高级检索

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

温度胁迫对白桦丛生苗次生产物合成及抗逆酶积累的影响

李可鑫 赵微 徐林琳 李影 张宝莲 詹亚光 尹静

李可鑫, 赵微, 徐林琳, 李影, 张宝莲, 詹亚光, 尹静. 温度胁迫对白桦丛生苗次生产物合成及抗逆酶积累的影响[J]. 北京林业大学学报, 2021, 43(7): 31-39. doi: 10.12171/j.1000-1522.20200241
引用本文: 李可鑫, 赵微, 徐林琳, 李影, 张宝莲, 詹亚光, 尹静. 温度胁迫对白桦丛生苗次生产物合成及抗逆酶积累的影响[J]. 北京林业大学学报, 2021, 43(7): 31-39. doi: 10.12171/j.1000-1522.20200241
Li Kexin, Zhao Wei, Xu Linlin, Li Ying, Zhang Baolian, Zhan Yaguang, Yin Jing. Effects of temperature stress on the accumulation of secondary metabolites and defensive enzymes in multiple shoots of Betula platyphylla[J]. Journal of Beijing Forestry University, 2021, 43(7): 31-39. doi: 10.12171/j.1000-1522.20200241
Citation: Li Kexin, Zhao Wei, Xu Linlin, Li Ying, Zhang Baolian, Zhan Yaguang, Yin Jing. Effects of temperature stress on the accumulation of secondary metabolites and defensive enzymes in multiple shoots of Betula platyphylla[J]. Journal of Beijing Forestry University, 2021, 43(7): 31-39. doi: 10.12171/j.1000-1522.20200241

温度胁迫对白桦丛生苗次生产物合成及抗逆酶积累的影响

doi: 10.12171/j.1000-1522.20200241
基金项目: 国家自然科学基金项目(31570589),中央高校基本科研业务费专项(2572020DY22),东北林业大学大学生创新项目(201910225082)
详细信息
    作者简介:

    李可鑫。主要研究方向:植物次生代谢调控。Email:1527552185@qq.com 地址:150040黑龙江省哈尔滨市和兴路26号东北林业大学生命科学学院

    责任作者:

    尹静,博士,教授。主要研究方向:植物次生代谢调控与细胞工程利用。Email:yinjing20135@163.com 地址:同上

  • 中图分类号: S718.43

Effects of temperature stress on the accumulation of secondary metabolites and defensive enzymes in multiple shoots of Betula platyphylla

  • 摘要:   目的  探究温度胁迫对白桦丛生苗三萜、黄酮及抗逆酶积累的影响,为提高白桦次生代谢产物积累以及工厂化生产奠定基础。  方法  利用不同浓度(0.5、1.0、2.0和5.0 mg/L)的6-苄氨基腺嘌呤(6-BA),诱导产生白桦丛生苗及建立悬浮培养体系,同时对丛生苗进行温度胁迫诱导次生产物合成。  结果  附加2.0 mg/L 6-BA的NT固体培养基可诱导白桦愈伤组织获得白桦丛生苗。2种温度(45和4 ℃)胁迫下,白桦丛生苗中总三萜含量均比对照(25 ℃)有所提高,其中45 ℃处理4 h,恢复培养6 h后,总三萜含量比对照提高5.03倍。4 ℃处理白桦丛生苗1 h,并在24 h时取样,齐墩果酸含量比对照提高了14.52倍,最高含量达2.33 mg/g。4 ℃处理白桦丛生苗4 h,在恢复培养48 h时,黄酮含量比对照提高38.37%。同时明确温度胁迫下,4 ℃胁迫4 h,恢复培养72 h,超氧化物歧化酶(SOD)活性最高,比对照提高69.77%。经4 ℃处理1 h的丛生苗在96 h 时,过氧化氢酶(CAT)活性达到最高峰,是对照的1.81倍。4 ℃处理1 h的丛生苗在恢复培养6 h时抗坏血酸过氧化物酶(APX)活性比对照提高55.29%。当胁迫时间延长至4 h,在45 ℃处理下,黄酮含量与APX酶,SOD与齐墩果酸含量均呈显著负相关(P < 0.05);经4 ℃处理,总三萜与APX(P < 0.01)、CAT(P < 0.05)分别呈极显著正相关和显著正相关;黄酮与SOD含量呈显著负相关(P < 0.05);CAT与APX(P < 0.01)、SOD(P < 0.05)相关性达极显著和显著水平,3种抗逆酶协调发挥作用共同参与次生产物合成。  结论  2.0 mg/L的6-BA能成功诱导白桦愈伤组织产生丛生苗,并建立悬浮培养系。短时间高温或低温胁迫均可以刺激3种防御酶发生显著变化,3种酶相互协调共同参与并促进白桦丛生苗总三萜和黄酮物质的合成与积累。

