張玉瓊，李勇,，周建輝，陳娜，王梅方，董召榮，高翠云，仲延龍 安徽農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院，安徽 合肥 006 安徽農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，安徽 合肥 006 廣西農(nóng)業(yè)科學(xué)院蔬菜研究所，廣西 南寧 50007

前體物對(duì)石蒜懸浮細(xì)胞生長(zhǎng)和生物堿積累的影響

張玉瓊1，李勇1,2，周建輝3，陳娜1，王梅方1，董召榮2，高翠云1，仲延龍1
1 安徽農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院，安徽 合肥 230036 2 安徽農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，安徽 合肥 230036 3 廣西農(nóng)業(yè)科學(xué)院蔬菜研究所，廣西 南寧 530007

為探究苯丙氨酸、酪氨酸和酪胺3種前體物對(duì)石蒜懸浮細(xì)胞系生長(zhǎng)和生物堿積累的影響。通過向培養(yǎng)基添加不同濃度的3種前體物，以及同時(shí)添加苯丙氨酸和酪氨酸，考察其對(duì)細(xì)胞生長(zhǎng)量及細(xì)胞中生物堿累積的影響。結(jié)果表明：苯丙氨酸對(duì)細(xì)胞的生長(zhǎng)和生物堿的積累影響不明顯；酪氨酸和酪胺作用顯著：添加200 μmol/L酪氨酸，細(xì)胞中生物堿的含量是對(duì)照組的2.56倍，其中力可拉敏和加蘭他敏含量為3.77 mg/g和4.46 mg/g，分別是對(duì)照組的6.61倍和6.97倍；添加200 μmol/L酪胺，細(xì)胞中生物堿含量是對(duì)照組的2.63倍，力可拉敏和加蘭他敏含量為4.45 mg/g和5.14 mg/g分別是對(duì)照組的9.08倍和9.18倍；在200 μmol/L酪氨酸的基礎(chǔ)上添加苯丙氨酸沒有明顯的增效作用。表明添加酪氨酸和酪胺對(duì)細(xì)胞生長(zhǎng)及生物堿生物合成具有顯著的促進(jìn)作用

石蒜，懸浮細(xì)胞系，前體物，生物堿

石蒜Lycoris radiata是石蒜科Amaryllidaceae石蒜屬Lycoris Herb.多年生單子葉植物，具有重要的藥用價(jià)值。石蒜中含有多種具有生物活性的生物堿，如石蒜倫堿 (Lycorenine)，加蘭他敏(Galanthamine)、表加蘭加敏 (Epigalanthamine)、普魯維因 (Pluviine)、力可拉敏 (Lycoramine)、小星蒜堿 (Hippeastrine)、石蒜堿 (Lycorine)、漳州水仙堿 (Tazettine) 等[1-2]。其中加蘭他敏是乙酰膽堿酯酶的可逆抑制劑，可以被用于治療阿爾茨海默病 (Alzheimers disease)[3-5]；石蒜堿具有抗癌活性和潛在的抗SAS-CoV病毒的活性[6]。這些藥用成分主要取自石蒜鱗莖，由于石蒜的生長(zhǎng)發(fā)育受環(huán)境影響較大，生長(zhǎng)緩慢[7-8]，且生物堿特別是加蘭他敏含量較低[9]。所以，大規(guī)模生產(chǎn)生物堿受制于石蒜野外資源。

