曼地亚红豆杉(Taxusmediacv “Hicksii”)栽培基质中适宜的锰、锌、铜浓度及采收时期研究

刘柿良， 杨秀利， 马明东， 但 方， 杨 君， 胡 菊， 邬梦希

(四川农业大学风景园林学院， 四川成都 611130)

曼地亚红豆杉(Taxusmediacv“Hicksii”)栽培基质中适宜的锰、锌、铜浓度及采收时期研究

刘柿良， 杨秀利， 马明东*， 但 方， 杨 君， 胡 菊， 邬梦希

(四川农业大学风景园林学院， 四川成都 611130)

曼地亚红豆杉； 紫杉醇； 锰； 锌； 铜; 生长发育； 采收季节； 采收年龄

本课题组已采用“Hicksii”带芽茎段外植体进行腋芽增殖再生, 成功实现其高效快速繁殖[12-13]。然而, 对获得组培苗最佳移植生长环境, 以产生更高含量紫杉醇的研究还未开展。药用植物的生长环境所含微量元素不仅对其生长有着直接的影响[14]，还与其疗效密切相关[15]。因此, 本研究选用3年和5年生“Hicksii”树苗为对象, 研究锰、锌和铜对两种树龄植株的生长及紫杉醇含量的影响, 为曼地亚红豆杉药用原料林培育、采收以及合理施用微肥确定提供借鉴。

1 材料和方法

1.1 研究材料与试验地概况

1.2 试验方法

盆栽基质为河砂和蛭石。河砂消毒后洗净、晾干；蛭石用稀盐酸(HCl)溶液浸泡 72 h 后用水反复冲洗(6次以上)后晾干。将河砂和蛭石各15 kg均匀装入盆中, 盆底垫铺尼龙纱。将树苗根系洗净，于2012年2月上旬移植到内径45 cm、高35 cm的花盆中(带托盘), 每盆栽植树苗2株, 幼苗间互不遮荫。试验大棚透光率为 80%; 大棚内外温度接近, 为20±3℃；相对湿度为70%。所有处理均用去离子水。

营养液为改进Hoagland (霍格兰氏)完全营养液[16]: KNO35 mmol/L、 Ca(NO3)25 mmol/L、 KH2PO41 mmol/L、 MgSO42 mmol/L、 NaFeEDTA 0.1 mmol/L、 HBO346 μmol/L、 NaMoO40.l μmol/L、 MnSO49 μmol/L、 ZnSO40.8 μmol/L、 CuSO40.3 μmol/L。铁(Fe)、硼(B)、钼(Mo)盐浓缩到原标准的1000倍混合放置, Mn、Zn、Cu盐浓缩1000倍单独放置; 其余的大量元素浓缩到100倍低温保存。施用时, 当添加1种不同浓度的微量元素时, 其他2种微量元素以改进的营养液为基础浓度作为基础溶液[10]，其他按配制的浓缩液量取、稀释。2012年2月13日起, 每盆植株浇施营养液500 mL, 每周更换1次营养液。

1.3 HPLC测定条件和参数

1.3.1 流动相选择 HPLC为岛津 LC-20AB 高效液相色谱仪(LC-20AB, Shimadzu Inc., Japan)。色谱柱为phenomenex Luna C 18 (4.6 mm × 250 mm, 5 μm), 流动相为乙腈 ∶水 ∶异丙醇=45 ∶ 55 ∶ 1 (v ∶ v ∶v), 检测波长为227 nm, 流速1.0 mL/min, 柱温30℃, 进样量为10 μL时, 保留时间30 min。

1.3.2 标准液制备 准确称取紫杉醇标准品10.0000 mg, 置于25 mL容量瓶中加甲醇溶解至刻度, 得到0.4 mg/mL 标准品储备液。

取标准品制备测定液，峰面积测定值相对标准偏差(RSD)值为0.57%, 表明试验仪器精密度良好。将标准品溶液于4℃保存, 于0、 1、 2、 4、 8、 16 h取样测定, 峰面积RSD为0.56%, 表明标准品溶液在16 h内稳定性良好。