     

  • 图  1  白桦丛生苗诱导进程

    A. 愈伤组织细胞开始分化出芽(T2处理);B. 分化出芽的愈伤组织(T1处理);C. 丛生苗的悬浮培养。A, callus cells that begin to differentiate and bud (T2 treatment); B, differentiated callus (T1 treatment); C, suspension culture of cluster seedlings.

    Figure  1.  Induction process of multiple shoots of birch

    图  2  悬浮培养的白桦丛生苗的生长曲线

    Figure  2.  Growth curve of birch multiple shoots in suspension culture

    图  3  悬浮培养的白桦丛生苗总三萜、齐墩果酸含量变化

    不同小写字母表示不同组织部位的基因表达差异显著性(P < 0.05)。Different lowercase letters indicate significant differences in gene expression at varied tissue sites (P < 0.05).

    Figure  3.  Contents of total triterpenoids and oleanolic acid in suspension cultured multiple shoots of birch

    图  4  温度胁迫下白桦丛生苗三萜和黄酮积累

    Figure  4.  Accumulation of triterpenoids and flavonoids in birch multiple shoots under temperature stress

    图  5  温度胁迫下白桦丛生苗SOD、CAT、APX活性的变化

    APX. 抗坏血酸过氧化物酶;SOD. 超氧化物歧化酶;CAT. 过氧化氢酶。APX, ascorbate peroxidase; SOD, superoxide dismutase; CAT, catalase.

    Figure  5.  Activity of SOD, CAT, APX of birch multiple shoots under temperature stress

    表  1  白桦愈伤组织不定芽分化

    Table  1.   Differentiation of adventitiousbuds from birch callus

    处理
    Treatment
    6-BA质量浓度
    6-BA mass
    concentration/
    (mg·L−1)
    不定芽诱导率
    Adventitious
    bud induction
    rate/%
    每块平均不定芽数
    Average number
    of adventitious
    buds per piece
    T21.046.673
    T32.063.336
    T45.053.334
    下载: 导出CSV

    表  2  45 ℃胁迫4 h下白桦丛生苗次生产物含量与抗逆酶活性相关分析

    Table  2.   Correlation analysis of secondary production content and anti-stress enzymeactivity of birch multiple shoots under 45 ℃ for 4 h

    项目
    Item
    总三萜
    Total triterpenoids
    齐墩果酸
    Oleanolic acid
    黄酮
    Flavonoid
    APXSODCAT
    总三萜 Total triterpenoids 1.00 −0.55 0.25 −0.54 0.26 −0.02
    齐墩果酸 Oleanolic acid 1.00 0.30 −0.10 −0.84* −0.41
    黄酮 Flavonoid 1.00 −0.79* −0.24 −0.69
    APX 1.00 0.30 0.08
    SOD 1.00 0.43
    CAT 1.00
    注:*表示处理间显著相关(P < 0.05)。下同。Notes: * means significant correlation between treatments (P < 0.05). Same as below.
    下载: 导出CSV

    表  3  4 ℃胁迫4 h处理下白桦丛生苗次生产物含量与抗逆酶活性相关分析

    Table  3.   Correlation analysis of secondary production content and anti-stress enzyme activity of birch multiple shoots at 4 ℃ for 4 h

