豆杉细胞活力[J]. 植物生理学通讯, 2001,37(6):537-539.
[10] 林丽, 王琰, 王福星, 等. 基于硫酸-蒽酮法对不同产地藏药线叶龙胆多糖的含量测定[J]. 中国中药杂志, 2014,39(14):2774-2776.
[11] 陈帅, 朱军莉, 潘伟春. 乳糖对鱿鱼中氧化三甲胺热分解反应动力学的影响[J]. 现代食品科技, 2017,33(3):116-121.
[12] 贾洪秀, 朱云勤, 朱必学, 等. 分光光度法测定轻质碳酸钙中硝酸根含量[J]. 无机盐工业, 2010,42(5):53-55.
[13] 夏庆兵, 王军, 朱鲁生, 等. 土壤微生物对邻苯二甲酸二(2-乙基己)酯胁迫的生态响应[J]. 农业环境科学学报, 2016,35(7):1344-1350.
[14] 王晓东, 李晓灿, 翟俏丽, 等. 真菌诱导子对白桦悬浮体系中N和P的吸收利用和三萜合成的影响[J]. 中草药, 2011,42(10):2119-2124.
[15] 薛文娇, 马赛箭, 常帆, 等. 利用动力学模型探讨碳源浓度对普鲁兰分批发酵过程的影响[J]. 化学工程, 2015,43(11):5-10, 40.
[16] 朱晓媛, 胡铁, 黎继烈, 等. 基于遗传算法的纤维素酶分批发酵动力学研究[J]. 中国食品学报, 2014,14(1):47-51.
[17] 张阳阳, 曾柏全. 曲酸产生菌米曲霉Co-26型分批发酵动力学研究[J]. 中国食品学报, 2013,13(11):43-47.
[18] Rahpeyma S A, Moieni A, Jalali Javaran M. Paclitaxel production is enhanced in suspension-cultured hazel (Corylus avellana L.) cells by using a combination of sugar, precursor, and elicitor [J]. Engineering in Life Sciences, 2015,15(2):234-242.
[19] 陈红贤, 于笑笑, 王晨阳, 等. 国槐槐角种胚细胞悬浮培养的动力学研究[J]. 浙江农林大学学报, 2016,33(2):272-279.
[20] 张珏, 尹欢, 阮利霞, 等. 高生物产量川芎细胞悬浮培养条件优化[J]. 成都大学学报:自然科学版, 2014,33(1):10-13.
[21] 黄卫文, 黎继烈, 戴梓茹, 等. 花生根细胞悬浮培养制备白藜芦醇条件的优化[J]. 食品科学, 2011,32(7):233-236.
[22] 王娟, 高文远, 尹双双, 等. 药用植物细胞悬浮培养的研究进展[J]. 中国中药杂志, 2012,37(24):3680-3683.
[23] 盛长忠, 王淑芳, 王勇, 等. pH对红豆杉愈伤组织生长、PAL活性和紫杉醇含量的影响[J]. 中草药, 2001,32(10): 929-931.
[24] 陈继光, 上官新晨, 尹忠平, 等. 青钱柳悬浮细胞的培养及其基质消耗的规律[J]. 现代食品科技, 2014,30(1):44-49.
[25] 王沐兰, 杨生超, 郁步竹, 等. 红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展[J]. 广西植物, 2016,36(9):1137-1146.
[26] Hirasuna T J, Pestchanker L J, Srinivasan V, et al. Taxol production in suspension cultures of Taxus baccata[J]. Plant Cell, 1996(44):95-102.
[27] Liu C, Guo C, Wang Y, et al. Effect of light irradiation on hairy root growth and artemisinin biosynthesis of Artemisia annua L.[J]. Process Biochemistry, 2002,38(4):581-585.
[28] 董杰, 詹亚光, 任健. 茶条槭悬浮培养的动力学[J]. 林业科学, 2012,48(10):18-23.
[29] Omar R, Abdullah M A, Hasan M A, et al. Kinetics and modelling of cell growth and substrate uptake in Centella asiatica cell culture[J]. Biotechnology and Bioprocess Engineering, 2006,11(3):223-229.