精确称取3年生曼地亚红豆杉样品粉末5份, 各1.000 g, 按样品溶液的制备方法制备待测溶液进行测定，5份样品峰面积相对标准偏差为0.84%, 表明试验重复性良好。

1.3.4 加样回收考察 取已施加Hoagland标准溶液的3年生曼地亚红豆杉样品粉末9份, 各1.0 g, 每3份为一组, 各加入紫杉醇标准品粉末, 按样品溶液的配制方法制取待测液10 μL, 进样分析并计算回收率。高、中、低三种水平的加样回收率平均值分别为99.69%、 99.02%和99.00%,RSD分别为1.22%、 0.24%和0.52%(n=3)，均在允许范围内, 说明该法测定曼地亚红豆杉枝叶紫杉醇含量结果可靠。

1.4 植株采样与测定

紫杉醇(%)=(S样×m标×V样×标准品纯度)/(S样×m标×V样) ×100

式中: S 指色谱图峰面积； m 指样品或标准品质量； V 指溶液体积。

1.5 生长指标测定

1.6 数据处理

采用SPSS 17.0 (SPSS, Chicago, USA)软件进行单因素方差分析(one-way ANOVA), 并用最小显著差数法(LSD)检验差异性。采用生长综合指标法(Overall Desirability, OD)对生长进行综合评定, OD即为同处理各生长指标测定值与参照测定值(本试验为Mn0、Zn0、Cu0处理)的比值之和[18]。

2 结果与分析

2.1 微量元素对曼地亚红豆杉“Hicksii”树苗生长的影响

移栽后未经处理的健康3年和5年生曼地亚红豆杉“Hicksii”树苗的基础生长值为: 3年生植株株高28.13 cm, 地径5.02 cm, 新梢长度1.38 cm, 枝叶鲜重24.67 g/plant, 根系鲜重10.28 g/plant, 枝叶干重2.36 g/plant, 根系干重1.24 g/plant; 5年生植株株高31.08 cm, 地径6.11 cm, 新梢长度0.74 cm, 枝叶鲜重31.27 g/plant, 根系鲜重22.32 g/plant, 枝叶干重3.15 g/plant, 根系干重2.28 g/plant。

表1 不同施锰量对曼地亚红豆杉“Hicksii”树苗生长的影响

注(Note): OD—综合指标 Overall desirability. 数值后不同大写字母表示相同处理下3年和5年生树苗间差异显著(P<0.05); 不同小写字母表示3年和5年生树苗在不同处理间差异显著(P<0.05) Values followed by different capital letters are significant in three-year and five-year old seedlings under the same treatments (P<0.05); Values followed by different small letters are significant among different treatments in three-year and five-year old seedlings (P<0.05).

2.1.2 锌 随着Zn施加量的增大, 两种树苗的生长指标值均呈先升高后降低的趋势。3年生“Hicksii”树苗的株高和地径在Zn 0.8 μmol/L最大, 而新梢长度、枝叶鲜重(干重)和根系鲜重(干重)在Zn 0.4 μmol/L最大, 且OD值为8.93 (表2)。5年生树苗的株高、新梢长度、根系鲜重(干重)在Zn 0.4 μmol/L处理时最大, OD值达到最高(8.71)。比较3年和5年生树苗可知, 5年生树苗的株高、地径及鲜重(干重)均显著高于3年生树苗(P<0.05), 而新梢长度却相反。

表2 不同施锌量对曼地亚红豆杉“Hicksii”树苗生长的影响

注(Note): OD—综合指标 Overall desirability. 数值后不同大写字母表示相同处理下3年和5年生树苗间差异显著(P<0.05); 不同小写字母表示3年和5年生树苗在不同处理间差异显著(P<0.05) Values followed by different capital letters are significant in three-year and five-year old seedlings under the same treatments (P<0.05); Values followed by different small letters are significant among different treatments in three-year and five-year old seedlings (P<0.05).

