虫草素高产菌株的筛选及不同添加物对虫草素产量的影响研究

通过对14株蛹拟青霉菌株进行摇瓶液体发酵培养试验,筛选虫草素产量最高的蛹虫草菌株,并确定了虫草素的最佳收获期;比较8种营养添加物对虫草发酵过程中虫草素积累的影响,筛选出最佳的营养添加物,以提高液体发酵生产虫草素的产量。结果表明：蛹虫草CM001号菌株的虫草素产量最高,蛹虫草菌在第10天细胞量达到最大值20.44mg/mL,虫草素含量在第13天达到最大值73.81mg/L,70%以上的虫草素分布在发酵液中。其中分别添加腺苷、腺嘌呤、丙氨酸、L-天冬氨酸、甘氨酸5种物质,对虫草素合成的促进作用均较明显,均能有效提高蛹虫草液体发酵虫草素的产量,特别是添加1g/L的腺嘌呤能使10号菌株的虫草素产量提高7.09倍。     

谷胱甘肽促进液体发酵体系中蛹虫草合成虫草素

虫草素是药用真菌蛹虫草Cordyceps militaris的主要活性成分,具有抗癌、抗肿瘤、抗病毒等多种生理活性。研究表明,氧化应激参与丝状真菌的次级代谢调控,然而在蛹虫草虫草素代谢中尚未见报道。本研究以蛹虫草深层液体发酵体系为研究对象,添加谷胱甘肽（glutathione,GSH）调节细胞氧化还原状态,考察其对虫草素代谢的影响。添加3.0g/L GSH,发酵20d虫草素产量达到(439.69±12.43)mg/L,较无添加对照组提高471.24%,同时谷胱甘肽过氧化物酶（glutathione peroxidase,GPX）活力提高414.82%;基因表达分析显示,添加GSH后虫草素生物合成关键基因Cns1和Cns2表达显著上调,发酵第15天其表达量分别是对照组的540.67倍和25.81倍。研究结果初步证实,氧化应激参与蛹虫草中虫草素代谢调控。     

真菌激发子对提高蛹虫草虫草菌素的作用

研究了不同真菌激发子菌株对提高蛹虫草生物量和虫草菌素含量的影响,其中一株疫霉（Phytophthora sp.)YL提高虫草菌素含量比对照高4倍。机械研磨和80℃高温细胞自溶相结合制备的真菌激发子诱导效果最佳。同一激发子不同浓度对虫草素的诱导实验表明,80μg/ml浓度碳水化合物的真菌激发子诱导蛹虫草产生虫草菌素效果最明显。     

真菌激发子对提高蛹虫草虫草菌素的作用*

研究了不同真菌激发子菌株对提高蛹虫草生物量和虫草菌素含量的影响,其中一株疫霉(Phytophthora sp.)YL提高虫草菌素含量比对照高4倍。机械研磨和80℃高温细胞自溶相结合制备的真菌激发子诱导效果最佳。同一激发子不同浓度对虫草素的诱导实验表明, 80mg/ml浓度碳水化合物的真菌激发子诱导蛹虫草产生虫草菌素效果最明显。     

不同培养条件和前体对蛹虫草液体发酵产虫草素的影响

蛹虫草能产生虫草素等多种活性物质。为考察不同液体发酵方式及添加前体物质对虫草素积累的影响,选用蛹虫草08Y1菌株,通过光照振荡、光照静置、黑暗振荡、黑暗静置四种培养条件和添加前体物质（腺嘌呤1g/L+甘氨酸16g/L）,发酵16d后检测虫草素和腺嘌呤含量。结果表明：08Y1菌株在黑暗振荡培养条件下,虫草素积累达1,015.0mg/L,腺嘌呤利用率达98.5%,说明黑暗振荡培养并添加前体物质是提高虫草素产量的有效方法。     

蛹虫草菌生物合成虫草素的研究进展

蛹虫草Cordyceps militaris是我国传统的药用真菌,虫草素是蛹虫草的主要活性成分,具有抗癌、抗肿瘤、抗病毒等多种生理功能。蛹虫草菌液体发酵是最有希望实现高效生产虫草素的途径,但现阶段生产强度低,亟需应用发酵工程及代谢工程手段提高虫草素产量。文中对液体发酵体系中培养基组分(碳/氮源、前体物质、金属离子等)和培养条件(pH、溶氧量、光照等)对虫草素产量的影响进行了总结,并对虫草素的分离纯化、生物合成基因簇及合成代谢途径进行了阐述,最后探讨了实现虫草素高效生产的关键环节。     

