核黄素发酵菌种改造研究进展

核黄素是一种水溶性维生素,与动植物的生长密切相关,人体不能合成核黄素,需从外部摄取,因此核黄素的生产具有重要意义。介绍了核黄素的生产发展历程和产核黄素的微生物种类。对枯草芽孢杆菌的核黄素代谢途径及其诱变育种进行了总结,重点介绍了菌种的基因重组改造方法,主要是提高核黄素操纵子表达以及提高Ru5P和GTP两种前体供应量合成途径通量,介绍了近些年新的改造方法,并对未来的发展方向进行了展望。     

核黄素基因工程研究进展

核黄素 (维生素B2 )为天然水溶性的B族维生素 ,是维持机体正常代谢所必须的物质 ,具有重要的生理功能。目前核黄素的生产方法主要有化学合成法和微生物发酵法。其中微生物发酵法是后来发展起来的一种十分经济有效的方法 ,并在核黄素主产中开始占据主导地位。为进一步获得核黄素高产菌株 ,人们对核黄素合成基因及其表达调控的机制做了深入细致的研究 ,并以此为依据 ,通过基因工程手段构建出了核黄素高产菌株 ,大大提高了核黄素的产量 ,其中尤以枯草芽孢杆菌最为成功。综述发酵法生产核黄素的现状、核黄素生物合成的分子生物学以及基因工程研究进展 ,讨论了其进一步的发展方向。     

代谢工程在核黄素生产上的应用

核黄素(维生素B2)为天然水溶性的B族维生素,是维持机体代谢所必须的营养物质。目前核黄素的工业化生产主要有微生物发酵法和化学半合成法两种,其中微生物发酵法以生产工艺简单、原料廉价、环境友好以及资源可再生等优点而倍受世界核黄素生产商的青睐。代谢工程是近二十年来发展起来的新型学科,主要利用分子生物学技术对细胞进行遗传修饰,从而改进产物生成或细胞特性。为进一步提高核黄素产量,通过代谢工程手段构建出了核黄素高产菌株,其中尤以枯草芽孢杆菌最为成功。要得到较高的核黄素产率,必须保证碳架、能量等价物以及氧化还原辅(酶)因子在细胞代谢过程中处于适当的比率。以枯草芽孢杆菌进行核黄素生产为例,主要从增强碳源和能源利用效率、增强核黄素生物合成途径代谢流以及解除核黄素生物合成过程中的反馈调节方面综述了代谢工程在指导核黄素生产方面的应用,并讨论了其未来的发展方向。     

蜡样芽孢杆菌ATCC14579核黄素操纵子的克隆及在枯草芽孢杆菌中的表达

利用BLAST从B．cereus ATCC14579的基因组中找到一段与枯草芽孢杆茵核黄素操纵子具有较高相似性的4．6kb大小的基因组DNA片段,该片段中含有完整的核黄素操纵子。该操纵子结构基因的编码产物的氨基酸序列与枯草芽孢杆菌核黄素操纵子相应结构基因的编码产物的氨基酸序列具有99％的同源性。该片段被克隆到大肠杆茵一枯草芽孢杆茵穿梭载体pHP13M中。表达分析的结果表明B．cereus ATCC14579核黄素操纵子可在大肠杆茵和枯草芽孢杆菌中表达。利用PCR方法用来自枯草杆菌的sac B基因的启动子替换B．cereus ATCC14579核黄素操纵子原有的启动子使其更好表达。替换启动子后的核黄素操纵子在本文使用的发酵条件下有较好的表达,核黄素产量从39．5mg／L增加到61．7mg/L.     

化学沉降法回收核黄素母液

核黄素提取过程中,结晶液分离后会产生大量的母液。母液中包含一定量的核黄素,其中一部分是溶解状态的核黄素,另一部分是不溶解的微小晶体。分离过程中无法将这两部分核黄素收回,造成收率偏低。通过在母液中加入连二亚硫酸钠,将母液中可溶态和结晶态的核黄素进行沉降,沉降后进行精制分离,最终得到核黄素产品,可以提高核黄素提取的收率。     

核黄素合酶的研究进展

核黄素合酶是核黄素生物合成中的关键酶,催化核黄素生物合成途径中的最后一步,产生核黄素.本文对近年来国内外有关不同生物中核黄素合酶及其编码基因的结构、特性、作用机制和应用进行综述.     

