缬草细胞培养合成缬草素的研究
摘要
缬草素（Valtrate）具有镇静安神、抗肿瘤、增加冠脉流量、抗心律失常、胃肠平滑肌解痉等突出的药理活性。缬草不易人工栽培，且药用成分含量很低，使得提取缬草素的成本高。本论文采用细胞组织培养、GC-MS、HPLC-PDA、Plackett–Burman主因素筛选、Box–Behnken响应面分析、RT-PCR等研究方法，对缬草细胞培养合成缬草素进行研究，分析了3种缬草的主要化学活性成分、建立了缬草素高产细胞培养体系、优化了生物合成缬草素条件并建立动力学模型、对缬草素生物合成调控因子优化，并分析了缬草素生物合成调控因子对dxr基因表达的影响，研究结果表明：
从缬草根部提取物中鉴定出4种环烯醚萜三酯（缬草素、Isovaltrate、Diavaltrate和Acevaltrate），其中最具药用活性和强极性的缬草素在黑水缬草中含量最高。在GC-MS分析，从黑水缬草中鉴定出43种化合物，所含的Isobornylacetate（27.6%）在已报道的缬草属植物挥发油中含量最高；北缬草中鉴定出44种化合物，含量最高的为α-Selinene（16.7%）；毛节缬草中鉴定出39种化合物，含量最高的为Bornyl acetate（39.1%）。
通过筛选外植体、基础培养基和激素组合，诱导了黑水缬草愈伤组织和不定根，分析了愈伤组织、固体不定根、悬浮细胞和悬浮不定根4种培养体系。发现悬浮不定根细胞系中缬草素含量最高，以其为基础建立了缬草素高产细胞培养体系。建立了悬浮不定根培养体系的组织生长、缬草素合成和基质消耗动力学模型，根据这3个动力学模型可定量评估产缬草素效果，优化培养工艺流程和控制培养条件。通过Plackett–Burman主因素筛选试验的结果表明：摇床转速、NH4NO3和KH2PO4浓度对于黑水缬草悬浮不定根培养体系有显著影响（P＜0.01）。进一步通过Box–Behnken设计的响应面分析，得到最佳水平为摇床转速=76r/min，NH4NO3浓度=0.0069mol/L，KH2PO4浓度=0.0436mol/L。在此条件下，实际获得缬草素含量最大值为8.36±0.20mg/g。
调控因子促进缬草素合成的试验结果表明，3种诱导子（茉莉酸甲酯、茉莉酸和壳聚糖）对缬草素积累具有显著的促进作用。应用香茅醛进行前体饲喂，发现在低浓度条件下，可以促进缬草素的合成。通过响应面分析，得到了3种诱导子的最优浓度组合：茉莉酸甲酯=419.6mg/L、茉莉酸=102.8mg/L、壳聚糖=29.0mg/L。在此条件下，缬草素含量的实际最大值为17.40±0.26mg/g。3种诱导子对缬草素的积累具有很强的协同作用。
克隆出的黑水缬草中萜类代谢关键酶—5-磷酸脱氧木酮糖还原异构酶（DXR）的cDNA片段基因编码区长为1425bp，编码474个氨基酸残基。原核表达证明，该基因编码蛋白的分子量约为50.98kD。dxr基因在缬草组织中的表达强弱为：不定根＞野生植株根和根茎＞野生植株叶片＞悬浮细胞。调控因子（茉莉酸甲酯、茉莉酸和壳聚糖）对dxr基因表达的影响与对缬草素合成量的影响呈正相关，说明对缬草素合成具有调控作用。3种调控因子对dxr基因表达强弱顺序为：茉莉酸甲酯＞茉莉酸＞壳聚糖。
在建立缬草素高产细胞培养体系基础上，优化了细胞培养合成缬草素条件及建立了力学模型，并通过调控缬草素合成前体物质及关键酶活性促进因子，使缬草素合成量与野生型相比提高了14.15倍。进一步研究表明，缬草素生物合成调控因子不但可以促进目标化合物的合成，并且可以促进dxr基因表达量。本研究为今后大量生物合成缬草素或其它环烯醚萜三酯类化合物奠定了坚实的基础；探寻缬草素生物合成的机理，具有重要的理论和实际意义。
Valtrate is a pharmacologically important compound. It’s well known thatpharmaceutically active functions of sedative, antineoplastic, increase coronary bloodflow, gastrointestinal smooth muscle, spasm spasmolysis analgesics and antiarrhythmic.Valeriana is difficult to artificial cultivation, and its wild sample has low content ofmedicine ingredients. The cost of traditional valtrate extract method is very expensive.In our research, biosynthesis of valtrate from in vitro culture system was studied bymethods of Cell Culture, GC-MS, HPLC-PDA, Plackett–Burman Design, Box–Behnken Design and RT-PCR. Therefore, we focused on analysis of the main chemicalconstituents of3kinds of Valeriana, establishment of valtrate high yield culture system,optimization of culture condition to enhance valtrate accumulation establishment ofkinetic model of adventitious roots suspension culture system, optimization ofregulation factors to enhance valtrate accumulation and analysis of the effect ofregulation factors on expression of dxr gene. The results are showed as follows:
Through GC-MS analysis,43kinds of chemicals were identified from the essentialoil of Valeriana amurensis Smir. ex Kom., the maximum content of Isobornyl acetatewas27.6%, which was the highest value in reported research.44kinds of chemicalswere identified from Valeriana fauriei Briq. essential oil, and α-Selinene (16.7%) wasthe main constituent.39kinds of chemical were identified from Valeriana alternifoliaBunge. essential oil, and Bornyl acetate (39.1%) was the main constituent.4kinds ofvalepotriates (Valtrate, Isovaltrate, Diavaltrate and Acevaltrate) were identified byHPLC. Valtrate was the main constituent, which has best medicinal properties.
