水杨酸、茉莉酸甲酯和乙烯利对野葛细胞悬浮培养生产葛根素的影响

野葛〔Ｐｕｅｒａｒｉａｌｏｂａｔａ(Ｗｉｌｌｄ.)Ｏｈｗｉ〕是我国及其他东亚国家传统的药用资源,其主要药用成分为葛根素,广泛用于治疗多种心血管疾病[1]。由于近年来生态环境的人为破坏,野生野葛资源已不能满足人们对葛根素药物的需求,通过细胞工程可以实现野葛资源的可持续性开发。本项目组已经建立了野葛组织培养和植株再生[2]及细胞悬浮培养体系[3],研究结果表明,悬浮细胞培养物能够生产葛根素,但产量仍有待于提高[3]。已知水杨酸(ｓａｌｉｃｙｌｉｃａｃｉｄ,ＳＡ)、乙烯和茉莉酸类化合物(ｊａｓｍｏｎａｔｅｓ,ＪＡｓ)能够在高…     

茉莉酸甲酯与二氢茉莉酮酸甲酯对悬浮培养的甘草细胞生长和黄酮积累的影响

建立了稳定的甘草细胞悬浮培养体系,在一个培养周期内,细胞的生长曲线呈"S"型,培养21 d的干重、鲜重和黄酮产量都达到最高值.甘草细胞悬浮培养体系中分別添加100 μmol·L-1 二氢茉莉酮酸甲酯和茉莉酸甲酯时,虽然对细胞生长有一定程度的抑制,但细胞中甘草黄酮产量仍有提高.添加二氢茉莉酮酸甲酯和茉莉酸甲酯的最适时间分别为细胞培养后的第5天和第10天.     

茉莉酸甲酯对茶条槭悬浮细胞没食子酸合成的影响

为了研究茶条槭悬浮细胞中没食子酸的合成,该研究进行了茉莉酸甲酯诱导试验。通过添加茉莉酸甲酯,利用HPLC检测诱导后细胞中没食子酸的含量变化情况,同时利用电导仪、酸度计、分光光度计法和激光共聚焦显微镜对培养液和细胞进行电导率、pH值、苯丙氨酸解氨酶(PAL)活性以及细胞形态等进行分析。结果表明:(1)相对于正常培养的细胞,添加100μmol·L~(-1)的茉莉酸甲酯诱导24 h时,没食子酸的含量达到最高为12.49 mg·g~(-1),其含量是对照的2倍左右。(2)茉莉酸甲酯的添加导致细胞培养液的pH值和电导率成波动趋势,细胞膜受损,通透性增大,细胞核分散,出现多个细胞核现象。(3)细胞内可溶性蛋白含量在诱导24 h、72 h和5 d时达到高峰,其含量分别是对照的1.4、1.67、2.07倍左右。(4)苯丙氨酸解氨酶活性在诱导24 h和5 d时分别出现一次高峰,其活性分别是对照的2倍和3.75倍。研究认为,茉莉酸甲酯处理短时间内促进了茶条槭细胞内没食子酸含量的积累,细胞内PAL活性和可溶性蛋白含量有所增加,对细胞液中的pH值和电导率影响不显著。     

茉莉酸甲酯处理雷公藤悬浮细胞的基因差异表达分析

以不同浓度茉莉酸甲酯(MeJA)为诱导子对雷公藤悬浮细胞进行处理,采用cDNA-AFLP技术对差异表达基因进行研究。结果表明,茉莉酸甲酯在50～400μmol/L浓度范围内对雷公藤悬浮细胞总碱的积累呈抑制作用。茉莉酸甲酯处理后,分析筛选出了19个雷公藤悬浮细胞内差异表达的基因。通过与NCBI蛋白质数据库比对,7个片段的功能得以预测,涉及植物细胞的信号转导、转录调控和能量代谢等。这些结果对今后利用生物技术手段提高雷公藤生物碱含量奠定了一定基础。     

茉莉酸甲酯(MeJA)对贯叶连翘悬浮细胞生长和贯叶金丝桃素产量的影响

培养基中添加100 μm01·L-1MeJA后,贯叶连翘悬浮细胞生物量增加量和贯叶金丝桃素(HF)的产量分别是未经MeJA处理的1.3倍和1.73倍;培养3 d时,培养基中添加100 μmol·L-1 MeJA能提高HF产量,是未经MeJA处理的2.4倍.     