枯草芽孢杆菌核黄素操纵子rib operon的克隆与表达

目的:构建产核黄素的枯草芽孢杆菌基因工程菌.方法:以穿梭载体pEB03构建核黄素操纵子的表达质粒载体pGJB13和pGJB14,与质粒pMX45分别转化产核黄素的枯草芽孢杆菌GJ07,并通过发酵摇瓶实验检测核黄素的产量.结果:得到产核黄素的工程菌GJ13 、GJ14和GJ08,在以蔗糖为碳源的发酵条件下,GJ08可产核黄素820mg/L,提高了约55%.结论:得到了产核黄素的高产菌种G J08.     

核黄素工业菌株高通量筛选方法的建立和应用

枯草芽孢杆菌是主要的核黄素工业生产菌之一,高通量筛选技术是选育获得高产核黄素菌株的关键环节。为实现工业菌种选育与高通量筛选技术相结合,对流式细胞分选、液滴微流控分选和96孔板筛选在核黄素工业菌株筛选中的应用进行了研究,并对96孔板筛选方法进行了优化。在流式细胞分析中,来源于同一株工业菌的低产菌株P1与高产菌株R1的细胞荧光与核黄素产量不成正相关。在液滴微流控分析中,P1和R1的发酵液上清荧光与核黄素产量成正相关,然而液滴分选后活细胞的数量很少。在96孔板筛选实验中,振荡培养后P1和R1的发酵液荧光分别为22 264 a.u.、28 647 a.u.;静置培养后荧光分别为7 095 a.u.、10 189 a.u.,核黄素产量与荧光成正相关,且二者荧光差异显著。利用96孔板静置培养的方法对工业菌株S1的突变体库进行筛选,得到的优选菌株核黄素产量为2.53 g/L,相比S1提高了15%。这些结果表明96孔板静置培养-荧光检测筛选可以应用于核黄素工业生产菌产量的提高。     

核黄素生物合成研究 1.微量培养中核黄菌(Eremothecium ashbyii)发育过程中的形态变化

从本试验观察到,核黄菌的生长全部过程是自配子或菌丝在培养基中开始发育成营养菌丝,部分菌丝形成配子囊形成配子,部分菌丝衰老自溶。当菌丝发育最盛时,核黄素的形成是最多,衰老时培养基核黄素逐渐丰富起来。核黄菌在有空气和缺乏空气时,它们的发育是有区别的,即是在缺乏空气(表面下生长)时,菌丝是细长不形成配子和产生少量核黄素。     

核黄素光敏损伤DNA的凝胶电泳

以琼脂糖凝胶电泳研究了波长为427~457nm可见光照射下核黄素(VB₂)光敏诱导的质粒DNA损伤,结果发现DNA光敏损伤主要与照光量、核黄素和DNA浓度比、溶液中的氧气等有关.在无氧条件下光敏损伤更为显著,证明VB₂光敏损伤DNA主要是通过VB₂激发三重态与DNA碱基组分间发生电荷转移导致其产生氧化性损伤实现的.表明核黄素光敏损伤DNA时最佳浓度比为bp∶VB₂ = 3.5∶1.对照结果表明,单链DNA(ssDNA)比双链DNA(dsDNA)的碱基更易被核黄素敏化发生损伤,这可能是由于ssDNA中碱基暴露的原因.     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

医疗机构合理用药评价模型的构建研究

目的 针对医疗机构的合理用药水平进行评价研究。方法 根据医疗机构合理用药的具体要求,构建医疗机构合理用药评价指标体系,采用基于模糊群决策的方法和多指标评价分析法构建医疗机构合理用药评价模型。结果 构建了基于模糊群决策的医疗机构合理用药评价模型,并通过实例分析证明了评价模型的可行性。结论 建立的基于模糊群决策的医疗机构合理用药评价模型能够对医疗机构的合理用药水平进行科学评价,为提高医疗机构合理用药水平奠定基础。     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.