石蒜生物堿產(chǎn)業(yè)化主要有兩條途徑：一是化學(xué)合成方法，如加蘭他敏合成方法[10-12]，但步驟復(fù)雜、得率低且成本高；二是利用植物組織或細(xì)胞培養(yǎng)調(diào)控次生代謝進(jìn)程，提高目標(biāo)化合物的產(chǎn)量。這一途徑具有生產(chǎn)周期較短,利于大規(guī)模培養(yǎng),可人為調(diào)控細(xì)胞生長(zhǎng)與獲得高產(chǎn)細(xì)胞株等優(yōu)點(diǎn)，已成為開發(fā)藥用植物資源的重要途徑。通過間歇浸入技術(shù) (Temporary immersion technology) 培養(yǎng)雪片蓮屬Leucojum aestivum L.芽組織，加蘭他敏和力可拉敏最高可達(dá)256 μg/RITA和1 699 μg/RITA[13]。培養(yǎng)基中添加蔗糖、植物生長(zhǎng)調(diào)節(jié)因子或茉莉酸甲酯 (MeJA)，可明顯促進(jìn)生物堿的生物合成[14-15]。此外，在培養(yǎng)體系中添加生物堿的前體能夠提升產(chǎn)物合成的水平。有研究顯示石蒜生物堿的合成前體為苯丙氨酸和酪胺[4-16]，D-苯丙氨酸和酪氨酸對(duì)培養(yǎng)物的次生代謝和生長(zhǎng)均有顯著的影響[17-19]。目前還沒有石蒜細(xì)胞懸浮培養(yǎng)體系及生物堿生物合成調(diào)控的報(bào)道，本實(shí)驗(yàn)在周建輝等[20]的基礎(chǔ)上建立石蒜懸浮細(xì)胞培養(yǎng)體系，探究苯丙氨酸、酪氨酸和酪胺3種前體物對(duì)石蒜懸浮細(xì)胞系的生長(zhǎng)和加蘭他敏、石蒜堿和力可拉敏合成的影響，以期獲得細(xì)胞生長(zhǎng)和目標(biāo)化合物較高的培養(yǎng)體系，同時(shí)為進(jìn)一步研究石蒜生物堿的合成途徑奠定基礎(chǔ)。

1 材料與方法

1.1 材料與儀器

石蒜懸浮細(xì)胞系：將疏松、增殖力強(qiáng)的石蒜愈傷組織夾碎轉(zhuǎn)接到液體培養(yǎng)基中，經(jīng)過多次繼代培養(yǎng)，待懸浮細(xì)胞系均一性較好時(shí)可作為試驗(yàn)研究材料。

儀器與試劑：Agilent 1 200高效液相色譜儀，Agilent 1 200系列泵，可變波長(zhǎng)檢測(cè)器，TUl8l0紫外可見分光光度計(jì)(北京普析通用儀器有限責(zé)任公司)，320-S pH計(jì)(Mettler Toledo)，HR-120電子天平(A&D公司)。加蘭他敏、力可拉敏和石蒜堿購(gòu)于株洲大有生物技術(shù)有限公司(純度≥98%)。甲醇和乙腈 (色譜級(jí)，美國(guó)Tedia公司)。

1.2 方法

1.2.1 細(xì)胞生物量的測(cè)定

將細(xì)胞培養(yǎng)液于真空泵下抽濾，去離子水洗滌2–3次，濾紙吸干表面水分，即為細(xì)胞培養(yǎng)物的鮮重 (Fresh weight, FW)。懸浮細(xì)胞的增長(zhǎng)率=(試驗(yàn)后的鮮重－初始鮮重)/初始鮮重× 100%。將細(xì)胞培養(yǎng)物在80 ℃烘干2 h，60 ℃干燥至恒重，冷卻稱重為干重 (Dry weight, DW)，重復(fù)3次取平均值。

1.2.2 石蒜懸浮細(xì)胞系生物堿含量的測(cè)定

按照李明凱等[9]的提取與檢測(cè)方并改進(jìn)優(yōu)化。將烘干的懸浮細(xì)胞研磨成粉末過篩，稱取0.5 g，加入95%乙醇5 mL，320 W微波爐處理1 min，浸取1 h，減壓濃縮至干，將濃縮物用5 mL 1 mol/L HCl溶解，用無(wú)水碳酸鈉調(diào)pH至9.5，用三氯甲烷萃取3次合并三氯甲烷相，總生物堿在234 nm測(cè)定總生物堿含量。三氯甲烷減壓濃縮至干后，溶于0.5 mL甲醇，0.22 μm微孔濾膜過濾即為待測(cè)生物堿樣品。

HPLC分析方法采用文獻(xiàn)[9]的方法。色譜柱為ZORBAX ODS-C18 (150 mm×4.6 mm，5 μm, Agilent公司)；流動(dòng)相：A：0.9%三乙胺水溶液(pH 8.0)，B：乙腈，采用梯度洗脫。流速為1 mL/min，進(jìn)樣量為10 μL，檢測(cè)波長(zhǎng)為234 nm，檢測(cè)溫度為室溫。標(biāo)準(zhǔn)曲線為：以加蘭他敏(X1)、力克拉敏 (X2) 和石蒜堿 (X3) 為對(duì)照品，得線性回歸方程為：加蘭他敏，Y1=29.17X1＋28.18，R1=0.9995；力可拉敏，Y2=21.83X2－1.105, R2=0.9995；石蒜堿，Y3=14.24X3－21.42, R3=0.9996。Y：峰面積，X：生物堿含量 (mg/L)。實(shí)驗(yàn)數(shù)據(jù)采用Microsoft Excel 2003軟件處理分析。

