前体物和诱导子对悬浮培养葡萄细胞多酚生物合成影响的研究
EFFECTS OF PRECURSOR AND ELICITORS ON THE PHENOLIC COMPOUNDS BIOSYNTHESIS IN SUSPENSION CULTURES OF GRAPE (VITIS ROMANETII ROMAN)
作者：徐国前 导师：张振文
西北农林科技大学 葡萄与葡萄酒学2011届博士
摘 要
利用植物细胞作为生物反应器生产高附加值次生代谢产物的技术，近些年来取得很大进展，该技术不但可以保护濒危物种，解决人类医药和保健问题，而且不占用耕地，不受地理环境和气候条件等的影响。本研究首先调整培养基与植物生长调节物质浓度配比，诱导葡萄茎和叶片外植体产生愈伤组织，通过筛选出高酚含量愈伤组织和优化细胞悬浮培养条件建立稳定的悬浮细胞系。以此悬浮细胞系为模式体系，添加前体物或诱导子，以Actin为内参基因，用实时定量PCR(RTq-PCR)方法检测苯丙氨酸解氨酶(PAL)、查尔酮合成酶(CHS)和二苯乙烯合成酶(STS)基因相对表达量变化；测定细胞生长量，分析PAL活性变化，定量分析总酚、原花色素和白藜芦醇的含量和产量，研究前体物和诱导子对悬浮培养葡萄细胞中多酚生物合成的影响，为葡萄次生代谢物实现大规模工业化生产提供理论依据。获得主要研究结果如下：
1. 筛选出中国野生葡萄高酚含量愈伤组织并建立稳定的悬浮细胞系
以中国野生的毛葡萄(V. quinquangularis Rehd)、秋葡萄(V. romanetii Roman)、刺葡萄(V. davidii Roman)和酿酒葡萄品种赤霞珠(V. vinifera L. cv. Cabernet Sauvignon)半木质化茎在附加30 g/L蔗糖、6 g/L琼脂、2.0 mg/L 6-BA和0.05 mg/L NAA，pH=6.0的B5培养基上诱导产生愈伤组织生长量最大，诱导率最高；赤霞珠产生愈伤组织较其他种容易；在同样消毒与培养条件下，半木质化茎诱导产生愈伤组织的能力较叶片强。对秋葡萄愈伤组织生长影响大小的因素依次是：培养基＞植物生长调节物质浓度＞植物生长调节物质组合＞培养基体积，最佳组合为每瓶35 mL B5培养基加入0.2 mg/L KT＋0.1 mg/L NAA。
以高酚含量秋葡萄愈伤组织培养稳定的悬浮细胞系；愈伤组织的继代次数、生长天数和初始接种量是悬浮培养成功的关键；继代培养时新旧培养基体积比控制在4:1～8:1之间，可以培养出稳定的悬浮细胞系。
2. 前体物对3、4和5 d龄悬浮细胞生长有促进作用，诱导子对悬浮细胞生长有抑制作用
悬浮细胞生长迟滞期随着蔗糖浓度增加而延长；40 g/L的蔗糖培养细胞的生长量最大。L-苯丙氨酸抑制1、2 d龄细胞生长，促进3、4和5 d龄细胞生长；4种诱导子对葡萄细胞生长均有抑制作用，且处理细胞龄越小，抑制作用越大。前体物和诱导子多因素协同作用时，各因素对悬浮培养细胞生长影响大小依次是：蔗糖浓度＞L-苯丙氨酸＞MJ＞真菌诱导子。其中最佳组合是40 gL-1蔗糖、0.2 mmol/L L-苯丙氨酸、10 μmol/L MJ和1.5 mL真菌诱导子。
3. 前体物和诱导子显著提高PAL、CHS和STS基因的表达量和PAL的活性
加入L-苯丙氨酸后，前期PAL、CHS和STS基因的表达量迅速增加，后期三个基因的表达量均下降。诱导子对PAL、CHS和STS三个基因表达具有较大的刺激作用，MJ、UV-B和UV-C在处理后，细胞生长初期对三个基因的表达影响较大，真菌诱导子则在后期影响较大。
L-苯丙氨酸促使细胞PAL活性在12 h时达到最大，而后下降。4种诱导子处理细胞的PAL活性呈双S形变化：第一个峰值出现在12 h时，第二个高峰则在3 d后的不同时间出现。前体物和诱导子联合处理后，PAL活性在12 h内迅速上升，然后持续下降；12 h时L-苯丙氨酸对PAL活性影响最大，到第3 d时MJ 对PAL活性影响大于L-苯丙氨酸。
4. 前体物和诱导子显著提高细胞总酚、原花色素和白藜芦醇的含量及产量
总酚、原花色素和白藜芦醇含量随着蔗糖浓度的增加而升高。40 g/L蔗糖培养细胞的总酚、原花色素和白藜芦醇产量最高，其总酚产量和其他处理之间的差异显著(p <0.05)；原花色素和白藜芦醇产量与其他处理之间的差异极显著(p <0.01)。30 g/L蔗糖添加水解酪蛋白(Casein hydrolysate,CH)的细胞生长量、总酚、原花色素和白藜芦醇的含量和最大产量都比无CH的高，但生长趋势相同，最大产量出现的时间不变。
L-苯丙氨酸处理3、4和5 d龄细胞总酚、原花色素和白藜芦醇的产量增加，但1、2 d龄细胞的产量都下降。4种诱导子对细胞的总酚、原花色素和白藜芦醇含量有促进作用，不同处理的最高产量之间差异达到极显著水平(p <0.01)，MJ处理的白藜芦醇产量在4种诱导子处理中最低，真菌诱导子处理的产量最高。
前体物和诱导子协同处理时，30 g/L蔗糖、1.2 mmol/L L-苯丙氨酸、50 μmol/L MJ和1.5 mL真菌诱导子处理细胞的总酚和白藜芦醇含量最高，与其他处理之间的差异极显著(p <0.01)。50 g/L蔗糖、1.2 mmol/L L-苯丙氨酸、30 μmol/L MJ和0.5 mL真菌诱导子处理的原花色素含量最高，与其他处理之间的差异极显著(p <0.01)。蔗糖、MJ和真菌诱导子分别对总酚、原花色素和白藜芦醇的产量影响较大。结果是40 g/L蔗糖、0.2 mmol/L L-苯丙氨酸、30 μmol/L MJ和1.5 mL真菌诱导子组合的总酚和原花色素产量最高；40 g/L蔗糖、0.7 mmol/L L-苯丙氨酸、30 μmol/L MJ和1.5 mL真菌诱导子组合白藜芦醇总产量最高。
本文的创新之处在于，以中国秋葡萄悬浮培养细胞为基础，通过添加前体物或诱导子处理，得到细胞PAL、CHS和STS三个基因的表达变化和PAL活性变化；该法显著提高葡萄内天然功能性成分原花色素和白藜芦醇含量。此项研究在未来天然食品添加剂工业化生产中具有较大的应用前景。
关键词 悬浮培养 葡萄细胞 前体物 诱导子 总酚 原花色素 白藜芦醇
Abstract
