前體物和誘導(dǎo)子對懸浮培養(yǎng)葡萄細(xì)胞多酚生物合成影響的研究

EFFECTS OF PRECURSOR AND ELICITORS ON THE PHENOLIC COMPOUNDS BIOSYNTHESIS IN SUSPENSION CULTURES OF GRAPE (VITIS ROMANETII ROMAN)

作者：徐國前　　導(dǎo)師：張振文

西北農(nóng)林科技大學(xué)　　葡萄與葡萄酒學(xué)2011屆博士

摘　要

利用植物細(xì)胞作為生物反應(yīng)器生產(chǎn)高附加值次生代謝產(chǎn)物的技術(shù)，近些年來取得很大進(jìn)展，該技術(shù)不但可以保護(hù)瀕危物種，解決人類醫(yī)藥和保健問題，而且不占用耕地，不受地理環(huán)境和氣候條件等的影響。本研究首先調(diào)整培養(yǎng)基與植物生長調(diào)節(jié)物質(zhì)濃度配比，誘導(dǎo)葡萄莖和葉片外植體產(chǎn)生愈傷組織，通過篩選出高酚含量愈傷組織和優(yōu)化細(xì)胞懸浮培養(yǎng)條件建立穩(wěn)定的懸浮細(xì)胞系。以此懸浮細(xì)胞系為模式體系，添加前體物或誘導(dǎo)子，以Actin為內(nèi)參基因，用實時定量PCR(RTq-PCR)方法檢測苯丙氨酸解氨酶(PAL)、查爾酮合成酶(CHS)和二苯乙烯合成酶(STS)基因相對表達(dá)量變化；測定細(xì)胞生長量，分析PAL活性變化，定量分析總酚、原花色素和白藜蘆醇的含量和產(chǎn)量，研究前體物和誘導(dǎo)子對懸浮培養(yǎng)葡萄細(xì)胞中多酚生物合成的影響，為葡萄次生代謝物實現(xiàn)大規(guī)模工業(yè)化生產(chǎn)提供理論依據(jù)。獲得主要研究結(jié)果如下：

1.　篩選出中國野生葡萄高酚含量愈傷組織并建立穩(wěn)定的懸浮細(xì)胞系

以中國野生的毛葡萄(V. quinquangularis Rehd)、秋葡萄(V. romanetii Roman)、刺葡萄(V. davidii Roman)和釀酒葡萄品種赤霞珠(V. vinifera L. cv. Cabernet Sauvignon)半木質(zhì)化莖在附加30 g/L蔗糖、6 g/L瓊脂、2.0 mg/L 6-BA和0.05 mg/L NAA，pH=6.0的B5培養(yǎng)基上誘導(dǎo)產(chǎn)生愈傷組織生長量最大，誘導(dǎo)率最高；赤霞珠產(chǎn)生愈傷組織較其他種容易；在同樣消毒與培養(yǎng)條件下，半木質(zhì)化莖誘導(dǎo)產(chǎn)生愈傷組織的能力較葉片強。對秋葡萄愈傷組織生長影響大小的因素依次是：培養(yǎng)基＞植物生長調(diào)節(jié)物質(zhì)濃度＞植物生長調(diào)節(jié)物質(zhì)組合＞培養(yǎng)基體積，最佳組合為每瓶35 mL B5培養(yǎng)基加入0.2 mg/L KT＋0.1 mg/L NAA。

以高酚含量秋葡萄愈傷組織培養(yǎng)穩(wěn)定的懸浮細(xì)胞系；愈傷組織的繼代次數(shù)、生長天數(shù)和初始接種量是懸浮培養(yǎng)成功的關(guān)鍵；繼代培養(yǎng)時新舊培養(yǎng)基體積比控制在4:1～8:1之間，可以培養(yǎng)出穩(wěn)定的懸浮細(xì)胞系。

2.　前體物對3、4和5 d齡懸浮細(xì)胞生長有促進(jìn)作用，誘導(dǎo)子對懸浮細(xì)胞生長有抑制作用

懸浮細(xì)胞生長遲滯期隨著蔗糖濃度增加而延長；40 g/L的蔗糖培養(yǎng)細(xì)胞的生長量最大。L-苯丙氨酸抑制1、2 d齡細(xì)胞生長，促進(jìn)3、4和5 d齡細(xì)胞生長；4種誘導(dǎo)子對葡萄細(xì)胞生長均有抑制作用，且處理細(xì)胞齡越小，抑制作用越大。前體物和誘導(dǎo)子多因素協(xié)同作用時，各因素對懸浮培養(yǎng)細(xì)胞生長影響大小依次是：蔗糖濃度＞L-苯丙氨酸＞MJ＞真菌誘導(dǎo)子。其中最佳組合是40 gL^-1蔗糖、0.2 mmol/L　L-苯丙氨酸、10 μmol/L MJ和1.5 mL真菌誘導(dǎo)子。

