红豆杉低温半致死温度和低温胁迫下紫杉烷含量

doi:10.13560/j.cnki.biotech.bull.1985.2022-0762

摘要/Abstract

摘要：

通过测定低温胁迫下红豆杉电阻抗参数和紫杉烷含量变化，确定适合红豆杉低温半致死温度测定的电阻抗参数，探讨低温对红豆杉紫杉烷生物合成的影响，为扩大红豆杉引种范围及利用低温条件提高红豆杉中紫杉醇含量奠定了理论基础。以云南红豆杉、南方红豆杉（山西长治）、中国红豆杉、曼地亚红豆杉和东北红豆杉为材料，分别采用电导率法和电阻抗法测定上述树种低温半致死温度，并利用LC-MS法测定低温胁迫下红豆杉叶片中紫杉烷含量和脱落酸（abscisic acid, ABA）含量。结果表明，5种红豆杉中，中国红豆杉、南方红豆杉（山西长治）、东北红豆杉和曼地亚红豆杉均能耐受-14.668- -9.106℃低温；云南红豆杉能耐受-8.802- -3.521℃低温，高于其他4种红豆杉。电阻抗τ_m参数与电导法测定的5种红豆杉一年生枝低温半致死温度显著相关，相关系数为0.912。低温胁迫下，红豆杉一年生叶片中紫杉醇含量高于当年生叶片。随着处理温度的下降，云南红豆杉和东北红豆杉一年生叶片中紫杉醇含量显著增加，中国红豆杉当年生叶片中巴卡亭Ⅲ含量和10-去乙酰基紫杉醇含量显著增加。此外，红豆杉叶片中ABA含量随温度的下降而增加，当年生叶片中ABA含量高于一年生叶片。与云南红豆杉相比，东北红豆杉、曼地亚红豆杉、南方红豆杉（山西长治）和中国红豆杉能耐受更低的温度。低温有利于云南红豆杉和东北红豆杉一年生叶片中紫杉醇的合成及中国红豆杉当年叶片中巴卡亭III和10-去乙酰基紫杉醇的合成。

关键词: 红豆杉, 低温胁迫, 电阻抗, 低温半致死温度, 紫杉烷

Abstract:

To determine the electrical impedance parameters suitable for the determination of semi-lethal low temperature of Taxus and explore the effects of low temperature on the taxane biosynthesis in Taxus, the electrical impedance parameters and taxane content changes of Taxus under low temperature stress were examined. It will lay a theoretical foundation for expanding the introduction range of Taxus and using low temperature conditions to increase the contents of taxane content in Taxus. Using T. yunnanensis, T. chinensis var. mairei （Changzhi, Shanxi）, T. chinensis, T. cuspidata and T. × media as materials, the semi-lethal low temperature of these trees were determined by conductivity method and electrical impedance method respectively. Moreover, the taxane contents and abscisic acid contents in the leaves of Taxus under low-temperature stress were determined by LC-MS method. The results showed that T. chinensis, T. chinensis var. mairei（Changzhi, Shanxi）, T. cuspidata and T. × media all tolerated low temperature of -14.668- -9.106℃. The T. yunnanensis tolerated -8.802- -3.521℃, which was higher than the ons of other four Taxus species. The semi-lethal low temperature of one-year-old branches of five Taxus measured by electrical impedance τ_m parameter was significantly correlated with that measured by conductivity method, and the correlation coefficient was 0.912. The taxol content in the leaves of one-year-old Taxus was higher than that in the current-year under low temperature stress. The taxol content in the leaves of one-year-old T. yunnanensis and T. cuspidata significantly increased with the decrease of treatment temperature, while the baccatin III content and 10-deacetyltaxol content in the leaves of one-year-old T. chinensis significantly increased. Moreover, ABA content in the leaves of Taxus increased with the decrease of temperature, and ABA content in the current-year leaves was higher than that in the one-year-old leaves. Compared with T. yunnanensis, T. chinensis, T. chinensis var. mairei（Changzhi, Shanxi）, T. cuspidata and T. × media tolerated lower temperature. Low temperature is beneficial to the synthesis of taxol in the leaves of one-year-old T. yunnanensis and T. cuspidata, and the synthesis of baccatin III and 10-deacetyltaxol in the leaves of current-year T. chinensis.