    项目
    Item
    总三萜
    Total triterpenoids
    齐墩果酸
    Oleanolic acid
    黄酮
    Flavonoid
    APXSODCAT
    总三萜 Total triterpenoids 1.00 0.57 −0.27 0.89** 0.49 0.81*
    齐墩果酸 Oleanolic acid 1.00 0.21 0.35 0.17 0.51
    黄酮 Flavonoid 1.00 −0.37 −0.80* −0.46
    APX 1.00 0.69 0.96**
    SOD 1.00 0.77*
    CAT 1.00
    注:**表示处理间极显著相关(P < 0.01)。Notes: ** means extremely significant correlation between treatments (P < 0.01).
    下载: 导出CSV
  • [1] Wang S, Yang C, Zhao X, et al. Complete chloroplast genome sequence of Betula platyphylla: gene organization, RNA editing, and comparative and phylogenetic analyses[J/OL]. BMC Genomics, 2018, 19(1): 950 (2018−12−20) [2020−06−28]. https://doi.org/10.1186/s12864-018-5346-x.
    [2] Malfa G A, Tomasello B, Acquaviva R, et al. Betula etnensis Raf. (Betulaceae) extract induced HO-1 expression and ferroptosis cell death in human colon cancer cells[J/OL]. International Journal of Molecular Sciences, 2019, 20(11): 2723 (2019−06−03) [2019−12−21]. https://doi.org/10.3390/ijms20112723.
    [3] Ríos J L, Máñez S. New pharmacological opportunities for betulinic acid[J]. Planta Medica, 2018, 84(1): 8−19. doi: 10.1055/s-0043-123472
    [4] Imran M, Rauf A, Abu-Izneid T, et al. Luteolin, a flavonoid, as an anticancer agent: a review[J/OL]. Biomedicine and Pharmacotherapy, 2019, 112: 108612 [2019−09−22]. https://doi.org/10.1016/j.biopha.2019.108612.
    [5] 盛艳, 吴泽柱. 桦树汁营养成分及功能利用研究进展[J]. 农产品加工, 2017, 7(14):49−52.

    Sheng Y, Wu Z Z. Research progress in nutrient composition function and utilization of birch sap[J]. Farm Products Processing, 2017, 7(14): 49−52.
    [6] Tan S N, Tee C S, Wong H L. Multiple shoot bud induction and plant regeneration studies of Pongamia pinnata[J]. Plant Biotechnology (Tokyo), 2018, 35(4): 325−334. doi: 10.5511/plantbiotechnology.18.0711a
    [7] 王思瑶, 李明阳, 邵占媛, 等. 不同培养基及激素处理下柽柳丛生芽总酚、三萜及黄酮物质的积累[J]. 北京林业大学学报, 2014, 36(5):74−81.

    Wang S Y, Li M Y, Shao Z Y, et al. Accumulation of total phenolics, triterpenoids and flavonoids in tufted buds of Tamarix chinensis under different medium and hormone treatment[J]. Journal of Beijing Forestry University, 2014, 36(5): 74−81.
    [8] 杨帆, 廉美兰, 李美兰, 等. 金线莲丛生芽培养及有效物质生产研究[J]. 中草药, 2016, 47(18):3284−3288. doi: 10.7501/j.issn.0253-2670.2016.18.024

    Yang F, Lian M L, Li M L, et al. Multiple shoot culture and effective material production of Anoectochilus roxburghii[J]. Chinese Traditional and Herbal Drugs, 2016, 47(18): 3284−3288. doi: 10.7501/j.issn.0253-2670.2016.18.024
    [9] 李球红, 宋华东, 周文怡, 等. 桂花幼胚离体培养形成丛生苗[J]. 杭州师范大学学报(自然科学版), 2015, 14(5):507−510.