不同浓度茉莉酸甲酯对悬浮培养的胀果甘草细胞合成甘草总黄酮的影响

研究了不同浓度的茉莉酸甲酯对胀果甘草悬浮培养细胞的生长和黄酮合成的影响,初步探讨了其影响甘草黄酮合成的机制.研究结果表明,一定浓度(10-200lanol/L)的茉莉酸甲酯对胀果甘草细胞的生长有抑制作用,但是能够促进甘草总黄酮产量的增加.此外,茉莉酸甲酯的添加导致细胞中过氧化氢含量升高,引起细胞中苯丙氨酸裂解酶、过氧化氢酶、过氧化物酶活性的增强和丙二醛含量升高,说明茉莉酸甲酯能够引起细胞产生防御反应,并提高防御反应的关键酶的活性,同时细胞膜在一定程度上仍发生过氧化,但最终促进了甘草总黄酮的合成,其最大产量达到对照的3.39倍.     

茉莉酸甲酯、水杨酸和一氧化氮诱导怀槐悬浮细胞合成异黄酮及细胞结构变化

本文对比研究了茉莉酸甲酯(MeJA)、水杨酸(SA)和一氧化氮(NO)三种激发子对怀槐悬浮培养物异黄酮合成及细胞结构变化的影响。结果表明,在三种激发子的作用下怀槐细胞异黄酮合成量显著提高:200μmol/L MeJA、100μmol/L SA及50μmol／L SNP处理培养细胞9d后,异黄酮含量分别为同期对照的417．18％、185．45％和222．45％。同时细胞内发现染色很深的电子致密小体(EDB),其数量随着异黄酮含量的升高而增加,亦在第九天达到最多,与异黄酮积累呈现正相关性。推测激发子可能诱导植物细胞结构变化来响应次生代谢产物的合成。     

葡萄子房胚性细胞悬浮系的建立及其生长特性研究

由葡萄栽培品种“蜜汁”子房诱导的愈伤组织,经不同浓度２,４－Ｄ试验表明,０．５ｍｇ／Ｌ的２,４－Ｄ最有利于形成胚性细胞;该浓度下形成的质地紧密适度的愈伤组织经２个月的悬浮振荡培养,成功地建立了分散性好、生长旺盛的胚性细胞悬浮系;并通过培养过程中生长曲线的测定,确定了细胞悬浮系的生长特性。     

红豆杉细胞悬浮培养生产紫杉醇研究进展

介绍了近年来由红豆杉细胞培养生产紫杉醇领域取得的进展,其中特别介绍了由紫杉醇生物合成途径及其代谢酶类的研究发展而来的用基因工程方法改良细胞系,为提高紫杉醇产量而采取添加前体物质,诱导子,抑制子等方面的研究。     

壳寡糖对烟草悬浮细胞茉莉酸合成基因转录的影响

采用RT-PCR方法研究了不同浓度壳寡糖对烟草悬浮细胞茉莉酸合成酶基因的转录调控。结果表明, 50 μg.mL-1壳寡糖能够明显诱导烟草悬浮细胞茉莉酸合成途径的关键酶——磷脂酶A2、13-脂氧合酶、丙二烯氧化物合成酶、丙二烯氧化物环化酶和12-氧-植物二烯酸还原酶基因的表达, 而且该浓度的壳寡糖对这些基因的诱导作用相同(似)。在实验设定时间内均诱导表达编码磷脂酶A2的基因, 对其它基因的诱导时间均为8小时, 表明50 μg.mL-1壳寡糖在诱抗过程中启动了茉莉酸合成途径。而200 μg.mL-1壳寡糖的处理对这些基因的表达无显著影响。表明不同浓度的壳寡糖对烟草悬浮细胞的作用模式存在差异, 且高浓度的壳寡糖在烟草悬浮细胞中启动的信号通路可能没有茉莉酸信号的参与。     

Regulation of polyphenol production in Vitis vinifera cell suspension cultures by sugars