Key words: Taxus, low temperature stress, electrical impedance, semi-lethal low temperature, taxane

蒋路园, 丰美静, 杜雨晴, 邸葆, 陈段芬, 邱德有, 杨艳芳. 红豆杉低温半致死温度和低温胁迫下紫杉烷含量[J]. 生物技术通报, 2023, 39(3): 232-242.

JIANG Lu-yuan, FENG Mei-jing, DU Yu-qing, DI Bao, CHEN Duan-fen, QIU De-you, YANG Yan-fang. Semi-lethal Low Temperature and Taxane Content of Taxus Under Low Temperature Stress[J]. Biotechnology Bulletin, 2023, 39(3): 232-242.

图/表 10

参考文献 35

[1]	中国科学院中国植物志编辑委员会. 中国植物志-第七卷[M]. 北京: 科学出版社, 1978: 438-443.
	Flora of China editorial committee of the Chinese academy of sciences. Flora of China volume 7[M]. Beijing: Science Press, 1978: 438-443.
[2]	汪群. 曼地亚红豆杉年生长节律研究[J]. 安徽农学通报, 2016, 22(16): 92-94.
	Wang Q. Study on the annual growth rhythm of Taxus × media[J]. Anhui Agric Sci Bull, 2016, 22(16): 92-94.
[3]	费永俊, 雷泽湘, 余昌均, 等. 中国红豆杉属植物的濒危原因及可持续利用对策[J]. 自然资源, 1997, 19(5): 59-63.
	Fei YJ, Lei ZX, Yu CJ, et al. The cause for endangerment of Taxus l and measures for its sustainable development in China[J]. Nat Resour, 1997, 19(5): 59-63.
[4]	贺川江, 张淑英, 李先荣, 等. 东北红豆杉抗寒性分析[J]. 安徽农业科学, 2016, 44(16): 174-176.
	He CJ, Zhang SY, Li XR, et al. Cold resistance analysis of Taxus cuspidate sieb. et zucc[J]. J Anhui Agric Sci, 2016, 44(16): 174-176.
[5]	祝佳媛. 东北红豆杉幼苗光合生理特性的季节动态研究[D]. 哈尔滨: 东北林业大学, 2013.
	Zhu JY. Seasonal dynamics study on photosynthetic physiological characteristics of Japanese yew(Taxus cuspidata)seedlings[D]. Harbin: Northeast Forestry University, 2013.
[6]	向邓云, 谢吉容, 谈锋. 自然降温过程中曼地亚红豆杉叶片膜保护系统的变化与低温半致死温度的关系[J]. 西南师范大学学报: 自然科学版, 2001, 26(4): 452-456.
	Xiang DY, Xie JR, Tan F. The relation between the membrane protective system and semilethal temperature of Taxus × media leaves as temperature fell[J]. Journalof Southwest China Norm Univ Nat Sci, 2001, 26(4): 452-456.
[7]	吕志鹏. 甘肃南部曼地亚红豆杉引种抗寒性研究[J]. 青海农林科技, 2014(2): 18-20, 25.
	Lv ZP. An anti-cold research of the introduced Taxus × media in southern Gansu[J]. Sci Technol Qinghai Agric For, 2014(2): 18-20, 25.
[8]	谢吉容, 向邓云, 梅虎, 谈锋. 南方红豆杉抗寒性的变化与内源激素的关系[J]. 西南师范大学学报: 自然科学版, 2002, 27(2): 231-234.
	Xie JR, Xiang DY, Mei H, et al. Relationship between changes of cold resistance and endogenous hormone of Taxus chinensis var. mairei chenget L.K.F[J]. Journalof Southwest China Norm Univ Nat Sci, 2002, 27(2): 231-234.
[9]	史喜兵, 刘杰, 张晓申, 等. 利用电导法确定五种耐阴植物的抗寒性[J]. 农业科技通讯, 2013(11): 100-101.
	Shi XB, Liu J, Zhang XS, et al. Determination of cold resistance of five shade-tolerant plants by conductivity method[J]. Bullenin Agric Sci Technol, 2013(11): 100-101.
[10]	张宏涛, 陈纹, 李小伟, 等. 低温胁迫下肋果沙棘试管苗黄酮类化合物合成关键酶的活性[J]. 北方园艺, 2015(10): 5-8.
	Zhang HT, Chen W, Li XW, et al. The activity of key enzymes related to flavonoids in test-tube plantlets of Hippophae neurocarpa under low temperature[J]. North Hortic, 2015(10): 5-8.