2 結(jié)果與分析

圖1 苯丙氨酸對(duì)細(xì)胞生長(zhǎng)和生物堿含量的影響Fig. 1 Effect of phenylalanine on the growth of Lycoris radiata suspension cells and the content alkaloids in the cells. The addition of phenylalanine had no significant effect the growth of the cells and the content alkaloids in the cells compared to the control group.

圖2 酪氨酸對(duì)細(xì)胞生長(zhǎng)和生物堿含量的影響Fig. 2 Effect of tyrosine on the growth of Lycoris radiata suspension cells and the content alkaloids in the cells. The low concentration of tyrosine significantly increased the growth of the cells and the content of alkaloids in the cells compared to the control group. And the best concentration of tyrosine for the cells was 200 μmol/L.

圖3 酪氨酸對(duì)石蒜懸浮細(xì)胞中的3種生物堿的含量的影響Fig. 3 Effect of tyrosine on the content of three kinds alkaloids in L. radiate suspension cells. Tyrosine had no effect on the accumulation of lycorine. Tyrosine at appropriate concentration (200 μmol/L) promoted the accumulation of lycoramine and galanthamine, but the effect of higher concentration of tyrosine on the accumulation of lycoramine and galanthamine weaken gradually.

2.1 苯丙氨酸對(duì)細(xì)胞生長(zhǎng)和生物堿含量的影響

實(shí)驗(yàn)結(jié)果表明，苯丙氨酸對(duì)石蒜懸浮細(xì)胞系的生長(zhǎng)和生物堿的合成都沒有明顯的影響(圖1)。添加不同濃度的苯丙氨酸，石蒜懸浮細(xì)胞的生長(zhǎng)情況與對(duì)照組的差異不明顯，苯丙氨酸不能夠促進(jìn)培養(yǎng)物的生長(zhǎng)。并且，處理組的生物堿的含量與對(duì)照組也沒有明顯的差異?？赡苁窃谑馍飰A合成途徑中，苯丙氨酸不是合成途徑中的關(guān)鍵化合物，合成途徑中存在著其他化合物調(diào)節(jié)著生物堿合成的進(jìn)行。

2.2 酪氨酸對(duì)細(xì)胞生長(zhǎng)和生物堿含量的影響

酪氨酸對(duì)懸浮細(xì)胞系的生長(zhǎng)和生物堿的積累有較大的影響 (圖2)。低濃度的酪氨酸明顯促進(jìn)細(xì)胞生長(zhǎng)。當(dāng)酪氨酸的濃度為200 μmol/L時(shí)懸浮細(xì)胞系的鮮重增加量達(dá)到最大，繼續(xù)增加酪氨酸的濃度，細(xì)胞鮮重增加幅度減小。低濃度的酪氨酸對(duì)生物堿積累有明顯的促進(jìn)作用，隨著酪氨酸的濃度增加，總生物堿含量減小，當(dāng)酪氨酸濃度為200 μmol/L時(shí)生物堿的積累量達(dá)到最大。

酪氨酸對(duì)石蒜懸浮細(xì)胞的3種生物堿積累的影響不同 (圖 3)。實(shí)驗(yàn)結(jié)果表明：酪氨酸對(duì)石蒜堿的含量沒有影響，對(duì)力可拉敏和加蘭他敏的含量影響比較明顯。當(dāng)酪氨酸濃度為200 μmol/L時(shí)，力可拉敏的含量可達(dá)3.77 mg/g，是對(duì)照組的6.61倍，加蘭他敏可達(dá)4.46 mg/g，是對(duì)照組的6.97倍，力可拉敏和加蘭他敏的含量都遠(yuǎn)高于對(duì)照組。

