黑曲霉提取物与水杨酸对长春花悬浮细胞生长及生物碱代谢作用的研究
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摘要
在植物细胞培养过程中，添加诱导子是提高植物次生代谢有效的方法之一。本文以黑曲霉提取物和水杨酸作为诱导子就长春花细胞悬浮培养过程中诱导子与其相互作用关系及诱导子对生物碱代谢的影响等方面进行初步探讨，研究结果如下：
毛霉、木霉、青霉、黑曲霉及酵母提取物5种真菌诱导子处理均能提高长春花悬浮培养细胞中生物碱的含量。在长春花悬浮培养细胞对数生长末期加入浓度为300μg/mL黑曲霉诱导子，生物碱含量达到了666μg/gFW，是对照的1.6倍。
真菌诱导子能激发长春花悬浮培养细胞的防御性应答反应。产生氧进发，细胞的氧化还原态势发生了明显的变化，培养基发生碱化现象，膜脂过氧化产物MDA含量大幅提高，蛋白质含量提高，POD、CAT的活力出现波动性变化，并具有一定的时序性。同时，与生物碱代谢有关的PAL的活力得到提高。
真菌诱导后加入200mmol/L氯化钙能够加强诱导子的诱导效果，生物碱含量进一步提高，达到730μg/gFw；而10mmol/L EGTA和1mmol/L LaCl。抑制真菌诱导子的诱导效果，对生物碱含量没有影响。
通过添加不同浓度的水杨酸(SA)，比较它们对长春花细胞POD、PAL、H_2O_2含量及对细胞生长和生物碱含量的影响，结果表明，SA可提高POD及PAL的活性，促进细胞内H_2O_2含量的上升并有利于生物碱的合成，其中10mg/L水杨酸对生物碱合成的促进效果明显，生物碱含量为对照组的1.3倍。
经过300μg/mL黑曲霉诱导子(FO)，10mg/L水杨酸(SA)，300μg/mL黑曲霉诱导子+10mg/L SA 3种处理，对长春花悬浮细胞膜脂过氧化和生物碱合成的影响。结果表明：FO和SA单独处理细胞均引起长春花细胞膜脂过氧化。SA+FO联合处理可以减轻FO单独处理细胞所引起的膜脂过氧化程度。SA+FO联合处理与真菌诱导子处理相比，较大地提高了过氧化物酶及苯丙氨酸解氨酶的活性。3种处理方法均可提高长春花细胞生物碱含量，特别以FO+SA处理含量最高，达到700μg/gFw。结果显示：在真菌诱导子诱导与水扬酸的联合作用下提高生物碱含量，可能与水杨酸减轻真菌诱导子所引起的细胞膜脂过氧化程度有关。
The addition of elicitor is a effectual method to improve the production of secondary metabolite in plant cell culture. This paper studied on the interaction of elicitor with Catharanthurs roseus Cell and discussed the effect of elicitor on the alkaloid metabolism, of Catharanthurs roseus Cells in suspension culture the results as follow:
The alkaloid biosynthesis in Catharanthurs roseus Cells in suspension culture was enhanced by treating with five kindsof fungal elicitors separately ,among them the Aspergillus niger elicitor had the highest inducing efficiency, The alkaloid yield was 1.6 folds of the control,reaching to 666ug /gFW. The best response of Catharanthurs roseus Cells to elicitor treatment was in the late exponential Growth phase.
Effects of fungal elicitor on cell redox status and alkaloid production were studied in suspension cultures of Catharanthurs roseus in the late exponential stage . the results show that fungal elicitor induced oxygen burst, changes of cell redox status , alka-linization of medium and fluctuation of the activity of redox enzymes with a sequence. The content of MDA and the content of protein representing the quantity of enzymes increased.The activity of POD could be kept at a higher level in contrast to the control.
The activity of CAT was inhibited at first by fungal elicitor and followed by an obvious increased, while the activity of PAL was promoted.