Great advances have been achieved in producing desirable plant secondary metabolites by the technology of plant cell bioreactors in recent years. It both protects endangered species and solves human healthcare and welfare issues,while neither impropriates cultivated land nor depends on geographical location,climatic conditions,seasonal variations and growth conditions. In this study,callus were induced from the stems and leaves of grape through adjusting the medium and the hormone concentrations,and a stable cell suspension culture system was developed by selecting callus with high phenolic content,optimizing the conditions of cell suspension culture. Precursor and elicitors were added into the model system that established cell suspension culture system. The relative expression changes of phenylalanine ammonia lyase (PAL),chalcone synthase(CHS) and stilbene synthase (STS) genes in cells were monitored by a real-time quantitative PCR. The PAL activity,cell biomass and the contents of total phenols,proanthocyanidins and resveratrol were also investigated during cell suspension culture. The study aims at examining the effects of precursors and elicitors on phenolic compounds biosynthesis in cell suspension cultures of grape (Vitis romanetii Roman),so as to provide theoretical foundation for the industrialized mass production of grape second metabolites in future. The main results are as follows:
1. A stable cell suspension culture system was developed by selecting callus with high phenolic content and optimizing the conditions of cell suspension culture
Callus were induced using semi-lignification stems as explants of four grape varieties (Cabernet Sauvignon,hairy grape,romanet grape and brier grape),and the best culture medium was B5 with sucrose 30 g/L,agar 6 g/L,6-BA 2.0 mg/L and NAA 0.05 mg/L,with the highest frequency above 85%. The induced callus of Cabernet Sauvignon was easily than other varieties and the better was with semi-ligification stems as explants than leaves. Factors influencing the growth of romanet grape callus were: medium >hormone concentrations >hormone combination >medium volume,and the optimum combination medium for its growth was 35 ml B5 medium per bottle with 0.2 mg/L KT and 0.1 mg/L NAA.
The Romanet Grapes Callus was chosen as the stable cell suspension culture system after comprehensively comparing the contents of total phenolics,proanthocyanidins and resveratrol in the four varieties. The key to succeeding suspension culture were callus passages,cell age and original inoculation sizes. The establishment of primary suspension cultural cell line of grape,required that the transferring–culturing callus should subculture at least 5 times,cell age 11 to 15-day and original inoculation amount 150 g/L and the volume ratio of the new and the old medium for subculture arranged from 4:1 to 8:1.