Sucrose was found to modulate polyphenol accumulation in Vitis vinifera cell cultures. The production of anthocyanins increased 12-fold after addition of 0.15 m sucrose, while that of stilbenes was only slightly affected. Sucrose did not play a physical role because metabolic sugars were required for the induction of polyphenol accumulation. Indeed, the polyols, mannitol and sorbitol, had no effect on this accumulation. We established a model system to investigate the mechanism of sucrose regulation of polyphenol production without inhibition of grape cell growth. After addition of sucrose to the culture medium, the major sugars accumulated in grape cells were glucose and fructose, reaching 40% of the dry weight. The increase in the level of these hexoses closely coincided with the increase in anthocyanin accumulation in grape cells. Received: 18 August 1997 / Revision received: 6 November 1997 / Accepted: 5 January 1998     

Jasmonates and Na-orthovanadate promote resveratrol production in Vitis vinifera cv. Barbera cell cultures

Here the effect of jasmonic acid, methyljasmonate and Na-orthovanadate on the production of resveratrol was studied in Vitis vinifera cv. Barbera cell suspension cultures. Na-orthovanadate at 0.1 mm and 1 mm concentration was efficient in promoting the production and/or accumulation and release in the culture medium of cis-resveratrol while trans-resveratrol levels were not affected by this treatment. Methyljasmonate was highly effective in stimulating both trans- and cis-resveratrol endogenous accumulation, as well as their release into the culture medium. Cis-resveratrol was absent or detected in very low amounts in the controls. Jasmonic acid was less efficient than methyljasmonate in promoting endogenous resveratrol accumulation, but it stimulated the release in the culture medium especially of cis-resveratrol. Gel analysis was performed on control and 10 microm MeJA treated cell suspensions. Results showed an up-regulation of the stilbene synthase demonstrating that MeJA stimulated the synthesis ex-novo of this protein.     

葡萄白藜芦醇合酶基因的克隆及序列分析

目的:从葡萄中克隆白藜芦醇合酶基因vrs1并对其序列进行生物信息学分析.方法:利用葡萄总RNA为模板,采用RT - PCR技术克隆白藜芦醇合酶基因vrs1并亚克隆进T- Vector.利用生物信息学工具对其核酸和蛋白序列进行分析.结果:测序结果显示其cDNA序列全长为1 257bp,含有一个1 179bp的开放阅读框.生物信息学分析表明葡萄白藜芦醇合酶基因编码392个氨基酸,分子量为42.9kDa,理论等电点为5.97,具有芪合酶家族固有的氨基酸保守结构域,二级结构主要由α-螺旋、无规则卷曲、延伸链和β-转角组成.结论:该基因的克隆、生物信息学分析为进一步研究其功能奠定了基础.     

Galloylated catechins and stilbene diglucosides in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were found to produce catechins and stilbenes. When cells were grown in a medium inducing polyphenol synthesis, (−)-epicatechin-3-O-gallate, dimeric procyanidin B-2 3′-O-gallate and two resveratrol diglucosides were isolated, together with a new natural compound that was identified as cis-resveratrol-3,4′-O-β-diglucoside by spectroscopical methods.     

Growth kinetics of vitis vinifera cell suspension cultures: I. Shake flask cultures

Vitis vinifera cell suspension cultures carried out in shake flasks were closely examined for biomass growth and cell division in relation to carbohydrate, NH(4), NO(3)PO(4), and dissolved oxygen (DO)consumption. After inoculation, the oxygen uptake rate of the cultures measured on-tine was observed to increase continuously to a maximum value of 3.8 mmol O(2)L(-1)h(-1) at day 7 when cell division ceased and dissolved oxygen reached its lowest level of 17% air saturation. During this first phase of growth, the specific oxygen uptake rate remained constant at approximately 0.6 mmol 02 O(2) g(-1) dw h(-1)or approximately 2.2 mumol O(2), (10(6) cells)(-1) h(-1) whereas dry biomass concentration increased exponentially from 1.5 to 6.0 g dw L(-1). Thereafter, dry biomass concentration increased linearly to approximately 14 g dw L(-1) at day 14 following nitrate and carbohydrate uptake. During this second phase of growth, the biomass wet-to-dry weight ratio was found to increase in an inverse relationship with the estimated osmotic pressure of the culture medium. This corresponded to inflection points in the dry and wet biomass concentration and packed cell volume curves. Furthermore, growth and nutrient uptake results suggest that extracellular ammonium or phosphate ion availability may limit cell division. These findings indicate that cell division and biomass production of plant cell cultures may not always be completely associated, which suggests important new avenues to improve their productivity. (c) 1995 John Wiley & Sons, Inc.     