2.3 酪胺對(duì)細(xì)胞生長(zhǎng)和生物堿含量的影響

酪胺對(duì)懸浮細(xì)胞系的生長(zhǎng)影響較大 (圖4)，低濃度的酪胺對(duì)懸浮細(xì)胞的生長(zhǎng)促進(jìn)作用明顯。酪胺對(duì)總生物堿的積累亦有較大的影響，隨著酪胺濃度的增加，總生物堿含量增加，當(dāng)酪胺濃度高于150 μmol/L，總生物堿積累增幅減小，酪胺濃度為250 μmol/L時(shí)，總生物堿含量是對(duì)照組的2.63倍。

酪胺對(duì)細(xì)胞中3種生物堿含量的影響不同，對(duì)石蒜堿的含量影響不大，對(duì)力可拉敏和加蘭他敏的含量影響較大。酪胺濃度為200 μmol/L時(shí)，力可拉敏的含量可達(dá)4.45 mg/g，是對(duì)照組的9.08倍。酪胺濃度為150 μmol/L時(shí)，加蘭他敏含量可達(dá)5.14 mg/g，是對(duì)照組的9.18倍，力可拉敏和加蘭他敏的含量都遠(yuǎn)高于對(duì)照組 (圖5)。

圖4 酪胺對(duì)石蒜懸浮細(xì)胞系的影響Fig. 4 Effect of tyramine on the growth of Lycoris radiata suspension cells and the content alkaloids in the cells. The low concentration of tyramine significantly increased the growth of the cells compared to the control group. And the best concentration of tyramine for the cells was 200 μmol/L.

圖5 酪胺對(duì)石蒜懸浮細(xì)胞中3種生物堿含量的影響Fig. 5 Effect of tyramine on the content of three kinds alkaloids in suspension cells. Tyramine also had no significant effect on accumulation of lycorine. The highest level of lycoramine and galanthamine was obtained by treated with 150 μmol/L and 200 μmol/L of tyramine, respectively.

2.4 苯丙氨酸和酪氨酸對(duì)細(xì)胞生長(zhǎng)和生物堿含量的影響

生物堿合成有共同的前體苯丙氨酸和酪氨酸[16]。試驗(yàn)發(fā)現(xiàn)，同時(shí)添加苯丙氨酸和酪氨酸對(duì)生長(zhǎng)和生物堿的合成影響較小，與單獨(dú)添加酪氨酸組相比差異不明顯 (表1)。

表1 苯丙氨酸和酪氨酸對(duì)細(xì)胞生長(zhǎng)和生物堿含量的影響Table 1 Effects of phenylalanine combined with tyrosine on the growth of Lycoris radiata suspension cells and the content alkaloids in the cells

3 討論

實(shí)驗(yàn)結(jié)果表明，苯丙氨酸對(duì)石蒜懸浮細(xì)胞的生長(zhǎng)和生物堿積累影響不明顯；酪氨酸和酪胺處理，細(xì)胞鮮重和3種生物堿含量要高于對(duì)照組。低濃度的酪氨酸和酪胺能夠促進(jìn)細(xì)胞生長(zhǎng)和生物堿合成。當(dāng)酪氨酸濃度大于200 μmol/L時(shí)，細(xì)胞鮮重增加的趨勢(shì)減緩，生物堿含量降低；當(dāng)酪胺濃度大于 150 μmol/L時(shí)，細(xì)胞鮮重和生物堿含量增加的趨勢(shì)減緩。但在相同的濃度下，酪胺促進(jìn)細(xì)胞生長(zhǎng)的作用要強(qiáng)于酪氨酸。酪氨酸可在相關(guān)酶的作用下轉(zhuǎn)化為其他物質(zhì)進(jìn)入其他途徑中[16-21]。在已發(fā)表的文獻(xiàn)中，精胺、MeJA、黃體酮、酪蛋白水解物、NO和酵母提取物均能促進(jìn)石蒜科植物芽的生長(zhǎng)，水楊酸和高濃度的茉莉酸甲酯則抑制生長(zhǎng)[22-23]。蔗糖能增加培養(yǎng)物存活率，2,4-D會(huì)降低存活率，但是將2,4-D和BA同時(shí)加入會(huì)促進(jìn)愈傷和根的生成[13]。