    Li Q H, Song H D, Zhou W Y, et al. Multiple shoots formed from in vitro culture for young embryo of Osmanthus fragrans[J]. Journal of Hangzhou Normal University (Natural Science Edition), 2015, 14(5): 507−510.
    [10] Gill S S, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J]. Plant Physiology and Biochemistry, 2010, 48(12): 909−930. doi: 10.1016/j.plaphy.2010.08.016
    [11] Mittler R. ROS are good[J]. Trends in Plant Science, 2017, 22(1): 11−19. doi: 10.1016/j.tplants.2016.08.002
    [12] 黄璐琦, 郭兰萍. 环境胁迫下次生代谢产物的积累及道地药材的形成[J]. 中国中药杂志, 2007, 32(4):277−280. doi: 10.3321/j.issn:1001-5302.2007.04.001

    Huang L Q, Guo L P. Secondary metabolites accumulating and geoherbs formation under enviromental stress[J]. China Journal of Chinese Materia Medic, 2007, 32(4): 277−280. doi: 10.3321/j.issn:1001-5302.2007.04.001
    [13] Mittler R, Vanderauwera S, Gollery M, et al. Reactive oxygen gene network of plants[J]. Trends in Plant Science, 2004, 9(10): 490−498. doi: 10.1016/j.tplants.2004.08.009
    [14] 杜蕙, 王春明, 郭建国, 等. 葡萄生单轴霉菌对葡萄几种防御酶活性的影响[J]. 江苏农业科学, 2019, 47(15):151−154.

    Du H, Wang C M, Guo J G, et al. Effects of Plasmopara viticola on activity of defense-related enzymes in grape[J]. Jiangsu Agricultural Sciences, 2019, 47(15): 151−154.
    [15] 郑金龙, 易克贤, 习金根, 等. 剑麻感染烟草疫霉菌后几种重要防御酶活性的变化[J]. 中国麻业科学, 2019, 41(5):210−216. doi: 10.3969/j.issn.1671-3532.2019.05.005

    Zheng J L, Yi K X, Xi J G, et al. Several cases of sisal (H. 11648) infection with Phytophthora nicotianae changes in important defense enzyme activities[J]. Plant Fiber Sciences in China, 2019, 41(5): 210−216. doi: 10.3969/j.issn.1671-3532.2019.05.005
    [16] Jumrani K, Bhatia V S. Interactive effect of temperature and water stress on physiological and biochemical processes in soybean[J]. Physiology Molecular Biology of Plants, 2019, 25(3): 671−681.
    [17] 常志凯, 白祎杰, 董恒, 等. 高温胁迫诱导对白桦悬浮细胞三萜积累及防御酶活性的影响[J]. 生物技术通报, 2015, 31(9):111−118.

    Chang Z K, Bai Y J, Dong H, et al. The effects of high temperature stress on the accumulation of triterpenoid and the activity of defense enzyme in the suspension cells of birch[J]. Biotechnology Bulletin, 2015, 31(9): 111−118.
    [18] Bhatia C, Pandey A, Gaddam S R, et al. Low Temperature-enhanced flavonol synthesis requires light-associated regulatory components in Arabidopsis thaliana[J]. Plant and Cell Physiology, 2018, 59(10): 2099−2112. doi: 10.1093/pcp/pcy132
    [19] 刘佳, 全雪丽, 姜明亮, 等. 低温胁迫对人参皂苷生物合成途径基因家族表达特性的影响研究[J]. 中草药, 2016, 47(11):1956−1961. doi: 10.7501/j.issn.0253-2670.2016.11.024

    Liu J, Quan X L, Jiang M L, et al. Effect of cold stress on expression characteristic of gene families of ginsenoside biosynthesis pathway[J]. Chinese Traditional and Herbal Drugs, 2016, 47(11): 1956−1961. doi: 10.7501/j.issn.0253-2670.2016.11.024
    [20] 乌凤章, 王柏臣, 刘桂丰, 等. 低温胁迫对白桦幼苗生长和生理的影响[J]. 东北林业大学学报, 2008, 36(9):8−10. doi: 10.3969/j.issn.1000-5382.2008.09.003

    Wu F Z, Wang B C, Liu G F, et al. Growth and physiological response of Betula platyphylla seedlings to low temperature stress[J]. Journal of Northeast Forestry University, 2008, 36(9): 8−10. doi: 10.3969/j.issn.1000-5382.2008.09.003
    [21] 曲桂芹. 白桦热激反应的分子生态学研究[D]. 哈尔滨: 东北林业大学, 2001.