Condensed tannin and anthocyanin production in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were cultured in different media in order to establish a model system for promoting high levels of phenolic substances identical with those found in wine. These media were: a low sucrose maintenance medium (MM) and four high sucrose media (differing mainly in sucrose and mineral contents) which were shown to induce secondary metabolism. In MM medium, polyphenol accumulation in the cells was low, and concentrations of 0.1 mg/gfw for condensed tannins and 0.3 mg/gfw for anthocyanins were reached within two weeks of cultivation. Values of 1.4 and 6.4 mg/gfw, respectively, were obtained with a low nitrate and high sucrose medium (HM1), but cell proliferation was reduced. To obtain a maximal production of polyphenols, we investigated the most effective conditions for cell growth and polyphenol production (a high mineral and high sucrose medium, IM1; inoculum dilution of 1.25:10). Under these conditions, the cells produced mainly anthocyanins (1100 mg/l), proanthocyanidins (300 mg/l) and catechins (25 mg/l).Abbreviations BuOH n-butanol - dw dry weight - fw fresh weight     

前体饲喂、诱导子和光照联合使用对葡萄细胞培养合成花青素的影响

苯丙氨酸前体饲喂分别和环糊精、葡聚糖、茉莉酸甲酯、黑曲霉和直喙镰孢菌提取液五种诱导子联合作用,其中以与茉莉酸甲酯的联合作用对葡萄细胞培养生产花青素的影响最大,可使单位鲜细胞花青素含量提高2.7倍,花青素产量提高3.4倍,实验证明两者在培养后第4天加入效果最好。在30μmol/L苯丙氨酸、218μmol/L茉莉酸甲酯和3000~4000lx光照条件下,不同花青素产量的细胞株都能显著提高花青素产量,但低产株VV06比高产株VV05具有更大的产率提高潜力。该条件下VV05和VV06花青素产量分别达到2975和4090CV/L,是对照组的2.5倍和5.2倍。     

A non-destructive determination of leaf chlorophyll in Vitis vinifera

A portable leaf greenness meter (SPAD-501) has been used to provide a rapid and non-destructive measurement of leaf chlorophyll in Vitis vinifera. Leaf extracted chlorophyll was related linearly to SPAD readings. It is suggested that separate linear equations should be developed for each cultivar so as to maximise the accuracy of estimating leaf chlorophyll content as a function of SPAD readings.     

Characterization of new microsatellite loci from Vitis vinifera and their conservation in some Vitis species and hybrids

We present here characterization data for seven new microsatellite markers designed from new microsatellite loci isolated from a microsatellite‐enriched DNA library from Vitis vinifera. The observed heterozygosity varied from 0.73 up to 0.93 and the number of alleles per locus ranged from 12 to 26. This high polymorphism makes these new markers interesting for use in genotyping studies and completing the set of microsatellite markers already available for V. vinifera. Additionally these seven new markers appear to be conserved in four other Vitis species and 15 Vitis hybrids used as rootstocks for V. vinifera cultivation.     

原位吸附和茉莉酸甲酯联合使用显著提高中国红豆杉细胞云南紫杉烷c的生产

本实验所用的中国红豆杉细胞悬浮培养体系中,云南紫杉烷c(Tc)是主要的次生代谢产物,该化合物有类神经生长因子活性,提高其产量是进一步规模化生产的前提。本研究考察了原位吸附和茉莉酸甲酯(MJA)联合调控提高Tc产量的可能性。在培养的第7天加入浓度为100μmol/L的MJA虽然会使细胞的生物量下降10%～30%,但是单位细胞内Tc含量和Tc产量均有显著提高,分别是对照的3.6和3.3倍。吸附剂XAD-7在不同时间加入对Tc的合成影响显著。在培养的第7天同时加入100μmol/L的MJA和100g/L的XAD-7会使细胞生物量增加,Tc产量显著提高。培养到第21天,Tc产量达477.4mg/L,为对照的6.3倍,为只加MJA的1.9倍,其中94%的Tc被树脂吸附。实验结果表明,在MJA诱导高表达的过程中,吸附剂XAD-7的加入使细胞内代谢产物外泌,浓度降低,减轻产物反馈抑制现象,从而大幅度提高代谢物产量,有较好的生产前景。