fcl基因对须糖多孢菌丁烯基多杀菌素生物合成及生长发育的影响

fcl基因编码的GDP-岩藻糖合成酶(GDP fucose synthetase,GFS),能催化由GDP-D-甘露糖合成GDP-L-岩藻糖过程中的两步差向异构酶和还原酶反应;还参与氨基糖和核糖的生物合成,是调控生物体糖代谢、核苷酸代谢的关键酶之一。通过前期基因组测序表明须糖多孢菌Saccharopolyspora pogona中存在fcl基因。利用基因工程技术构建了fcl基因的过表达菌株S. pogona-fcl和敲除菌株S. pogona-Δfcl。结果表明该基因对菌株生长发育、蛋白表达及其转录水平、杀虫活性、丁烯基多杀菌素的生物合成均存在影响。经HPLC分析显示,S. pogona-Δfcl的丁烯基多杀菌素产量增加为野生型菌株的130%,S.pogona-fcl的丁烯基多杀菌素产量降低了25%。生测结果显示,与野生型菌株相比S.pogona-Δfcl对棉铃虫的杀虫活性明显增强,而S.pogona-fcl的杀虫活性降低。利用扫描电镜观察发现,S. pogona-Δfcl菌丝体表面出现褶皱,呈现短棒状,S. pogona-fcl菌丝形态与野生型菌株一致。以上结果表明,fcl基因的敲除影响菌丝体的生长发育,能促进丁烯基多杀菌素的生物合成和增强杀虫活性,该基因的过表达抑制了丁烯基多杀菌素的生物合成和降低了杀虫活性。SDS-PAGE结果表明,三株菌株在96 h时蛋白表达差异最为明显。对差异蛋白通过实时荧光定量聚合酶链式反应结果显示,三菌株蛋白的转录水平存在显著表达差异。通过研究结果构建了网络代谢调控图,分析fcl基因对须糖多孢菌生长发育及丁烯基多杀菌素生物合成代谢调控网络途径的影响,初步构建了fcl基因调控的代谢途径,为揭示丁烯基多杀菌素生物合成的调控机制及相关后续研究提供了实验依据。     

微生物核糖体工程研究进展

摘要：微生物获得特定类型的抗性突变,不仅反映了其核糖体或RNA多聚酶上相关靶位点结构的改变,也对突变菌株次级代谢产物（抗生素等）的生物合成能力产生深刻影响,因此筛选抗性突变株可作为微生物推理选育的途径之一。“核糖体工程”是利用微生物的各类抗性突变为筛选标记,高效获得次生代谢产物合成能力提高的突变株的推理育种新方法。本文综述了微生物“核糖体工程”的概念、各类突变的作用机理,并着重介绍组合抗性突变在提高出发菌株次级代谢产物产量方面的应用。     

LytSR和PdtaSR双组分系统对须糖多孢菌丁烯基多杀菌素生物合成的影响

【目的】须糖多孢菌(Saccharopolysporapogona)基因组中存在一些双组分系统(two-component system,TCS),包括一个LytR调控因子和一个PdtaR转录抗终止调节因子,我们旨在通过基因工程技术改造分析这两个双组分系统蛋白在须糖多孢菌中的作用。【方法】本研究利用融合PCR和属间接合转移技术,构建了S. pogona-?lytR和S. pogona-PdtaR工程菌株,研究它们对须糖多孢菌丁烯基多杀菌素生物合成的影响。【结果】HPLC检测和质谱鉴定表明,与原始菌相比,S. pogona-?lytR中丁烯基多杀菌素产量有所提高,而S. pogona-PdtaR中产量下调,表明lytR和pdtaR对丁烯基多杀菌素生物合成有负调控作用。此外,生长曲线测定和表型分析结果表明,S. pogona-?lytR和S. pogona-PdtaR对须糖多孢菌的生长发育和孢子形成也产生了一定的影响。【结论】本研究为进一步阐明丁烯基多杀菌素生物合成的调控机制奠定了理论基础。     