Through screening of explants, culture medium and hormone combination, callusand adventitious roots were induced. Then, callus, suspended cell, solid and suspendedadventitious roots culture system were established. The highest content of valtrate (1.77times than wild root sample) was gotten from suspended adventitious roots culturesystem which was chosen as the high-yield cultlure system. Plackett–Burman designcriterion was applied to identify the significant effects of various cultural parameters.Among the various variables screened, the rotating speed, NH4NO3and KH2PO4concentrations were most significant factors. Through the Box–Behnken design, thespecific optimum levels of these significant parameters were determined as follows: the rotate speed (76r/min), NH4NO3concentration (0.0069mol/L) and KH2PO4concentration (0.0436mol/L). And the maximum valtrate content (8.46mg/g,6.79times than wild root sample) was gotten under this optimized conditions. Using theOrigin software to non-linear fit the experimental data, the kinetics model of tissuegrowth, valtrate production and substrate consumption were established. Thismathematical model can interpret and reflect the change of key parameters in theprocess of suspension adventitious root culture.
We assayed the effects of different regulation factors on the production of valtrate.Methyl jasmonate, Jasmonic acid and Chitosan showed the significant promotion effecton valtrate production. The test results of precursor feeding indicated that citronellal hada modest promotion effect on valtrate production. Using the response surfacemethodology, the optimal combination of methyl jasmonate, jasmonic acid and chitosanwas shown to have strong synergistic effect on valtrate production, which wasconfirmed by actual experiments. After the test of best time of induction treatment, wegot the maximum valtrate content of17.40±0.26mg/g, which was14.15times thanwild root sample, and this was the highest value in reported research.
DXR is a key enzyme in terpenoid metabolic pathways. One dxr gene was clonedfrom cell of V. amurensis by RTPCR. The cDNA fragment code region of dxr was1425bp long, encoding474amino acids. According to the test results of prokaryoticexpression, a fragment about50.98kD was achieved as expect. This order of geneexpressed strength from different tissues of V. amurensis is: adventitious roots＞wildroot&rhizome＞wild leaf＞suspended cell. The effect of regulatory factor on theexpression of dxr gene has positive correlation with the effect of regulatory factor onproduction of valtrate. The results show dxr gene is an effective gene target in thesynthesis pathway of valtrate. Through the research of establishment of valtrate highyield culture system, the culture condition was optimized, the kinetic model ofadventitious roots suspension culture system was established, the regulation factors wasoptimized, and the valtrate production level was significantly enhanced. Further studieshave shown that the biosynthesis regulatory factors can not only promote the synthesisof the target compound, but also enhance the level of dxr gene expression. The results ofthis study provide the theoretical basis for large-scale biosynthesis of varltrate, and arehighly significant to reveal the mechanism of varltrate biosynthesis.