[11]	Dong YP, Qu Y, Qi R, et al. Transcriptome analysis of the biosynthesis of anthocyanins in Begonia semperflorens under low-temperature and high-light conditions[J]. Forests, 2018, 9(2): 87. doi: 10.3390/f9020087 URL
[12]	谢腾, 王升, 张山山, 等. 低温胁迫对新疆紫草悬浮细胞次生代谢物的影响[J]. 中草药, 2015, 46(10): 1525-1532.
	Xie T, Wang S, Zhang SS, et al. Effects of low temperature stress on secondary metabolites of Arnebia euchroma suspension cells[J]. Chin Tradit Herb Drugs, 2015, 46(10): 1525-1532.
[13]	Lange BM, Conner CF. Taxanes and taxoids of the genus Taxus-A comprehensive inventory of chemical diversity[J]. Phytochemistry, 2021, 190: 112829. doi: 10.1016/j.phytochem.2021.112829 URL
[14]	Yang YH, Mao JW, Tan XL. Research progress on the source, production, and anti-cancer mechanisms of paclitaxel[J]. Chin J Nat Med, 2020, 18(12): 890-897.
[15]	王亚飞, 王强, 阮晓, 等. 红豆杉属植物资源的研究现状与开发利用对策[J]. 林业科学, 2012, 48(5): 116-125.
	Wang YF, Wang Q, Ruan X, et al. Research status and utilization strategies of rare medicinal plants in Taxus[J]. Sci Silvae Sin, 2012, 48(5): 116-125.
[16]	Ryyppö A, Repo T, Vapaavuori E. Development of freezing tolerance in roots and shoots of Scots pine seedlings at nonfreezing temperatures[J]. Can J For Res, 1998, 28(4): 557-565. doi: 10.1139/x98-022 URL
[17]	张钢, 肖建忠, 陈段芬. 测定植物抗寒性的电阻抗图谱法[J]. 植物生理与分子生物学学报, 2005, 31(1): 19-26.
	Zhang G, Xiao JZ, Chen DF. Electrical impedance spectroscopy method for measuring cold hardiness of plants[J]. Acta Photophysiol Sin, 2005, 31(1): 19-26.
[18]	Repo T, Lappi J. Estimation of standard error of impedance-estimated frost resistance[J]. Scand J For Res, 1989, 4(1/2/3/4): 67-74. doi: 10.1080/02827588909382547 URL
[19]	李亚青, 张钢, 郤书鹏, 等. 白皮松茎和针叶的电阻抗参数与抗寒性的相关性[J]. 林业科学, 2008, 44(4): 28-34.
	Li YQ, Zhang G, Xi SP, et al. Relation between electrical impedance spectroscopy parameters and frost hardiness in stems and needles of Pinus bungeana[J]. Sci Silvae Sin, 2008, 44(4): 28-34.
[20]	王爱芳, 张钢, 魏士春, 等. 不同发育时期樟子松(Pinus sylvestris L.var. mongolica Litv.)的电阻抗参数与抗寒性的关系[J]. 生态学报, 2008, 28(11): 5741-5749.
	Wang AF, Zhang G, Wei SC, et al. Relation between frost hardiness and parameters of electrical impedance spectroscopy in saplings of different development stage of Pinus sylvestris L. var. mongolica Litv[J]. Acta Ecol Sin, 2008, 28(11): 5741-5749.
[21]	Repo T, Zhang G, Ryyppö A, et al. The relation between growth cessation and frost hardening in Scots pines of different origins[J]. Trees, 2000, 14(8): 456-464. doi: 10.1007/s004680000059 URL
[22]	杨雪, 张殿生, 张钢. 抗寒锻炼期间几种梨、苹果枝条的抗寒性和电阻抗图谱比较[J]. 西北农业学报, 2014, 23(4): 60-67.
	Yang X, Zhang DS, Zhang G. Frost hardiness and electrical impedance spectroscopy of shoots in several pear and apple varieties during hardening[J]. Acta Agric Boreali Occidentalis Sin, 2014, 23(4): 60-67.