不同碳源对须糖多孢菌生长发育及丁烯基多杀菌素生物合成的影响

本文研究了不同碳源对须糖多孢菌生长以及丁烯基多杀菌素生物合成的影响,通过寻找优势碳源优化发酵培养基配方,促进须糖多孢菌丁烯基多杀菌素的生物合成。试验共设11个处理,1个对照,通过单因素试验比较不同处理组菌体OD600值和丁烯基多杀菌素产量,筛选获得最优碳源及其发酵培养基配方。结果表明,除可溶性淀粉和木糖外,须糖多孢菌在9种碳源中都能进行生长,对不同构型碳源显示较好的利用率。在以半乳糖、葡萄糖、果糖和甘露糖作为碳源时具有较好的生长速率,而以甘露糖为碳源时能显著促进丁烯基多杀菌素的合成。选择甘露糖最佳添加浓度为5 g/L,须糖多孢菌最高菌体浓度和丁烯基多杀菌素产量分别是初始配方条件的1. 32倍和1. 78倍,显著提高了丁烯基多杀菌素的产量。上述结果为培养基碳源对丁烯基多杀菌素生物合成影响机制的研究及丁烯基多杀菌素大规模工业化发酵生产提供了科学依据和新的技术途径。     

放线菌Streptomyces sp．FJ3的核糖体工程改良与活性产物的分离

[目的]利用核糖体工程抗性筛选技术,获得有抗菌活性突变株,并对突变株新产生活性物质进行研究.[方法]以三峡库区筛选出的无抗菌活性放线菌野生株为出发菌,通过单菌落挑选与平板划线培养,分离筛选具有链霉素和利福平抗性突变株;通过摇瓶发酵和对发酵液进行纸片法活性测定,获得抗金葡菌活性突变株;采用高效液相色谱法(HPLC)分析其发酵液组分,通过LC-MS对变化峰进行分析;进行16S rDNA及形态学鉴定.[结果]链霉素和利福平对放线菌菌株FJ3的MIC分别为0.5μg/mL和110μg/mL;在FJ3突变菌株中,共获得24株链霉素突变菌株和20株利福平突变菌株,抗菌活性筛选显示6株具有抗菌活性,其中2株链霉素突变菌株对金葡菌有强抑菌活性,采用Doskochilova溶剂系统纸层析结果表明,该活性物质为一种核酸类抗生素,HPLC和LC-MS显示该活性物质可能为硫藤黄菌素.[结论]利用核糖体工程技术可以改变放线菌的次级代谢,获得具有生物活性的突变株,拓展药源放线菌活性菌株新资源.     

利用CRISPR-Cas9系统与核糖体工程获得新型可利霉素产生菌

可利霉素 (Carrimycin,CAM) 是将异戊酰基转移酶基因 (Isovaleryltransferase gene,ist) 导入到螺旋链霉菌中产生的以异戊酰螺旋霉素 (Isovalerylspiramycin,ISP) 为主组分的抗生素。原工程菌中的ist基因与螺旋霉素 (Spiramycin,SP) 生物合成基因簇相距较远,且具有两种抗性基因,难以对其进行基因改造,因此需要构建新型CAM工程菌株。文中通过CRISPR-Cas9基因编辑系统靶向切割2个位点,将ist和其正调控基因acyB2通过同源重组插入到SP生物合成基因簇附近且不参与SP合成的orf54基因下游,获得2种无外源抗性基因插入的CAM产生菌54IA-1和54IA-2,经发酵产物检测发现54IA-2菌株中的ISP产量明显高于54IA-1菌株。通过实时定量PCR (Quantitative real-time PCR,qPCR) 检测证实54IA-2菌株中ist和acyB2基因以及部分SP生物合成基因的表达量均高于54IA-1菌株。为进一步获得高产菌株,以54IA-2为出发菌株,利用核糖体工程的方法筛选利福平 (Rifampicin,RFP) 抗性菌株,在RFP浓度为40 μg/mL的抗性菌株中,ISP的产量明显提高,最高可达842.9 μg/mL,比原始菌株提高约6倍。对其中7株菌的rpoB基因进行测序分析,每株菌的第576位丝氨酸都突变为丙氨酸,在其他错义突变中产量最高的菌株RFP40-6-8在第424位的谷氨酰胺突变为亮氨酸。综上所述,本研究应用CRISPR-Cas9系统成功构建了无任何抗性标记的新型CAM工程菌株54IA-1和54IA-2,并通过核糖体工程技术筛选获得了新型CAM高产菌株RFP40-6-8,为CAM工程菌株的进一步优化改造奠定了基础。     