    Qu G Q. Study on molecular ecology of heat shock response in Betula platyphylla[D]. Harbin: Northeast Forestry Universiey, 2001.
    [22] 王博. 促进白桦(Betula platyphylla)培养物中三萜物质积累的初步研究[D]. 哈尔滨: 东北林业大学, 2008.

    Wang B. Primary studies on aecumulation of triterpenoids in culture medium of Betula platyphylla[D]. Harbin: Northeast Forestry Universiey, 2008.
    [23] 谭朝阳, 袁宏佳. HPLC法测定龙芽楤木药材中齐墩果酸的含量[J]. 中国中医药信息杂志, 2010, 17(1):46−47. doi: 10.3969/j.issn.1005-5304.2010.01.021

    Tan C Y, Yuan H J. Determination of oleanolic acid in cortex Aralia elatae by HPLC[J]. Chinese Journal of Information on Traditional Chinese Medicine, 2010, 17(1): 46−47. doi: 10.3969/j.issn.1005-5304.2010.01.021
    [24] 施特尔马赫·B. 酶的测定方法[M]. 钱嘉渊, 译. 北京: 中国轻工业出版社, 1992.

    Stellmach B. Method for measuring enzyme[M]. Qian J Y, trans. Beijing: China Light Industry Press, 1992.
    [25] 李合生. 植物生理生化实验原理和技术[M]. 北京: 高等教育出版社, 2000.

    Li H S. Principle and technology of plant physiological and biochemical experiments[M]. Beijing: Higher Education Press, 2000.
    [26] 赵微. 温度及其与MeJA互作处理对白桦三萜合成的影响研究[D]. 哈尔滨: 东北林业大学, 2013.

    Zhao W. The effect of temperature and its interaction with MeJA treatment on the synthesis of triterpene in Betula platyphylla [D]. Harbin: Northeast Forestry University, 2013.
    [27] 马泓思, 潘亚婕, 王艳, 等. Ca2+在介导MeJA诱导白桦悬浮培养三萜合成中的作用[J]. 植物研究, 2015, 35(1):117−126. doi: 10.7525/j.issn.1673-5102.2015.01.018

    Ma H S, Pan Y J, Wang Y, et al. Effect of Ca2 + on mediating the MeJA-induced synthesis of triterpenoid in suspension cells of Betula platyphylla Suk.[J]. Bulletin of Botanical Research, 2015, 35(1): 117−126. doi: 10.7525/j.issn.1673-5102.2015.01.018
    [28] 苏欣, 尹静, 孙建广, 等. 白桦植株中三萜前体及不同三萜分支产物积累特征[J]. 中草药, 2013, 44(24):3534−3539.

    Su X, Yin J, Sun J G, et al. Accumulative characteristics of triterpenoid precursor and different triterpenoid branch products in Betula platyphylla[J]. Chinese Traditional and Herbal Drugs, 2013, 44(24): 3534−3539.
    [29] 毛美琴. 诱导子对朱砂根愈伤组织生长及三萜皂苷合成的影响[D]. 温江: 四川农业大学, 2018.

    Mao M Q. Effect of elicitor on callus growth and triterpenoid saponins synthesis in Ardisia crenata Sims allus[D]. Wenjiang: Sichuan Agricultural University, 2018.
    [30] 姜艳, 刘奇, 冯尚国, 等. 环境因子对杭白菊黄酮类化合物和绿原酸含量的影响[J]. 中国现代应用药学, 2018, 35(2):225−230.

    Jiang Y, Liu Q, Feng S G, et al. Effects of environmental factors on the content of flavonoids and chlorogenic acid in Chrysanthemum morifolium Ramat[J]. Chinese Journal of Modern Applied Pharmacy, 2018, 35(2): 225−230.
    [31] 林彦萍, 张美萍, 王康宇, 等. 人参皂苷生物合成研究进展[J]. 中国中药杂志, 2016, 41(23):4292−4302.

    Lin Y P, Zhang M P, Wang K Y, et al. Research achievements on ginsenosides biosynthesis from Panax ginseng[J]. China Journal of Chinese Materia Medica, 2016, 41(23): 4292−4302.
    [32] 姜明亮. 低温对人参愈伤组织皂苷积累的影响[D]. 延边: 延边大学, 2016.