[23]	Stoll M, Loveys B, Dry P. Hormonal changes induced by partial rootzone drying of irrigated grapevine[J]. J Exp Bot, 2000, 51(350): 1627-1634. pmid: 11006312
[24]	Wilkinson S, Davies WJ. Ozone suppresses soil drying- and abscisic acid(ABA)-induced stomatal closure via an ethylene-dependent mechanism[J]. Plant Cell Environ, 2009, 32(8): 949-959. doi: 10.1111/pce.2009.32.issue-8 URL
[25]	赵树亮. 越橘越冬期枝条生理特性研究[D]. 哈尔滨: 东北农业大学, 2014.
	Zhao SL. Research of physiological and biochemical characteristics in blueberry annual branches during overwintering[D]. Harbin: Northeast Agricultural University, 2014.
[26]	芦站根, 赵昌琼, 谈锋. 不同光照条件下曼地亚红豆杉抗寒力的变化及内源激素调控[J]. 西南师范大学学报: 自然科学版, 2003, 28(1): 122-125.
	Lu ZG, Zhao CQ, Tan F. The change of leaf cold resistance abilities of Taxus × media cv. Hicksii grown at different light condition and regulation of endogenetic hormone[J]. J Southwest China Norm Univ Nat Sci, 2003, 28(1): 122-125.
[27]	于少帅. 南方红豆杉活性成分含量差异分析及其与生态因子的关系研究[D]. 芜湖: 安徽师范大学, 2013.
	Yu SS. Investigation on difference of active compounds contents in Taxus chinensis var. mairei and its relationship with ecological factors[D]. Wuhu: Anhui Normal University, 2013.
[28]	余龙江, 朱敏, 周莹, 等. 茉莉酸甲酯对紫杉醇生物合成的诱导作用[J]. 天然产物研究与开发, 1999, 11(5): 1-7.
	Yu LJ, Zhu M, Zhou Y, et al. The induction effect of methyl-jasmonate on taxol biosynthesis[J]. Nat Prod Res Dev, 1999, 11(5): 1-7.
[29]	Du H, Liu H, Xiong L. Endogenous auxin and jasmonic acid levels are differentially modulated by abiotic stresses in rice[J]. Front Plant Sci, 2013, 4: 397. doi: 10.3389/fpls.2013.00397 pmid: 24130566
[30]	Croteau R, Ketchum REB, Long RM, et al. Taxol biosynthesis and molecular genetics[J]. Phytochem Rev, 2006, 5(1): 75-97. doi: 10.1007/s11101-005-3748-2 pmid: 20622989
[31]	梁研, 郑珩, 吴亮, 等. 类胡萝卜素等物质对灰葡萄孢霉菌产脱落酸的影响[J]. 药物生物技术, 2004, 11(2): 96-98.
	Liang Y, Zheng H, Wu L, et al. Effect of carotenoids on abscisic acid production of Botrytis cinerea[J]. Pharm Biotechnol, 2004, 11(2): 96-98.
[32]	李永波, 樊庆琦, 王宝莲, 等. 植物法呢基焦磷酸合酶基因(FPPS)研究进展[J]. 农业生物技术学报, 2012, 20(3): 321-330.
	Li YB, Fan QQ, Wang BL, et al. Advances in the study of plant farnesyl pyrophosphate synthase genes(FPPS)[J]. J Agric Biotechnol, 2012, 20(3): 321-330.
[33]	Hoffman A, Shock C, Feibert E. Taxane and ABA production in yew under different soil water regimes[J]. Hort Science, 1999, 34(5): 882-885.
[34]	Xu MJ, Jin HH, Dong JF, et al. Abscisic acid plays critical role in ozone-induced taxol production of Taxus chinensis suspension cell cultures[J]. Biotechnol Prog, 2011, 27(5): 1415-1420. doi: 10.1002/btpr.660 URL
[35]	张孟夏, 王燕燕, 于放. 脱落酸调节长春花中单萜吲哚生物碱的生物合成[J]. 分子植物育种, 2019(10): 3371-3377.
	Zhang MX, Wang YY, Yu F. Regulation of abscisic acid on the biosynthesis of monoterpenoid indole alkaloids in Catharanthus roseus[J]. Mol Plant Breed, 2019(10): 3371-3377.