亚硝基胍消除链霉菌FR-008的线性质粒

【目的】利用亚硝基胍(NTG)消除链霉菌FR-008线性质粒以简化其基因组,获得背景清晰的菌株,作为抗生素异源生物合成的底盘细胞。【方法】NTG溶液处理链霉菌FR-008孢子悬液,从存活的诱变株中筛选砷敏感的突变株,再通过脉冲场凝胶电泳(PFGE)检测线性质粒是否被消除;用生测实验定性检测各个线性质粒消除突变株杀念菌素合成的能力,最后通过HPLC定量比较突变株和野生型产生杀念菌素的差异。【结果】从103个诱变株中筛选到3株砷敏感的突变株(10#、59#、115#)。PFGE检测发现它们均丢失了大线性质粒p SSFR1,此外,42#突变株的小线性质粒p SSFR2被消除,在此基础上,第二轮NTG诱变获得了双质粒消除的突变株。大线性质粒p SSFR1消除率约为3%,小线性质粒p SSFR2消除率约为1%。发酵结果显示:10#、115#突变株杀念菌素有效组分III产量分别提高了40%和30%。【结论】首次发现NTG是一种有效消除链霉菌线性质粒的诱变剂,2株大线性质粒消除的突变株杀念菌素的产量得到提高。此方法可以用来消除特定链霉菌菌株中的巨型线性质粒以高效简化其基因组,因而是一种有效的抗生素遗传育种的方法。     

乙基多杀菌素抗性小菜蛾代谢解毒酶酶活性研究

【目的】阐明小菜蛾Plutella xylostella(L.)对乙基多杀菌素的代谢抗性机理,为延缓小菜蛾对乙基多杀菌素抗药性发展及抗性治理技术提供支持。【方法】通过酶动力学方法测定了小菜蛾对乙基多杀菌素高抗、中抗和敏感种群的谷胱甘肽-S-转移酶、羧酸酯酶、乙酰胆碱酯酶和多功能氧化酶4种代谢解毒酶的比活力。【结果】乙基多杀菌素高抗小菜蛾种群的谷胱甘肽-S-转移酶、羧酸酯酶、乙酰胆碱酯酶的比活力分别为15.38、3.15和7.30 OD·min~(-1)·mg~(-1)pro,显著高于敏感种群;但乙酰胆碱酯酶在中抗种群和敏感种群中比活力差异不显著;多功能氧化酶在高抗、中抗和敏感种群中的比活力分别为4.97、4.08和4.23 OD·min~(-1)·mg~(-1)pro,差异不显著。【结论】谷胱甘肽-S-转移酶、羧酸酯酶和乙酰胆碱酯酶的酶活随着小菜蛾对乙基多杀菌素抗性的增强而增强,而多功能氧化酶的酶活在抗性种群与敏感种群间差异不显著,因此小菜蛾对乙基多杀菌素的代谢抗性机理研究应重点关注这3种酶。     

核糖体工程技术选育ε-聚赖氨酸高产菌株

【目的】利用核糖体工程技术选育Streptomyces albulus AS3-14的链霉素和利福平双重抗性突变株,以提高其ε-聚赖氨酸合成能力。【方法】通过链霉素抗性筛选,获得链霉素抗性的ε-聚赖氨酸产量提高突变株;在此基础上,继续筛选其利福平抗性突变株,实现链霉素和利福平双重抗性ε-聚赖氨酸高产菌选育。【结果】获得的双重抗性高产突变株Streptomyces albulus WG-608的ε-聚赖氨酸摇瓶产量达到3.7 g/L,5 L发酵罐补料分批发酵ε-聚赖氨酸产量达到53.0 g/L,较出发菌株分别提高了42.3%和32.5%。【结论】链霉素和利福平双重抗性选育能够显著提高ε-聚赖氨酸产生菌Streptomyces albulus的产物合成能力。     

放线菌核糖体工程的发展与应用

核糖体工程(ribosome engineering)是一项利用靶点位于细菌RNA聚合酶及核糖体功能因子的抗生素诱导细菌产生抗性突变,进而提升菌株次级代谢生产潜能的技术。该方法无需依赖菌株完善的遗传操作体系,可应用于发掘几乎所有放线菌菌株中潜在的宝贵活性次级代谢产物,并广泛应用于放线菌基因组挖掘和次级代谢产物增产优化。核糖体工程效果显著,迄今为止,已从百余种放线菌菌株中发掘了10余种新结构分子和提升近30种活性次级代谢产物的生产效价。鉴于此,文中从核糖体工程的发展角度出发,对该技术的建立与优化,及其作用机制的阐明进行了系统的归纳与总结;同时也全面分析探讨了该技术在放线菌次级代谢产物开发中的推广应用,以期为核糖体工程的发展完善及放线菌次级代谢产物的综合开发提供借鉴与参考。     

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.     

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.     

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.     

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.     

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。     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

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.