西洋参细胞悬浮培养中皂甙生物合成的代谢调节

七种真菌菌丝体诱导因子中,六种促进培养细胞的皂甙生物合成。尤其葡枝根霉作用更为明显,提高皂甙含量二倍。人参寡糖素是一种既能诱导皂甙生物合成,又能促进细胞生长的新型诱导因子,浓度为50mg／L寡糖素既能维持细胞较好生长又能提高皂甙含量,因而得到较高的皂甙产率。用四种条件培养液进行培养,结果均促进悬浮培养细胞的皂甙生物合成,其中丹参条件培养液使悬浮培养细胞的皂甙含量提高二倍多。加入皂甙生物合成主途径的三种前体,都不同程度地促进皂甙的生物合成,前体中以角鲨烯作用最为明显。     

促进黄花蒿发根青蒿素合成的内生真菌诱导子的制备

应用酸解法对黄花蒿(ArtemisiaannuaL.)内生胶孢炭疽菌(Colletotrichumgloeosporioides)菌丝体进行提取,在黄花蒿发根培养系统中比较了各制备提取物的青蒿素诱导活性。活性提取物经过SephadexG25层析后,部分纯化的内生菌寡糖提取物(MW<2500)可显著促进发根青蒿素的合成,培养23d的发根经诱导子(0.4mg/mL)处理4d后,青蒿素产量可达13.51mg/L,比同期对照产量提高51.63%,诱导作用与诱导子浓度、作用时间相关。内生菌寡糖诱导子的制备和使用,在青蒿素生物技术生产研究中为首次应用。     

诱导子对雷公藤不定根生长和次生代谢产物含量的影响

为探讨真菌诱导子以及硝酸银等非生物诱导子对雷公藤不定根生长及次生代谢产物含量的影响。以雷公藤不定根为材料,通过向培养基中添加不同种类的真菌诱导子以及硝酸银等非生物诱导子,采用高效液相色谱检测不定根中雷公藤甲素和生物碱含量。结果表明:各种真菌诱导子不影响不定根的生长,但对次生代谢产物含量有显著影响,其中,苹果炭疽和柿子炭疽诱导子的加入不仅使不定根中雷公藤甲素的含量分别提高了2.24和1.93倍,生物碱的含量也各提高了2.02和2.07倍。苹果炭疽诱导子浓度为50μg/m L时比较适合雷公藤不定根生长及雷公藤甲素和生物碱的积累。硝酸银抑制不定根的生长和生物碱的积累,但促进雷公藤甲素的积累。硝酸银浓度为25μmol/L时雷公藤甲素含量为对照的1.71倍。茉莉酸甲酯浓度为50μmol/L时,不定根增长量为对照的1.04倍,雷公藤甲素和生物碱含量分别为对照的1.64倍和2.12倍。酵母提取物浓度为2 g/L时,雷公藤甲素含量为对照的1.48倍。表明培养基中添加硝酸银和酵母提取物对不定根中雷公藤甲素的合成具有明显的促进作用,苹果炭疽和茉莉酸甲酯的协同作用既能促进雷公藤甲素的合成又能促进雷公藤生物碱的合成。     

内生真菌Colletotrichum sp. B501的寡糖提取物对黄花蒿发根中青蒿素生物合成的诱导

在黄花蒿(Artemisia annua L.)发根液体培养中,黄花蒿内生炭疽菌(Colletotrichum sp. B501)细胞壁寡糖提取物可促进发根青蒿素的合成.经寡糖诱导子(20 mg/L)处理4 d后,发根青蒿素含量达1.15 mg/g, 比对照高出64.29%.诱导作用与诱导子浓度、作用时间相关.诱导处理1 d后,X射线能谱分析表明黄花蒿发根细胞中Ca2+积累量显著增高,电镜观察发现液泡内出现高电子致密物,具活性氧清除作用的过氧化物酶表现出高活性(6.5 unit*min-1*g-1 FW).诱导处理第三天,细胞核DNA呈梯度条带降解,部分细胞出现程序化死亡.内生菌细胞壁寡糖提取物引起的生理反应有利于细胞中青蒿素的生物合成.     