编号 Code number	种名 Species	拉丁名 Latin name	来源 Resource
TY	云南红豆杉	T. yunnanensis	云南腾冲
TCV	南方红豆杉	T. chinensis var. mairei	山西长治
TC	中国红豆杉	T. chinensis（Pilger）Rehd.	甘肃陇南
TM	曼地亚红豆杉	T. × media cv. Hicksii	山东济南
TCU	东北红豆杉	T. cuspidata Sieb. et Zucc	黑龙江牡丹江

编号 Code number	种名 Species	拉丁名 Latin name	来源 Resource
TY	云南红豆杉	T. yunnanensis	云南腾冲
TCV	南方红豆杉	T. chinensis var. mairei	山西长治
TC	中国红豆杉	T. chinensis（Pilger）Rehd.	甘肃陇南
TM	曼地亚红豆杉	T. × media cv. Hicksii	山东济南
TCU	东北红豆杉	T. cuspidata Sieb. et Zucc	黑龙江牡丹江

叶/枝Leaves/branches	种Species	回归方程Logistic equation	决定系数R²	半致死温度 Semi-lethal low temperature LT₅₀/℃
当年生叶 Current-year leaves	云南红豆杉T. yunnanensis	y=75.01/（1+e^-（-3.00-^x^））+24.12	0.989	-3.521
	中国红豆杉T. chinensis（Pilger）Rehd	y=63.37/（1+e^-（-12.00-^x^））+34.25	0.993	-11.600
	南方红豆杉T. chinensis var. mairei	y=72.81/（1+e^-（-12.00-^x^））+21.70	0.996	-11.426
	东北红豆杉 T. cuspidata Sieb. et Zucc	y=63.35/（1+e^-（-12.00-^x^））+36.18	0.992	-9.611
一年生叶 One-year-old leaves	云南红豆杉T. yunnanensis	y=72.15/（1+e^-（-7.00-^x^））+26.41	0.998	-8.802
	中国红豆杉T. chinensis（Pilger）Rehd	y=82.18/（1+e^-（-12.00-^x^））+16.05	0.997	-9.673
	南方红豆杉T. chinensis var. mairei	y=76.03/（1+e^-（-12.50-^x^））+22.12	0.999	-11.225
	东北红豆杉 T. cuspidata Sieb. et Zucc	y=72.79/（1+e^-（-12.00-^x^））+23.22	0.997	-10.937
	曼地亚红豆杉T. × media cv. Hicksii	y=71.34/（1+e^-（-12.00-^x^））+46.00	0.963	-9.106
当年生枝 Current-year branches	云南红豆杉T. yunnanensis	y=30.41/（1+e^-（-3.00-^x^））+69.59	0.981	-6.806
	中国红豆杉T. chinensis（Pilger）Rehd	y=18.59/（1+e^-（-12.00-^x^））+77.98	0.998	-11.103
	南方红豆杉T. chinensis var. mairei	y=16.08/（1+e^-（-10.00-^x^））+78.74	0.994	-11.385
	东北红豆杉 T. cuspidata Sieb. et Zucc	y=16.32/（1+e^-（-12.00-^x^））+82.36	0.940	-12.731
一年生枝 One-year-old branches	云南红豆杉T. yunnanensis	y=29.94/（1+e^-（-2.00-^x^））+68.86	0.915	-7.479
	中国红豆杉T. chinensis（Pilger）Rehd	y=25.11/（1+e^-（-12.00-^x^））+74.89	0.967	-9.626
	南方红豆杉T. chinensis var. mairei	y=17.52/（1+e^-（-11.00-^x^））+82.48	0.987	-12.536
	东北红豆杉 T. cuspidata Sieb. et Zucc	y=19.94/（1+e^-（-12.00-^x^））+80.06	0.934	-14.191
	曼地亚红豆杉T. × media cv. Hicksii	y=24.47/（1+e^-（-15.00-^x^））+73.12	0.917	-14.572