2.2 微量元素对曼地亚红豆杉“Hicksii”树苗紫杉醇含量的影响

移栽后未经处理的健康 3 年和5年生曼地亚红豆杉“Hicksii”树苗的紫杉醇基础值为: 3 年生植株枝叶紫杉醇含量为 2.16%, 5 年生植株枝叶紫杉醇含量为 3.02%。

表3 不同施Cu量对曼地亚红豆杉“Hicksii”树苗生长的影响

注(Note): OD—综合指标 Overall desirability. 数值后不同大写字母表示相同处理下3年和5年生树苗间差异显著(P<0.05); 不同小写字母表示3年和5年生树苗在不同处理间差异显著(P<0.05) Values followed by different capital letters are significant in three-year and five-year old seedlings under the same treatments (P<0.05); Values followed by different small letters are significant among different treatments in three-year and five-year old seedlings (P<0.05).

图1 不同施锰量曼地亚红豆杉“Hicksii” 3年(a) 和5年(b) 生树苗紫杉醇含量Fig.1 Taxol content in 3-year (a) and 5-year (b) “Hicksii” scalps dependent on Mn application rates

图2 不同施锌量曼地亚红豆杉“Hicksii” 3年(a)和5年(b)生树苗紫杉醇含量Fig.2 Taxol content in three-year (a) and five-year old (b) Hicksii scalpels dependent on Zn application rates

图3 不同施铜量对曼地亚红豆杉“Hicksii” 3年(a) 和5年(b)生树苗紫杉醇含量Fig.3 Taxol content in three-year (a) and five-year old (b) scaples of T. media cv “Hicksii” dependent on Cu application rate

2.2.3 铜 铜处理紫杉醇积累量6月最低值3年生幼树为 1.953.43，5年生为2.463.68。3年和5年生树苗枝叶紫杉醇积累量6月份比3月份显著下降39.02%68.69% 和 32.79%44.97% (P<0.05)。

3 讨论与结论

3.1 适量微量元素能显著提高曼地亚红豆杉“Hicksii”树苗的生长

3.2 适量微量元素能显著提高树苗紫杉醇的积累

红豆杉细胞合成紫杉醇的能力除了受遗传特性影响之外, 外界逆境信号激活细胞的防御反应是改变细胞合成紫杉醇速率和积累的重要途径[24]。本试验中, 3种微量元素对树苗紫杉醇含量影响效果的顺序为Mn>Zn>Cu，与王建安等[31]的研究结论相似, 盾叶薯蓣(Dioscoreazingiberensis)薯蓣皂苷元对施Mn肥的敏感性高于Zn。可能原因是Mn对代谢合成酶具有高度专一性, 促进植物吸收土壤中氮和磷, 高Mn可抑制吲哚乙酸(IAA)氧化酶活性, 从而促进产量增加[32]。Zn通过参与叶绿素合成, 改变光能利用率, 影响有机物合成[14,17]。Liu[20]指出, Zn2+可能首先与细胞膜上受体的结合, 改变膜离子通道, 促使诱导过程迅速完成。Zn2+与其受体高度亲合, 引起位于原生质膜上的离子通道改变, 引起 Ca2+内流并迅速发生 H2O2, 胞内依赖 Ca2+的蛋白质磷酸化作用, 激活核内防御基因(如TCH基因), 引起防御反应, 诱导合成植保素酶合成植保素而完成信号传递作用。当氧迸发(OXB)达到最大时, 胞内苯丙氨酸鲜氨酶(PAL)活性、Taxol 和酚的合成开始激活[20-21,23]。因此, 防御应答的强弱可能与 Taxol 的合成有密切的正相关性。而Cu影响紫杉醇积累主要是通过与过氧化氢酶(Catalase, CAT)活性中心结合, 影响酶与底物的结合能力[33]。同时, 高浓度的 Cu2+可能会导致细胞活性氧(ROS)的积累。ROS积累和次生代谢产物都是植物在响应外界刺激过程中强有力的化学武器, Cu2+诱导次生代谢产物的合成而抗氧化剂的加入则阻止次生代谢物的生成[15,23]。ROS可能作为第二信使调控防卫基因的表达和启动与植保素合成基因相关的基因转录, 因此推断ROS 对植保素的调控作用可能发生在转录水平上, 也可能使 mRNA 稳定性有所提高[14]。