真菌诱导子对青蒿发根细胞生长和青蒿素积累的影响

３种真菌诱导子（大菌丽花轮枝孢（Ｖｅｒｔｉｃｉｌｌｉｕｍ ｄａｈｉａｅ Ｋｌｅｂ．）、葡枝根霉（Ｒｈｉｚｏｐｕｓ ｓｔｏｌｏｎｉｆｅｒ（Ｅｈｒｅｎｂ．ｅｘＦｒ．）Ｖｕｉｌｌ）和束状刺盘孢（Ｃｏｌｌｅｔｏｒｉｃｈｕｍ ｄｅｍａｔｉｕｍ（Ｐｅｒｓ．）Ｇｒｏｖｅ）处理青蒿（Ａｒｔｅｍｉｓｉａ ａｎｎｕａＬ．）的发根,均能促进发根中青蒿素的积累,其中以大丽花轮枝孢的诱导效果最好;对细胞生长均没有明显影响,     

内生真菌Colletotrichum sp. B501的寡糖提取物对黄花蒿发根中青蒿素生物合成的诱导(英文)

在黄花蒿 (ArtemisiaannuaL .)发根液体培养中 ,黄花蒿内生炭疽菌 (Colletotrichumsp .B5 0 1)细胞壁寡糖提取物可促进发根青蒿素的合成。经寡糖诱导子 (2 0mg/L)处理 4d后 ,发根青蒿素含量达 1.15mg/g ,比对照高出6 4 .2 9%。诱导作用与诱导子浓度、作用时间相关。诱导处理 1d后 ,X射线能谱分析表明黄花蒿发根细胞中Ca2 积累量显著增高 ,电镜观察发现液泡内出现高电子致密物 ,具活性氧清除作用的过氧化物酶表现出高活性 (6 .5unit·min-1·g-1FW)。诱导处理第三天 ,细胞核DNA呈梯度条带降解 ,部分细胞出现程序化死亡。内生菌细胞壁寡糖提取物引起的生理反应有利于细胞中青蒿素的生物合成。     

丹参植株内生菌的分离纯化及其抗病性研究

为探明丹参(Salvia miltiorrhiza Bge)内生菌的种类,筛选拮抗农作物病害菌的生防菌株,从健康丹参植株中进行内生菌分离,依照形态特征以及16SrDNA序列对菌株进行初步分类鉴定,采用琼脂块法进行拮抗菌株筛选,并选取拮抗活性较强的菌株液体发酵进行体外抑菌试验。结果表明:(1)从丹参植株中共分离得到69株内生菌(真菌62株、放线菌7株),其中23株内生真菌属于无孢类群或现有条件不适合产孢,其余真菌菌株中串珠镰孢菌属(Fusarium moniliforme Sheld.)和交链孢菌属(Alternaria sp)为优势菌群;7株内生放线菌均为链霉菌属。(2)病原菌拮抗性实验表明,有44株内生菌对病原菌具有不同程度的拮抗活性,其中12株内生菌对2种及以上靶标病原菌具有拮抗活性,表明丹参内生菌具有一定的广谱抗菌性。(3)放线菌菌株A232的次级代谢产物对白色假丝酵母(Canidia albicans)和苹果腐烂菌(Valsa mali)都表现出较强的拮抗活性,通过形态、培养特征以及16SrDNA序列分析,鉴定其为Streptomyces luteoverticillatus。     

不同寄主来源的串珠镰孢生物学性状及其在无性后代的遗传与变异

自安徽、山东、湖北、江苏等地多点采集棉花红腐病、水稻恶苗病、玉米穗腐病的病组织,经分离、鉴定和纯化,获得107个串珠镰孢（Fusarium monilifoFine）菌株。对上述来源于棉花、水稻、玉米的串珠镰孢的菌落形态、生长速率和产孢量等生物学性状及其在无性后代的遗传与变异进行了研究。结果表明,不同寄主来源的串珠镰孢菌株的菌丝生长温度范围和最适温度大致相同,但在菌落形态特别是色素方面存在明显差异,生长速率和产孢量也存在显著差异。棉花菌株的平均生长速率最大,玉米菌株生长速率最小,水稻菌株生长速率居中,相同群体的不同菌株间生长速率有极显著差异;玉米菌株产孢量最大,棉花菌株产孢量最小,水稻菌株产孢量居中。方差分析显示,不同寄主菌株群体间产孢量存在显著差异,而同一寄主群体的不同菌株间产孢量均无显著差异,说明菌株产孢量大小主要与其寄主种类有关,而与地区来源关系不大。遗传测定结果表明,分离自棉花、玉米和水稻的串珠镰孢的菌落形态和生长速率在单分生孢子后代均可稳定遗传;产孢量性状遗传有两种情况：分离自棉花和水稻的串珠镰孢菌株Fm1和Fm31的产孢量性状在单分生孢子后代均可稳定遗传,而分离白玉米的串珠镰孢菌株Fm19的产孢量性状在单分生孢子第一代（CG1）发生变异。     