锌富集对蛹虫草菌丝体内虫草素、腺苷含量的影响

为了解蛹虫草菌丝体对锌的富集特性,研究锌富集对蛹虫草菌丝体内虫草素、腺苷含量的影响,通过在液体培养基中添加不同质量浓度的锌离子(0~35 g/L),探讨其对蛹虫草菌丝生物量、菌丝体内锌积累量,以及锌的富集对菌丝体内虫草素、腺苷含量产生的影响。结果表明:在0~35 g/L锌离子的梯度范围内,蛹虫草菌丝生物量与锌质量浓度呈显著负相关,锌质量浓度35 g/L为蛹虫草菌丝生长极限浓度。锌质量浓度40.0 g/L及以上菌丝生长受到完全抑制。菌丝体内锌的积累量随锌质量浓度的增加而显著升高,锌质量浓度为35.0 g/L时锌积累量可达到193.87 mg/g(干重)。蛹虫草菌丝体内腺苷的含量随锌质量浓度的增加而降低,在锌质量浓度为5 g/L时降幅显著,腺苷含量仅为对照组的17.24%,之后腺苷含量变化趋势趋于水平。腺苷含量的降低可能是因为锌的富集干扰了蛹虫草菌丝体内初生代谢的正常进行。虫草素的含量随锌质量浓度的增加而显著降低,可能是由于其直接前体腺苷含量的降低,或是Zn离子的加入,使得某些被刺激的酶和基因通过转录因子影响了虫草素的合成。     

低能离子束修饰蛹虫草菌株高产虫草素

虫草素具有抗肿瘤、免疫调节、抗炎等多种功效。为了更好地开发蛹虫草资源,选择合适剂量的低能离子束注入蛹虫草,优化虫草素的提取工艺条件,采用紫外分光光度法检测注入前后菌株中虫草素的含量。结果表明:最佳注入剂量为2.60×1015ions/cm2,虫草素最佳的微波-超声波提取工艺为:乙醇浓度70%,提取功率200W,提取时间110s,料液比1:240。选育出虫草素含量较高的15株菌株,最高含量达(11.924±0.063)mg/g,比原始菌株增长了近30%。     

蛹虫草耐铁特征研究

培养富铁蛹虫草研究其耐铁特征,发现:蛹虫草菌丝耐受铁的浓度范围为0~1.6 g/L;富铁蛹虫草培养基最佳添加铁浓度为0.06 g/L;富铁蛹虫草液体深层发酵72 h达到最大生物量和次高富集铁。     

拟青霉A20140822发酵生产虫草素工艺的响应面优化研究

用响应面分析法研究优化拟青霉A20140822发酵虫草素工艺。通过单因素试验找出影响发酵虫草素得率的主要因素。并按照响应面分析方法设计试验方案,分析得出影响虫草素得率的主要因素的最佳参数。拟青霉在温度21℃、时间38 h、维生素B_1添加量0.08 g/L时,虫草素含量最高达到1.465 g/L。拟青霉A20140822发酵生产虫草素的量比已报道的拟青霉菌种高20%~50%,其发酵条件受温度、时间、维生素B1添加量等因素影响。     

小麦成熟胚愈伤组织诱导及分化研究

以2个小麦品种成熟胚为外植体进行离体培养,研究了不同预处理、不同2,4-D浓度及与KT组合、不同蔗糖浓度等因素对愈伤组织诱导及分化的影响。结果表明:4℃低温预处理可提高愈伤组织的出愈率及再生苗率,2个材料的出愈率及再生苗率均达到90%和30%以上;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,2,4-D浓度为1~2 mg/L更有利于愈伤组织诱导及分化;附加KT能缓解高浓度2,4-D对再生苗率的抑制作用,而对于在1、2 mg/L 2,4-D的培养基中附加KT则不表现这种作用;蔗糖浓度则在30 g/L条件下更有利于愈伤组织诱导。因此通过4℃低温预处理,在MS基本培养基中附加1~2mg/L 2,4-D及30 g/L蔗糖亦可促进小麦成熟胚愈伤组织的诱导和分化。     

葡萄风信子叶片及花器官组织培养研究

以4种葡萄风信子Muscari armeniacum、Muscari aucheri ‘Dark Eyes’、Muscari aucheri ‘Ocean Magic’、Muscari azureum的叶片以及M.aucheri ‘Dark Eyes’的花器官为外植体,进行诱导愈伤组织分化的研究,建立了葡萄风信子的组织培养体系。研究结果表明：葡萄风信子抽梗期叶片最适升汞灭菌时间为7 min;筛选出四种品种的叶片最佳诱导愈伤组织培养基均为：MS+2,4-D 0.5 mg·L^-1+TDZ 0.1 mg·L^-1;同一叶片不同部位的愈伤组织诱导率由高到低依次是叶片基部、叶片中部、叶片尖端;M.aucheri ‘Dark Eyes’品种的花器官出愈率由高到低依次是花梗、花瓣、花药;筛选出M.aucheri ‘Dark Eyes’花药的最佳诱导培养基：MS+2,4-D 0.1 mg·L^-1+6-BA 0.5 mg·L^-1。     