加蘭他敏和力可拉敏的積累量最高時(shí)(5.14 mg/g，4.45 mg/g) ，酪胺濃度分別為150 μmol/L、200 μmol/L。同時(shí)添加苯丙氨酸(20 μmol/L) 和酪氨酸 (200 μmol/L) 時(shí)，總生物堿含量最高。Schumann等[24]和Ivanov等[13]獲得的加蘭他敏分別為2.40 mg/g DW和256 μg/RITA，后者獲得的石蒜堿高達(dá)1 699 μg/RITA。Georgiev等[25]培養(yǎng)的雪片蓮芽組織中加蘭他敏含量1.7 mg/L，石蒜堿為8.3 mg/L。茉莉酸甲酯、NO和酵母提取物均能促進(jìn)加蘭他敏的合成，茉莉酸甲酯和酵母提取物對(duì)石蒜堿的合成也有促進(jìn)作用，然而水楊酸、NO和酵母提取物對(duì)力可拉敏的合成均有抑制作用[22]。25 μmol/L的茉莉酸甲酯處理后，水仙細(xì)胞和培養(yǎng)基中的加蘭他敏的含量約8.1 mg/g DW和5.0 mg/g DW[14]。在本實(shí)驗(yàn)中，懸浮細(xì)胞中的生物堿含量與已發(fā)表的文獻(xiàn)中生物堿含量相比均要高出很多。因此，在本實(shí)驗(yàn)和前人研究的基礎(chǔ)上可以建立用于大規(guī)模制備生物堿的培養(yǎng)體系。

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(本文責(zé)編 陳宏宇)

Effect of precursor on growth and accumulation of alkaloids of Lycoris radiata suspension cells

Yuqiong Zhang1, Yong Li1,2, Jianhui Zhou3, Na Chen1, Meifang Wang1，Zhaorong Dong2, Cuiyun Gao1, and Yanlong Zhong1
1 School of Life Sciences, Anhui Agricultural University, Hefei 230036, Anhui, China 2 School of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China 3 Vegetable Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, Guangxi, China

In order to investigate the effects of phenylalanine, tyrosine and tyramine on the growth of Lycoris radiata suspension cells and the accumulation of alkaloids, the growth quantity of the cells as well as the content of alkaloids in cells were determined, which were treated with above three kinds of precursors alone and phenylalanine combined with tyrosine respectively. The results indicate that the addition of phenylalanine alone and addition of phenylalanine on the basis of tyrosine at high concentration (200 μmol/L) had no significant effect on the growth of Lycoris radiata suspension cells and the content of alkaloids in cells; whereas tyrosine and tyramine promoted the growth of the cells and alkaloids accumulation. Treated with tyrosine at high concentration (200 μmol/L), the content of alkaloids of the cells was 2.56-fold higher than that of the control group, the amounts of lycoramine (3.77 mg/g) and galanthamine (4.46 mg/g) were 6.61-fold and 6.97-fold higher than that of the control group, respectively. When treated with tyramine (200 μmol/L), the amount of alkaloids in Lycoris radiata suspension cells was 2.63-fold higher than that of the control group, and the amounts of lycoramine (4.45 mg/g) and galanthamine (5.14 mg/g) were 9.08-fold and 9.18-fold higher than that of the control group, respectively. The above results demonstrate that adding tyrosine and tyramine in the media significantly promoted the growth of the Lycoris radiata suspension cells and alkaloids accumulation in the cells

Lycoris radiata, suspension cell line, precursors, alkaloids

July 9, 2013; Accepted: September 5, 2013

Zhaorong Dong. Tel: +86-551-65786955; E-mail: d3030@163.com

張玉瓊, 李勇, 周建輝, 等. 前體物對(duì)石蒜懸浮細(xì)胞生長(zhǎng)和生物堿積累的影響. 生物工程學(xué)報(bào), 2014, 30(2): 247–254.

Zhang YQ, Li Y, Zhou JH, et al. Effect of precursor on growth and accumulation of alkaloids of Lycoris radiata suspension cells. Chin J Biotech, 2014, 30(2): 247–254.

Supported by: Key Technologies Research and Development Program of China (No. 2013BAJ10B12), Natural Science Foundation of Anhui Province (No. 1208085mc48).

國(guó)家科技支撐計(jì)劃項(xiàng)目(No. 2013BAJ10B12)，安徽省自然科學(xué)基金 (No. 1208085mc48) 資助。

時(shí)間：2013-10-17 網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/11.1998.Q.20131017.1242.004.html