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Suitable Mn, Zn and Cu concentrations in the culture media and proper harvest time forTaxusmediacv“Hicksii”

LIU Shi-liang, YANG Xiu-li, MA Ming-dong*, DAN Fang, YANG Jun, HU Jü, WU Meng-xi

(CollegeofLandscapeArchitecture,SichuanAgriculturalUniversity,Chengdu,Sichuan611130,P.R.China)

【Objectives】 Paclitaxel (Taxol®) shows excellent antitumor activity against breast cancer, ovarian cancer and non-small cell lung cancer, is one of the best chemotherapeutic agents developed from plant sources. Taxus production has

attention as an important natural material for extracting taxol. The purpose of this study was to study the effects of mircoalements (Mn, Zn and Cu) on the growth and paclitaxel accumulation in seedlings of Taxus, providing a basis for the optimum harvest period and the rational application of micronutrient fertilizer.【Methods】 A sand culture experiment was conducted and the seedlings of 3-year-old and 5-year-oldTaxusmediacv“Hicksii” were growen. Five concentration of Mn 0, 4.50, 9.00, 13.50 and 18.00 μmol/L, Zn 0, 0.15, 0.30, 0.45 and 0.60 μmol/L, and Cu: 0, 0.40, 0.80, 1.20 and 1.60 μmol/L were designed, each treatment replicated three times. Branches and leaves of plants were collected monthly from March 15, 2012 to October 15, 2012. The paclitaxel accumulation contents was determined with high-performance liquid chromatography (HPLC) method, the seedling growth were observed in November 15, 2012, the biomass were weighed at the same time. 【Result】 1) Mn, Zn and Cu treatment levels significantly affect the growth and paclitaxel contents, increase the plants heights, crossing diameters and shoot length, increase the weights of branches, leaves and roots of the saplings. For the growth of seedlings, the optimum treatment is Cu 0.15 μmol/L for 5-year-old seedlings and 0.30 μmol/L for 3-year-old seedlings, Mn 9.00 μmol/L and Zn 0.40 μmol/L for the two ages of seedlings. For paclitaxel accumulation, the optimum level is Zn 0.40 μmol/L and Cu 0.30 μmol/L for the both ages, and Mn 9.00 μmol/L for the 3-year-old and 4.50 μmol/L for 5-year-old saplings. When the micronutrient treatment levels exceeded the optimum one, the paclitaxel accumulation will be inhibited and the higher the treatment levels, the stronge the inhibition. 2) The paclitaxel content in 5-year-old plants are significantly higher than in 3-year-old ones under all the treatments, and the differences are significant at harvest. 3) The paclitaxel content start to decrease from March to June, and is lowest in June, then increase and keep relatively stabile in September and October. The effects of Mn on the paclitaxel accumulation is higher than Zn and higher than Cu, no matter their treatment concentrations.【Conclusions】 The paclitaxel content in 5-year-old saplings is significantly higher than the 3-year-old ones, the proper harvest time for obtaining high paclitaxel content of saplings is September and October. The optimum treatment level is Mn 9.00 μmol/L, Zn 0.40 μmol/L and Cu 0.15-0.30 μmol/L.

Taxusmediacv “Hicksii”; paclitaxel; Mn; Zn; Cu; ontogenetic development; gathering season; gathering year

2014-01-06 接受日期： 2014-02-25

四川省教育厅重点攻关项目“曼地亚红豆杉快繁技术研究”(2003A023)资助。

刘柿良(1986—)，男，四川南充人，博士研究生，主要从事植物营养及生理生态等方面研究。E-mail： liushiliang9@163.com * 通信作者 E-mail： mmingdong1958@gmail.com

S791.49; S725.5

A

1008-505X(2015)02-0439-10