从缬草根部提取物中鉴定出4种环烯醚萜三酯（缬草素、Isovaltrate、Diavaltrate和Acevaltrate），其中最具药用活性和强极性的缬草素在黑水缬草中含量最高。在GC-MS分析，从黑水缬草中鉴定出43种化合物，所含的Isobornylacetate（27.6%）在已报道的缬草属植物挥发油中含量最高；北缬草中鉴定出44种化合物，含量最高的为α-Selinene（16.7%）；毛节缬草中鉴定出39种化合物，含量最高的为Bornyl acetate（39.1%）。
通过筛选外植体、基础培养基和激素组合，诱导了黑水缬草愈伤组织和不定根，分析了愈伤组织、固体不定根、悬浮细胞和悬浮不定根4种培养体系。发现悬浮不定根细胞系中缬草素含量最高，以其为基础建立了缬草素高产细胞培养体系。建立了悬浮不定根培养体系的组织生长、缬草素合成和基质消耗动力学模型，根据这3个动力学模型可定量评估产缬草素效果，优化培养工艺流程和控制培养条件。通过Plackett–Burman主因素筛选试验的结果表明：摇床转速、NH4NO3和KH2PO4浓度对于黑水缬草悬浮不定根培养体系有显著影响（P＜0.01）。进一步通过Box–Behnken设计的响应面分析，得到最佳水平为摇床转速=76r/min，NH4NO3浓度=0.0069mol/L，KH2PO4浓度=0.0436mol/L。在此条件下，实际获得缬草素含量最大值为8.36±0.20mg/g。
调控因子促进缬草素合成的试验结果表明，3种诱导子（茉莉酸甲酯、茉莉酸和壳聚糖）对缬草素积累具有显著的促进作用。应用香茅醛进行前体饲喂，发现在低浓度条件下，可以促进缬草素的合成。通过响应面分析，得到了3种诱导子的最优浓度组合：茉莉酸甲酯=419.6mg/L、茉莉酸=102.8mg/L、壳聚糖=29.0mg/L。在此条件下，缬草素含量的实际最大值为17.40±0.26mg/g。3种诱导子对缬草素的积累具有很强的协同作用。
克隆出的黑水缬草中萜类代谢关键酶—5-磷酸脱氧木酮糖还原异构酶（DXR）的cDNA片段基因编码区长为1425bp，编码474个氨基酸残基。原核表达证明，该基因编码蛋白的分子量约为50.98kD。dxr基因在缬草组织中的表达强弱为：不定根＞野生植株根和根茎＞野生植株叶片＞悬浮细胞。调控因子（茉莉酸甲酯、茉莉酸和壳聚糖）对dxr基因表达的影响与对缬草素合成量的影响呈正相关，说明对缬草素合成具有调控作用。3种调控因子对dxr基因表达强弱顺序为：茉莉酸甲酯＞茉莉酸＞壳聚糖。
在建立缬草素高产细胞培养体系基础上，优化了细胞培养合成缬草素条件及建立了力学模型，并通过调控缬草素合成前体物质及关键酶活性促进因子，使缬草素合成量与野生型相比提高了14.15倍。进一步研究表明，缬草素生物合成调控因子不但可以促进目标化合物的合成，并且可以促进dxr基因表达量。本研究为今后大量生物合成缬草素或其它环烯醚萜三酯类化合物奠定了坚实的基础；探寻缬草素生物合成的机理，具有重要的理论和实际意义。
Valtrate is a pharmacologically important compound. It’s well known thatpharmaceutically active functions of sedative, antineoplastic, increase coronary bloodflow, gastrointestinal smooth muscle, spasm spasmolysis analgesics and antiarrhythmic.Valeriana is difficult to artificial cultivation, and its wild sample has low content ofmedicine ingredients. The cost of traditional valtrate extract method is very expensive.In our research, biosynthesis of valtrate from in vitro culture system was studied bymethods of Cell Culture, GC-MS, HPLC-PDA, Plackett–Burman Design, Box–Behnken Design and RT-PCR. Therefore, we focused on analysis of the main chemicalconstituents of3kinds of Valeriana, establishment of valtrate high yield culture system,optimization of culture condition to enhance valtrate accumulation establishment ofkinetic model of adventitious roots suspension culture system, optimization ofregulation factors to enhance valtrate accumulation and analysis of the effect ofregulation factors on expression of dxr gene. The results are showed as follows:
Through GC-MS analysis,43kinds of chemicals were identified from the essentialoil of Valeriana amurensis Smir. ex Kom., the maximum content of Isobornyl acetatewas27.6%, which was the highest value in reported research.44kinds of chemicalswere identified from Valeriana fauriei Briq. essential oil, and α-Selinene (16.7%) wasthe main constituent.39kinds of chemical were identified from Valeriana alternifoliaBunge. essential oil, and Bornyl acetate (39.1%) was the main constituent.4kinds ofvalepotriates (Valtrate, Isovaltrate, Diavaltrate and Acevaltrate) were identified byHPLC. Valtrate was the main constituent, which has best medicinal properties.