叶/枝Leaves/branches	种Species	回归方程Logistic equation	决定系数R²	半致死温度 Semi-lethal low temperature LT₅₀/℃
当年生叶 Current-year leaves	云南红豆杉T. yunnanensis	y=75.01/（1+e^-（-3.00-^x^））+24.12	0.989	-3.521
	中国红豆杉T. chinensis（Pilger）Rehd	y=63.37/（1+e^-（-12.00-^x^））+34.25	0.993	-11.600
	南方红豆杉T. chinensis var. mairei	y=72.81/（1+e^-（-12.00-^x^））+21.70	0.996	-11.426
	东北红豆杉 T. cuspidata Sieb. et Zucc	y=63.35/（1+e^-（-12.00-^x^））+36.18	0.992	-9.611
一年生叶 One-year-old leaves	云南红豆杉T. yunnanensis	y=72.15/（1+e^-（-7.00-^x^））+26.41	0.998	-8.802
	中国红豆杉T. chinensis（Pilger）Rehd	y=82.18/（1+e^-（-12.00-^x^））+16.05	0.997	-9.673
	南方红豆杉T. chinensis var. mairei	y=76.03/（1+e^-（-12.50-^x^））+22.12	0.999	-11.225
	东北红豆杉 T. cuspidata Sieb. et Zucc	y=72.79/（1+e^-（-12.00-^x^））+23.22	0.997	-10.937
	曼地亚红豆杉T. × media cv. Hicksii	y=71.34/（1+e^-（-12.00-^x^））+46.00	0.963	-9.106
当年生枝 Current-year branches	云南红豆杉T. yunnanensis	y=30.41/（1+e^-（-3.00-^x^））+69.59	0.981	-6.806
	中国红豆杉T. chinensis（Pilger）Rehd	y=18.59/（1+e^-（-12.00-^x^））+77.98	0.998	-11.103
	南方红豆杉T. chinensis var. mairei	y=16.08/（1+e^-（-10.00-^x^））+78.74	0.994	-11.385
	东北红豆杉 T. cuspidata Sieb. et Zucc	y=16.32/（1+e^-（-12.00-^x^））+82.36	0.940	-12.731
一年生枝 One-year-old branches	云南红豆杉T. yunnanensis	y=29.94/（1+e^-（-2.00-^x^））+68.86	0.915	-7.479
	中国红豆杉T. chinensis（Pilger）Rehd	y=25.11/（1+e^-（-12.00-^x^））+74.89	0.967	-9.626
	南方红豆杉T. chinensis var. mairei	y=17.52/（1+e^-（-11.00-^x^））+82.48	0.987	-12.536
	东北红豆杉 T. cuspidata Sieb. et Zucc	y=19.94/（1+e^-（-12.00-^x^））+80.06	0.934	-14.191
	曼地亚红豆杉T. × media cv. Hicksii	y=24.47/（1+e^-（-15.00-^x^））+73.12	0.917	-14.572

种 Species	EL测得的半致死温度 Semi-lethal temperature by EL/℃	EIS-r_e得到的半致死温度 Semi-lethal temperature by EIS-r_e/℃	EIS-τ_m得到的半致死温度 Semi-lethal temperature by EIS-τ_m/℃	EIS-β得到的半致死温度 Semi-lethal temperature by EIS-β/℃
云南红豆杉T. yunnanensis	-7.479	-7.538	-7.287	-8.346
中国红豆杉T. chinensis（Pilger）Rehd	-9.626	-9.923	-9.305	-10.493
南方红豆杉T. chinensis var. mairei	-12.536	-12.073	-12.312	-11.603
东北红豆杉T. cuspidata Sieb. et Zucc	-14.191	-12.043	-11.061	-17.285
曼地亚红豆杉T. × media cv. Hicksii	-14.572	-10.290	-12.087	-11.422
相关系数Correlation coefficent	1	0.801	0.912*	0.734