The paper showed that after fungal elicitation , the addition of 200mmol/L CaCl2 could furthermore strengthen the elicitation effect, and get more alkaloid production than fungal elicitation;
While the addition of 10mmol/L EGTA or 1mmol/L LaCl3 after fungal elicitation restrained the elicitation effects
The activity of peroxidase (POD), phenylalanine ammonia-lyase (PAL) the content of hydrogen peroxide (H2O2) and the biosynthesis of alkaloid in Catharanthurs roseus suspension culture exposed to various concentrations of salicylic acid was investigated. The results indicated that the addition of SA was followed by an increase in POD, PAL activity and it also promoted the content of H2O2 and alkaloid, alkaloid productivity increased 1. 3-fold when the cultures were exposed to 10mg/L SA. It suggested that SA could
be used as an elicitor to improve the alkaloid in catharanthurs roseus cell culture.
Effects of the treatments of 300ug/mL fungal elicitor (FO), 10mg/L salicylic acid(SA) and 300ug/mL F0+10mg/L SA on membrane -lipid peroxidat-ion and alkaloid production in catharanthurs roseussusupension cell cultures were studied . the results showd either FO or SA caused cell to membrane-1ipid peroxidation . combined treatment of SA and FO could decrease the degree of cell membrane-lipid peroxidation caused by only FO treament .combined treatment induced higher PAL,POD activity and obtained more biomass than that of only FO treament did. Among three treatments, the combined treatment get the most alkaloid production. The results suggested that combinedof FO and SA obviously improving alkaloid production be possibly related to addition of SA relieving membrane-lipid peroxidation induced by treatment of fungal elicitor.
毛霉、木霉、青霉、黑曲霉及酵母提取物5种真菌诱导子处理均能提高长春花悬浮培养细胞中生物碱的含量。在长春花悬浮培养细胞对数生长末期加入浓度为300μg/mL黑曲霉诱导子，生物碱含量达到了666μg/gFW，是对照的1.6倍。
真菌诱导子能激发长春花悬浮培养细胞的防御性应答反应。产生氧进发，细胞的氧化还原态势发生了明显的变化，培养基发生碱化现象，膜脂过氧化产物MDA含量大幅提高，蛋白质含量提高，POD、CAT的活力出现波动性变化，并具有一定的时序性。同时，与生物碱代谢有关的PAL的活力得到提高。
真菌诱导后加入200mmol/L氯化钙能够加强诱导子的诱导效果，生物碱含量进一步提高，达到730μg/gFw；而10mmol/L EGTA和1mmol/L LaCl。抑制真菌诱导子的诱导效果，对生物碱含量没有影响。
通过添加不同浓度的水杨酸(SA)，比较它们对长春花细胞POD、PAL、H_2O_2含量及对细胞生长和生物碱含量的影响，结果表明，SA可提高POD及PAL的活性，促进细胞内H_2O_2含量的上升并有利于生物碱的合成，其中10mg/L水杨酸对生物碱合成的促进效果明显，生物碱含量为对照组的1.3倍。
经过300μg/mL黑曲霉诱导子(FO)，10mg/L水杨酸(SA)，300μg/mL黑曲霉诱导子+10mg/L SA 3种处理，对长春花悬浮细胞膜脂过氧化和生物碱合成的影响。结果表明：FO和SA单独处理细胞均引起长春花细胞膜脂过氧化。SA+FO联合处理可以减轻FO单独处理细胞所引起的膜脂过氧化程度。SA+FO联合处理与真菌诱导子处理相比，较大地提高了过氧化物酶及苯丙氨酸解氨酶的活性。3种处理方法均可提高长春花细胞生物碱含量，特别以FO+SA处理含量最高，达到700μg/gFw。结果显示：在真菌诱导子诱导与水扬酸的联合作用下提高生物碱含量，可能与水杨酸减轻真菌诱导子所引起的细胞膜脂过氧化程度有关。
The addition of elicitor is a effectual method to improve the production of secondary metabolite in plant cell culture. This paper studied on the interaction of elicitor with Catharanthurs roseus Cell and discussed the effect of elicitor on the alkaloid metabolism, of Catharanthurs roseus Cells in suspension culture the results as follow:
The alkaloid biosynthesis in Catharanthurs roseus Cells in suspension culture was enhanced by treating with five kindsof fungal elicitors separately ,among them the Aspergillus niger elicitor had the highest inducing efficiency, The alkaloid yield was 1.6 folds of the control,reaching to 666ug /gFW. The best response of Catharanthurs roseus Cells to elicitor treatment was in the late exponential Growth phase.