Through screening of explants, culture medium and hormone combination, callusand adventitious roots were induced. Then, callus, suspended cell, solid and suspendedadventitious roots culture system were established. The highest content of valtrate (1.77times than wild root sample) was gotten from suspended adventitious roots culturesystem which was chosen as the high-yield cultlure system. Plackett–Burman designcriterion was applied to identify the significant effects of various cultural parameters.Among the various variables screened, the rotating speed, NH4NO3and KH2PO4concentrations were most significant factors. Through the Box–Behnken design, thespecific optimum levels of these significant parameters were determined as follows: the rotate speed (76r/min), NH4NO3concentration (0.0069mol/L) and KH2PO4concentration (0.0436mol/L). And the maximum valtrate content (8.46mg/g,6.79times than wild root sample) was gotten under this optimized conditions. Using theOrigin software to non-linear fit the experimental data, the kinetics model of tissuegrowth, valtrate production and substrate consumption were established. Thismathematical model can interpret and reflect the change of key parameters in theprocess of suspension adventitious root culture.
We assayed the effects of different regulation factors on the production of valtrate.Methyl jasmonate, Jasmonic acid and Chitosan showed the significant promotion effecton valtrate production. The test results of precursor feeding indicated that citronellal hada modest promotion effect on valtrate production. Using the response surfacemethodology, the optimal combination of methyl jasmonate, jasmonic acid and chitosanwas shown to have strong synergistic effect on valtrate production, which wasconfirmed by actual experiments. After the test of best time of induction treatment, wegot the maximum valtrate content of17.40±0.26mg/g, which was14.15times thanwild root sample, and this was the highest value in reported research.
DXR is a key enzyme in terpenoid metabolic pathways. One dxr gene was clonedfrom cell of V. amurensis by RTPCR. The cDNA fragment code region of dxr was1425bp long, encoding474amino acids. According to the test results of prokaryoticexpression, a fragment about50.98kD was achieved as expect. This order of geneexpressed strength from different tissues of V. amurensis is: adventitious roots＞wildroot&rhizome＞wild leaf＞suspended cell. The effect of regulatory factor on theexpression of dxr gene has positive correlation with the effect of regulatory factor onproduction of valtrate. The results show dxr gene is an effective gene target in thesynthesis pathway of valtrate. Through the research of establishment of valtrate highyield culture system, the culture condition was optimized, the kinetic model ofadventitious roots suspension culture system was established, the regulation factors wasoptimized, and the valtrate production level was significantly enhanced. Further studieshave shown that the biosynthesis regulatory factors can not only promote the synthesisof the target compound, but also enhance the level of dxr gene expression. The results ofthis study provide the theoretical basis for large-scale biosynthesis of varltrate, and arehighly significant to reveal the mechanism of varltrate biosynthesis.
引文
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