3.　前體物和誘導(dǎo)子顯著提高PAL、CHS和STS基因的表達(dá)量和PAL的活性

加入L-苯丙氨酸后，前期PAL、CHS和STS基因的表達(dá)量迅速增加，后期三個基因的表達(dá)量均下降。誘導(dǎo)子對PAL、CHS和STS三個基因表達(dá)具有較大的刺激作用，MJ、UV-B和UV-C在處理后，細(xì)胞生長初期對三個基因的表達(dá)影響較大，真菌誘導(dǎo)子則在后期影響較大。

L-苯丙氨酸促使細(xì)胞PAL活性在12 h時達(dá)到最大，而后下降。4種誘導(dǎo)子處理細(xì)胞的PAL活性呈雙S形變化：第一個峰值出現(xiàn)在12 h時，第二個高峰則在3 d后的不同時間出現(xiàn)。前體物和誘導(dǎo)子聯(lián)合處理后，PAL活性在12 h內(nèi)迅速上升，然后持續(xù)下降；12 h時L-苯丙氨酸對PAL活性影響最大，到第3 d時MJ 對PAL活性影響大于L-苯丙氨酸。

4.　前體物和誘導(dǎo)子顯著提高細(xì)胞總酚、原花色素和白藜蘆醇的含量及產(chǎn)量

總酚、原花色素和白藜蘆醇含量隨著蔗糖濃度的增加而升高。40 g/L蔗糖培養(yǎng)細(xì)胞的總酚、原花色素和白藜蘆醇產(chǎn)量最高，其總酚產(chǎn)量和其他處理之間的差異顯著(p <0.05)；原花色素和白藜蘆醇產(chǎn)量與其他處理之間的差異極顯著(p <0.01)。30 g/L蔗糖添加水解酪蛋白(Casein hydrolysate,CH)的細(xì)胞生長量、總酚、原花色素和白藜蘆醇的含量和最大產(chǎn)量都比無CH的高，但生長趨勢相同，最大產(chǎn)量出現(xiàn)的時間不變。

L-苯丙氨酸處理3、4和5 d齡細(xì)胞總酚、原花色素和白藜蘆醇的產(chǎn)量增加，但1、2 d齡細(xì)胞的產(chǎn)量都下降。4種誘導(dǎo)子對細(xì)胞的總酚、原花色素和白藜蘆醇含量有促進(jìn)作用，不同處理的最高產(chǎn)量之間差異達(dá)到極顯著水平(p <0.01)，MJ處理的白藜蘆醇產(chǎn)量在4種誘導(dǎo)子處理中最低，真菌誘導(dǎo)子處理的產(chǎn)量最高。

前體物和誘導(dǎo)子協(xié)同處理時，30 g/L蔗糖、1.2 mmol/L L-苯丙氨酸、50 μmol/L MJ和1.5 mL真菌誘導(dǎo)子處理細(xì)胞的總酚和白藜蘆醇含量最高，與其他處理之間的差異極顯著(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真菌誘導(dǎo)子處理的原花色素含量最高，與其他處理之間的差異極顯著(p <0.01)。蔗糖、MJ和真菌誘導(dǎo)子分別對總酚、原花色素和白藜蘆醇的產(chǎn)量影響較大。結(jié)果是40 g/L蔗糖、0.2 mmol/L L-苯丙氨酸、30 μmol/L MJ和1.5 mL真菌誘導(dǎo)子組合的總酚和原花色素產(chǎn)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真菌誘導(dǎo)子組合白藜蘆醇總產(chǎn)量最高。

本文的創(chuàng)新之處在于，以中國秋葡萄懸浮培養(yǎng)細(xì)胞為基礎(chǔ)，通過添加前體物或誘導(dǎo)子處理，得到細(xì)胞PAL、CHS和STS三個基因的表達(dá)變化和PAL活性變化；該法顯著提高葡萄內(nèi)天然功能性成分原花色素和白藜蘆醇含量。此項研究在未來天然食品添加劑工業(yè)化生產(chǎn)中具有較大的應(yīng)用前景。

關(guān)鍵詞　懸浮培養(yǎng)　葡萄細(xì)胞　前體物　誘導(dǎo)子　總酚　原花色素　白藜蘆醇

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 12^th 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 12^th 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 3^rd 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