在自然衰老和诱导条件下棉花悬浮细胞程序性死亡的发生

棉花胚性悬浮细胞在MS 0.1mg/L2,4-D 0.1mg/LKT培养基中培养生长良好,但在不继代的情况下会自然衰老。培养至第17天,细胞生活力开始下降;至第21天可检测到核小体大小倍增的DNA梯(DNALadder)存在。42±3℃热激、10μmol/L喜树碱、20μmol/L串珠镰孢菌毒素和50mmol/L放线菌酮等胁迫诱导可分别引起MS 0.1mg/L2,4-D 0.1mg/LKT培养基中的棉花悬浮细胞发生程序性死亡。在MS 0.1mg/L2,4-D 0.1mg/LKT和MS 0.1mg/LIBA 0.1mg/LKT培养基中悬浮培养的棉花胚性细胞处于不同的生理状态,两种不同状态的棉花悬浮细胞对热激、喜树碱、串珠镰孢菌毒素等胁迫因子的反应不同。     

两种基因型大豆根分泌物对大豆根腐病菌的化感作用

采用生物模拟试验、化学分析等方法,研究了两种大豆基因型(9536、吉林30)的根分泌物中的糖、氨基酸、有机酸组分对大豆根腐病菌的化感作用.结果表明,两种大豆基因型(9536、吉林30)根分泌物糖组分表现出低浓度显著促进、高浓度显著抑制半裸镰孢菌、尖镰孢菌生长的规律,对粉红粘帚菌的生长影响不明显;氨基酸组分对上述三种病原菌所表现出的促进、抑制规律不同,9536基因型根分泌物氨基酸组分的中、高浓度处理对半裸镰孢菌、粉红粘帚菌、尖镰孢菌的生长表现出显著的抑制作用,而吉林30多表现出显著的促进作用;有机酸组分对半裸镰孢菌、粉红粘帚菌、尖镰孢菌生长都有显著的抑制作用.两种基因型大豆根分泌物(糖、氨基酸、有机酸组分)与根腐病害发生密切相关,大豆基因型不同,根分泌物对根腐病菌的化感促进或抑制作用有差异.     

Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate reductoisomerase

Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g^?1 DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag⁺ than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g^?1 DW by YE, Ag⁺, and YE-Ag⁺ treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.     

Methyl jasmonate elicitation enhances glycyrrhizin production in Glycyrrhiza inflata hairy roots cultures

Hairy roots were induced by infecting stems and leaves of Glycyrrhiza inflata with Agrobacterium rhizogenes ATCC 15834. The optimization of growth and glycyrrhizin accumulation of G. inflata hairy roots was studied. Sucrose (6%, w/v) was optimal for growth and glycyrrhizin accumulation in G. inflata hairy roots. Effects of elicitors like chitosan, methyl jasmonate, and yeast extract on glycyrrhizin production were studied. Methyl jasmonate (100 microM) was most efficient in enhancing glycyrrhizin production up to almost 109 microg/g dry weight on day 5 of elicitation. These results indicate that application of elicitors can enhance the capacity of G. inflata hairy roots to produce glycyrrhizin.     

Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed-batch culture of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots

Hyperosmotic stress (OS, created with 50 g/L sorbitol) and a yeast elicitor (YE, polysaccharide fraction of yeast extract) applied to Salvia miltiorrhiza hairy root cultures had a synergistic effect on the diterpenoid tanshinone production. With a single OS+YE treatment and nutrient feeding, the total tanshinone content of roots was increased by sevenfold (from 0.2 to 1.6 mg/g dry weight (dw)) and the volumetric yield by 13-fold (from 1.95 to 27.4 mg/L) compared to the batch control culture. With repeated feeding of OS and nutrient medium in an extended fed-batch culture process (i.e., 10 mL fresh medium with 50 g/L sorbitol 25 mg/L YE, every 5 days from day 21 to day 60), the total tanshinone content of roots was increased to 18.1 mg/g dw (or 1.8 wt.%) and the volumetric tanshinone yield to 145 mg/L, which were about 100-fold and 70-fold of those, respectively, in the batch control. Another interesting finding was the presence of root fragments (fine particles) with extremely high tanshinone content in the OS+YE treated cultures. It was also possible to reuse the sorbitol medium for the hairy root growth and tanshinone production to reduce the medium expenses.     