2. The precursors promoted the growth of the suspension cultural cells that growth 3,4,5 days,while the elicitors inhibited the growth of suspension cultural cells
The growth lag phases of suspension culture cells extended with the increasing concentrations of sucrose. The largest cell biomass was achieved when grown in B5 medium with 40 g/L sucrose. The cell growth was inhibited by L-phenylalanine when the cell at 1,2-day-cell age,but promoted when the cell at 3-,4- and 5-day-cell age. The grape cell growth was inhibited by four elicitors,and the greater inhibition existed in the cell with smaller age. Different factors affecting the cell biomass of grape suspension culture were: sucrose>L-phenylalanine>MJ>fungal elicitor,when synergistic effects of precursors and elicitors were considered. The best combination of precursors and elicitors was: 40 g/L sucrose,0.2 mmol/L L-phenylalanine,10 μmol/L MJ and 1.5 ml fungal elicitor.
3. The precursors and elicitors significantly promoted the PAL activity and the gene expression of PAL,CHS and STS
The addition of L-phenylalanine enhanced the expression of PAL,CHS and STS genes in the 12 hours,then PAL and STS began to decrease,while the expression of CHS gene increased continuously until reached the peak at the 7th day. Three genes’ expression was decreased with the cell entering a decline phase after 7 days. The elicitors showed marked effects on the expression of the three genes: MJ,UV-B and UV-C had significant influence in the early stage,but the fungal elicitor was markedly in the late stage.
The PAL activity reached the peak at 12th hour after added L-phenylalanine,and then decreased. The PAL activity with the treatment time showed a double sigmoid curve after induced with the four elicitors: the first peak was at the 12th hour and the second peak occurred at different times after 3 days. When the synergistic effects between precursors and elicitors were studied,the PAL activity increased rapidly within 12 hours and then decreased,which was influenced most at 12 hour by L-phenylalanine. The effect of MJ on PAL activity was more than L-phenylalanine’s at the 3rd day.
4. The precursor and elicitors significantly increased the production of total polyphenol,proanthocyanidins and resveratrol in the suspension cultural cells
The contents of total polyphenol,proanthocyanidins and resveratrol in the cells increased with the increase of sucrose concentration. Total polyphenol,proanthocyanidins and resveratrol all had highest yields when sucrose content in medium at 40 gl-1,and the total polyphenol yield reached significant differences between treatments (p <0.05). The production of proanthocyanidins and resveratrol had extremely significant differences between treatments (p <0.01). The cell biomass and the contents of total polyphenol,proanthocyanidins and resveratrol were higher than those without CH (Casein hydrolysate),but the growth trends seemed similar and the time with the maximum yield appeared unchanged,when sucrose concentration was 30 g/L.
The production of total polyphenol,proanthocyanidins and resveratrol was increased when adding L-phenylalanine at 3-,4- and 5-day-cell age,but the production was decreased at 1- and 2-day-cell age. Four elicitors had greatly positive influence on the contents of total polyphenol,proanthocyanidins and resveratrol,and the differences between the maximum yields of different treatments seemed significant (p <0.01). MJ treatment induced resveratrol production was the lowest,while the fungal elicitor treatment had the highest yield among the four treatments.
Synergistic effect between the precursors and elicitors,the highest content of total polyphenol and resveratrol was achieved with 30 g/L sucrose,1.2 mmol/L L-phenylalanine,50 μmol/L MJ and 1.5 mL fungal elicitor,and the total polyphenol content was 3.5 times of the control with significant differences between treatments (p <0.01);the highest content of proanthocyanidin was obtained with 50 g/L sucrose,1.2 mmol/L L-phenylalanine,30 μmol/L MJ and 0.5 ml fungal elicitor,and significant differences were also found between treatments (p <0.01). Meanwhile,the factors affecting the content of total polyphenol were: sucrose>fungal elicitor>MJ >L-phenylalanine,ones affecting proanthocyanidin production were: MJ >sucrose>L-phenylalanine >fungal elicitor,and those affecting resveratrol production were: fungal elicitor>sucrose>MJ >L-phenylalanine. As a result,the conditions with the highest production of total polyphenol and proanthocyanidin were: 40 g/L sucrose,0.2 mmol/L L-phenylalanine,30 μmol/L MJ and 1.5 ml fungal elicitor. The conditions with the highest production of resveratrol were: 40 g/L sucrose,0.7 mmol/L L-phenylalanine,30 μmol/L MJ and 1.5 ml fungal elicitor.
The innovation of this paper is that obtained expression changes of PAL,CHS and STS genes and PAL activity,based on the suspension cultural cells by adding the precursors and elicitors;and significantly improved the proanthocyanidins and resveratrol production in the suspension cultural cells,Thus possessing broad application foreground in the natural food additives.
Key words Suspension culture Grape cell Precursor Elicitor Total polyphenol Proanthocyanidin Resveratrol
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