Effects of fungal elicitor on cell redox status and alkaloid production were studied in suspension cultures of Catharanthurs roseus in the late exponential stage . the results show that fungal elicitor induced oxygen burst, changes of cell redox status , alka-linization of medium and fluctuation of the activity of redox enzymes with a sequence. The content of MDA and the content of protein representing the quantity of enzymes increased.The activity of POD could be kept at a higher level in contrast to the control.
The activity of CAT was inhibited at first by fungal elicitor and followed by an obvious increased, while the activity of PAL was promoted.
The paper showed that after fungal elicitation , the addition of 200mmol/L CaCl2 could furthermore strengthen the elicitation effect, and get more alkaloid production than fungal elicitation;
While the addition of 10mmol/L EGTA or 1mmol/L LaCl3 after fungal elicitation restrained the elicitation effects
The activity of peroxidase (POD), phenylalanine ammonia-lyase (PAL) the content of hydrogen peroxide (H2O2) and the biosynthesis of alkaloid in Catharanthurs roseus suspension culture exposed to various concentrations of salicylic acid was investigated. The results indicated that the addition of SA was followed by an increase in POD, PAL activity and it also promoted the content of H2O2 and alkaloid, alkaloid productivity increased 1. 3-fold when the cultures were exposed to 10mg/L SA. It suggested that SA could
be used as an elicitor to improve the alkaloid in catharanthurs roseus cell culture.
Effects of the treatments of 300ug/mL fungal elicitor (FO), 10mg/L salicylic acid(SA) and 300ug/mL F0+10mg/L SA on membrane -lipid peroxidat-ion and alkaloid production in catharanthurs roseussusupension cell cultures were studied . the results showd either FO or SA caused cell to membrane-1ipid peroxidation . combined treatment of SA and FO could decrease the degree of cell membrane-lipid peroxidation caused by only FO treament .combined treatment induced higher PAL,POD activity and obtained more biomass than that of only FO treament did. Among three treatments, the combined treatment get the most alkaloid production. The results suggested that combinedof FO and SA obviously improving alkaloid production be possibly related to addition of SA relieving membrane-lipid peroxidation induced by treatment of fungal elicitor.
引文
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2. Yeoman M M. Manipulating secondary metabolism in cultured plant cell[J] . New Phytol.,1996,(134): 553～563.
3. Cheng J, Guo Y. Research progress in the production of pla nt cell-derived drugs[J] , jiangxi Sci(江西科学),2000,18(1):60～62.
4．吴晓玲．邓光存．植物细胞次级代谢产物生产条件的调控[J]．生物学通报．2002，37(12)：17～18．
5．吴兆亮，元英进，王传贵．植物细胞培养—分离藕合过程研究进展[J]．天然产物研究与开发，1998，10(1)：79～83．
6．刘春朝，王玉春．植物组织培养生产有用次生代谢产物的研究进展[J] 。生物技术通报，1997，1：1～6．
7．张丽华，李建明．植物冠瘿生长调控及相关基因研究进展[J]．西北植物学报．2002，22(5)：1282～1288．
8． 黄建安，刘仲华．毛状根培养与植物次生代谢物的生产[J]．微生物学杂志．2003，23(5)：35～39．
9． 高丽萍，夏涛．茶树次生代谢产物的细胞工程技术研究进展[J]．中国茶叶加工，2000，(2)：26～30．
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