外源激素对何首乌毛状根生长及蒽醌类成分生物合成的影响

研究了外源激素对液体培养何首乌毛状根生长及其蒽醌类化合物生物合成的影响。结果表明外源激素2,4-D、NAA和6-BA对何首乌毛状根的生长及蒽醌生物合成有较大的影响。在MS培养基中添加2,4-D对何首乌毛状根的生长和蒽醌类化合物的生物合成有很强的抑制作用,而添加适量浓度NAA和6-BA则可促进何首乌毛状根的生长以及蒽醌类物质的生物合成。     

若干因子对鸡冠花悬浮培养中花色素苷积累的影响

本文研究几种植物生长调节剂和蔗糖浓度对鸡冠花细胞悬浮培养中花色素苷积累的影响。结果表明,细胞分裂素KT使花色素苷积累明显高于6-BA,且KT在2 μmol/L时积累量最高;2,4-D在2 μmol/L时对花色素苷积累效果明显,其它浓度的2,4-D和NAA对花色素苷积累效果不明显。高浓度蔗糖有利于花色素苷积累;MS+2,4-D(2 μmol/L)+KT(2 μmol/L)+蔗糖(292 mmol/L)为鸡冠花悬浮细胞培养生产花色素苷的最佳培养基。研究中还发现,在黑暗条件培养下无花色素苷积累,推断光是诱导花色素苷积累的主要因素。随着继代次数的增加,花色素苷含量明显增高,但到第4代时基本稳定。     

Somatic embryogenesis,organogenesis and callus growth kinetics of flax

The effects of plant growth regulators (PGR) on calli induction, morphogenesis and somatic embryogenesis of flax were studied. The organogenic and callus formation capacity were assessed for different types of source explants. Root and shoot explants were equally good material for calli production but the former produced calli without shoot regeneration capacity. Under the experimental conditions tested, 2,4-dichlorophenoxyacetic acid (2,4-D) + zeatin was the most efficient PGR combination on calli induction and biomass production. The calli were green but with no rhizogenic capacity. In contrast, and at similar concentrations, indole-3-butyric acid (IBA) + kinetin induced white or pale green friable calli with a good root regeneration capacity (60%). A factorial experiment with different combinations of 2,4-D + zeatin + gibberellic acid (GA₃) levels revealed that the direction of explant differentiation was determined by specific PGR interactions and concentrations. The results from these experiments revealed that the morphogenetic pathway (shoot versus root differentiation) can be manipulated on flax explants by raising the 2,4-D level from 0.05 to 3.2 mg l^?1 in the induction medium. The induction and development of somatic embryos from flax explants was possible in a range of 2,4-D + zeatin concentrations surrounding 0.4 mg l^?1 2,4-D and 1.6 mg l^?1 zeatin, the most efficient growth regulator combination.     

曼陀罗茎段愈伤组织诱导和再生植株的研究

本试验以曼陀罗茎段为外植体,在附加不同植物激素组合的培养基中对愈伤组织的诱导和植株再生进行研究。结果表明:采用修改的MS培养基(除去甘氨酸,维生素B1含量增加至0.5mg/L,pH5.5)附加2mg/L2,4-D可由曼陀罗茎段诱导大量胚性愈伤组织;愈伤组织继代选用0.5mg/L2,4-D为宜;不定芽的诱导采用MS培养基(20g蔗糖,8g琼脂,0.1g水解干酪素) 6-BA(0.5mg/L);幼苗进一步转接至1/2MS IBA(0.2mg/L)生根培养基中,可完成曼陀罗茎段愈伤组织诱导和再生植株的组织培养过程。     

Effects of macronutrients on growth and rosmarinic acid formation in cell suspension cultures of Anchusa officinalis