    Jiang M L. The effect of chilling on ginseoside accumulation in Panax ginseng cells[D]. Yanbian: Yanbian University, 2016.
    [33] 杨颖丽, 吕丽荣, 徐玉玲, 等. 胞间活性氧产生对盐胁迫下两种小麦叶抗氧化反应的影响[J]. 兰州大学学报(自然科学版), 2019, 55(4):476−484.

    Yang Y L, Lü L R, Xu Y L, et al. Effects of apoplastic reactive oxygen species on the antioxidant responses in the leaves of two wheat seedlings under salt stress[J]. Journal of Lanzhou University (Natural Sciences), 2019, 55(4): 476−484.
    [34] Reddy Y P, Yadav R K, Tripathi K, et al. Isolation and characterization of high temperature tolerant mutant from the cyanobacterium Anabaena doliolum[J]. Journal of Basic Microbiology, 2019, 59(3): 314−322. doi: 10.1002/jobm.201800447
    [35] 赵远伟, 刘小京, 李存东, 等. 温度对盐胁迫小麦抗氧化机制的影响[J]. 中国生态农业学报, 2014, 22(12):1460−1468.

    Zhao Y W, Liu X J, Li C D, et al. Effect of temperature on antioxidation mechanism of wheat (Triticum aestivum L.) seedlings under salt stress[J]. Chinese Journal of Eco-Agriculture, 2014, 22(12): 1460−1468.
    [36] Wu Z, Jiang Q, Yan T, et al. Ammonium nutrition mitigates cadmium toxicity in rice (Oryza sativa L.) through improving antioxidase system and the glutathione-ascorbate cycle efficiency[J/OL]. Ecotoxicology and Environmental Safety, 2020, 189: 110010 (2019−11−29) [2020−05−19]. https://doi.org/10.1016/j.ecoenv.2019.110010.
    [37] 张涛. 人参及其皂苷生物合成对低温的生理生态响应机制研究[D]. 长春: 吉林农业大学, 2019.

    Zhang T. Physiological and ecoligical response mechanism of Panax ginseng and its saponins biosynthesis to low temperature[D]. Changchun: Jilin Agricultural University, 2019.
    [38] 王斌. 干旱、H2O2及Na2S2O4对黄芩悬浮细胞代谢的影响[D]. 哈尔滨: 黑龙江中医药大学, 2020.

    Wang B. The effects of drought, H2O2 and Na2S2O4 stress on cell metabolism in Scutellaria baicalensis georgi suspension cells[D]. Harbin: Heilongjiang University of Chinese Medicine, 2020.
    [39] 刘文盈. 低温胁迫对黑果枸杞愈伤组织的影响[J]. 基因组学与应用物, 2018, 37(1):408−412.

    Liu W Y. The effects of low temperature stress on the callus of Lycium ruthenicum Murr.[J]. Genomics and Applied Biology, 2018, 37(1): 408−412.
    [40] Li S, Yu J, Li Y, et al. Heat-responsive proteomics of a heat-sensitive spinach variety[J/OL]. International Journal of Molecular Sciences, 2019, 20(16): 3872 (2019−08−08) [2020−07−05]. https://doi.org/10.3390/ijms20163872.
    [41] 高慧如, 王佳慧, 关瑜, 等. 高温对甘草黄酮类成分的影响[J]. 中药材, 2019, 42(3):524−529.

    Gao H R, Wang J H, Guan Y, et al. Effect of high temperature on flavonoids of Glycyrrhiza uralensis[J]. Journal of Chinese Medicinal Materials, 2019, 42(3): 524−529.
  • 加载中
图(5) / 表(3)
计量
  • 文章访问数:  176
  • HTML全文浏览量:  86
  • PDF下载量:  24
  • 被引次数: 0
出版历程
  • 收稿日期:  2020-08-31
  • 修回日期:  2020-12-18
  • 网络出版日期:  2021-06-21
  • 刊出日期:  2021-07-25

目录

    /

    返回文章
    返回