Induction and potentiation of diterpenoid tanshinone accumulation in Salvia miltiorrhiza hairy roots by β-aminobutyric acid

The non-protein amino acid -aminobutyric acid (BABA) is a proven inducer of plant defense against pathogens. This work examines its effect on the production of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures, both separately and in combination with a yeast elicitor (YE, the carbohydrate fraction of yeast extract). In the absence of YE, BABA at 0.1, 1 and 2 mM caused a dose-dependent enhancement of tanshinone accumulation, with up to a 4.5-fold increase (from 0.24 to 1.09 mg/g DW) in total content of three major tanshinones (cryptotanshinone, tanshinone I and tanshinone IIA) in the hairy roots. The combination of BABA with YE treatment further enhanced tanshinone production, but only when the BABA treatment was applied to the culture a few days before the YE treatment. Compared with methyl jasmonate, BABA was more effective in enhancing tanshinone production. A 3-day pretreatment with 1 mM BABA followed by YE-treatment, increased the total tanshinone content of roots by 9.4 times to 2.26 mg/g cells, and the volumetric tanshinone yield of culture by 6.3 times (from 3.2 to 20.1 mg/l). The results suggest that BABA can strongly potentiate elicitor-induced secondary metabolism in plant tissue cultures.     

Accumulation of cell wall-bound phenolic metabolites and their upliftment in hairy root cultures of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.)

Alkaline hydrolysis of cell wall material of tomato hairy roots yielded ferulic acid as the major phenolic compound. Other phenolics were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. The content of phenolics was much higher at the early stage of hairy root growth. The ferulic acid content decreased up to 30 days and then sharply increased to 360 microg/g at 60 days of growth. Elicitation of hairy root cultures with Fusarium mat extract (FME) increased ferulic acid content 4-fold after 24 h. As the pathogen-derived elicitors have specific receptors in plants, FME may thus be used for inducing resistance against Fusarium oxysporum f. sp. lycopersici.     

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Valerenic acid (VA) is a pharmacologically-active sesquiterpene found in valerian (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L^?1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.     

丹参冠瘿组织高产株系选择和丹参酮的产生

丹参(Salvia miltiorrhiza)是治疗心血管系统疾病的重要中草药。前文我们已报道了丹参冠瘿组织培养的部分研究结果^[1].利用根癌农杆菌(Agrobacterium tumefaciens)Ti 质粒转化产生的冠瘿组织进行高产株系选择的研究还很少,本文报道丹参冠瘿组织高产株系选择和丹参酮产生的研究结果。     

Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation

In Salvia miltiorrhiza hairy root cultures, the desired secondary metabolites diterpenoid tanshinones are normally produced at low yields and stored within the roots. To enhance tanshinone production and the secondary product recovery, we employed three means, elicitation with a yeast elicitor (YE), in situ adsorption of tanshinones with a hydrophobic polymeric resin (X-5) and semi-continuous mode of operation. YE treatment stimulated the tanshinone biosynthesis, increasing the total tanshinone (TT) content of root by about two-fold, from 0.46 to 1.37 mg/g dry weight (dw) (TT content=total content of three major tanshinones, cryptotanshinone, tanshinone I and tanshinone IIA). The addition of X-5 resins to the culture only increased the tanshinone yield slightly, but recovered more than 80% of tanshinones from the roots. With the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement, starting at the late exponential growth phase, the root biomass was increased to 30.5g dw/l (versus 8-10g dw/l in batch mode) and the volumetric tanshinone yield to 87.5mg/l (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/lday, more than 7.4 times that of the batch culture. The results demonstrate that the integration of multiple elicitation, in situ adsorption and semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures.     

The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza

Hairy root cultures of Salvia miltiorrhiza transformed with Agrobacterium rhizogenes ATCC 15834 produced a tiny amount of tanshinones and a constituent level of phenolic acids under normal growth conditions. Upon elicitation with yeast elicitor, the production of both phenolic acids and tanshinones was enhanced. For example, the contents of two phenolic acids, rosmarinic acid and lithospermic acid B were elevated from 1.24% and 2.59% to 2.89% and 2.98% of dry wt, respectively while the intracellular content of cryptotanshinone increased from 0.001% to as much as 0.096% of dry wt. Yeast elicitor also improved the growth of hairy roots (from 3.9 g/l to 7.3 g/l on a dry wt basis). Liquid chromatography-mass spectrometry (LC-MS) was developed for simultaneous detection and identification of phenolic acids and tanshinones in the extracts of S. miltiorrhiza. Rosmarinic acid, lithospermic acid B, cryptotanshinone, tanshinone I, tanshinone IIA and tanshinone IIB were identified by comparison with standards available. Dihydrotanshinone I and methylenetanshiquinone were tentatively identified by the molecular weights and the elution comparable with the literature. An unknown compound with a molecular weight of 280 was found in yeast-elicitor treated hairy root cultures, which was one of the major tanshinones induced.