The influence of various macronutrients on growth and RA formation in cell suspension cultures of Anchusa officinalis has been investigated. Factors tested included sucrose concentration, alternate carbon sources, nitrate, phosphate and calcium concentration. The optimum concentration of sucrose was 3%. Fructose, glucose or their 1:1 mixture were also suitable carbon sources. The optimum concentrations of nitrate (15 mM), phosphate (3 mM) and calcium (0.25 mM) were, respectively, 3/5, 3x, and 1/4 those in normal B5 medium, when tested separately. These concentrations improved not only the yield of RA but also cell growth to a similar degree (10%–50%). Studies on the combined effects of these optimum macronutrient concentrations in B5 medium showed that RA production is inhibited by 2,4-D-containing revised medium but stimulated by NAA-containing revised medium.Abbreviations RA rosmarinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-napthaleneacetic acid     

Somatic embryo maturation from long-term suspension cultures of white spruce (Picea glauca)

Summary The production of cotyledonary somatic embryos of white spruce from cultures grown long-term as suspensions was investigated. We report the effects of removal of 2,4-dichlorophenoxyacetic acid (2,4-D) from the maintenance medium (ordinarily containing both 2,4-D and benzyl adenine) before (±)-ABA-stimulated maturation. In particular the use of a 1-wk culture period without 2,4-D was found to improve the production of normal-looking cotyledonary somatic embryos. Using high performance liquid chromatography analyses of culture supernatants, it was determined that this affect was not related to altered ABA metabolism. Germination of cotyledonary somatic embryos from cultures pretreated by the 1-wk culture period without 2,4-D was improved compared with similar embryos from cultures that had not been pretreated.     

2,4-D Resistance in a Tobacco Cell Culture Variant II. Effects of 2,4-D on Nucleic Acid and Protein Synthesis and Cell Respiration

The effects of 2,4-D on nucleic acid and protein synthesis andcell respiration were compared between a 2,4-D-resistant variantand its wild-type cell lines of tobacco (Nicotiana tabacum L.).The variant continued cell division and growth in the presenceof 100 µM 2,4-D which was strongly inhibitory to the wild-typecell lines. Among the macromolecular syntheses studied, DNAsynthesis was the most sensitive and protein synthesis was theleast sensitive to inhibitory concentrations of 2,4-D. The variantdisplayed threefold higher resistance to 2,4-D than the wild-typecell line based on the 50% inhibitory concentrations of 2,4-Don DNA synthesis. No significant differences which could explainthe 2,4-D resistance were found between the variant and thewild-type cell lines in 2,4-D concentrations required to inhibitRNA and protein synthesis. The effect of 2,4-D on cell respirationwas detectable without a noticeable lag. The resistance of thevariant based on the effect on cell respiration also was apparentimmediately after 2,4-D addition. According to the 50% inhibitoryconcentrations of 2,4-D on cell respiration, the variant showeda level of resistance similar to that estimated by DNA synthesis.These results indicate that the resistance of the variant isdue to a modification which reduces the cellular sensitivityto phyto-toxic concentrations of 2,4-D with respect to, at least,DNA synthesis and respiration. (Received August 6, 1985; Accepted November 27, 1985)     

Effects of brassinosteroid, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants

Inflorescence stalks produced the highest amount of ethylene in response to IAA as compared with other plant parts tested. Leaf age had an effect on IAA-induced ethylene with the youngest leaves showing the greatest stimulation. The highest amount of IAA-induced ethylene was produced in the root or inflorescence tip with regions below this producing less. Inflorescence stalks treated with IAA, 2,4-D, or NAA over a range of concentrations exhibited an increase in ethylene production starting at 1 microM with increasingly greater responses up to 100 microM, followed by a plateau at 500 microM and a significant decline at 1000 microM. Both 2,4-D and NAA elicited a greater response than IAA at all concentrations tested in inflorescence stalks. Inflorescence leaves treated with IAA, 2,4-D, or NAA exhibited the same trend as inflorescence stalks. However, they produced significantly less ethylene. Inflorescence stalks and leaves treated with 100 microM IAA exhibited a dramatic increase in ethylene production 2 h following treatment initiation. Inflorescence stalks showed a further increase 4 h following treatment initiation and no further increase at 6 h. However, there was a slight decline between 6 h and 24 h. Inflorescence leaves exhibited similar rates of IAA-induced ethylene between 2 h and 24 h. Light and high temperature caused a decrease in IAA-induced ethylene in both inflorescence stalks and leaves. Three auxin-insensitive mutants were evaluated for their inflorescence's responsiveness to IAA. aux2 did not produce ethylene in response to 100 microM IAA, while axr1-3 and axr1-12 showed reduced levels of IAA-induced ethylene as compared with Columbia wild type. Inflorescences treated with brassinolide alone had no effect on ethylene production. However, when brassinolide was used in combination with IAA there was a dramatic increase in ethylene production above the induction promoted by IAA alone.