钝顶螺旋藻表达载体p215 t-spp的构建及转化

用合适的限制性内切酶消化载体p215t,胶回收获得含有gfp基因及amp基因大片段.设计引物,采用PCR扩增无菌钝顶螺旋藻A9藻株的强启动子片段;在T4连接酶的作用下进行体外连接重组,构建了带有螺旋藻启动子和gfp报告基因的新型表达载体p215t-spp.将该载体转入钝顶螺旋藻,利用报告基因的表达,在荧光显微镜下观察并记录转化藻细胞,p215t-spp质粒显著提高转化率.研究了不同PEG浓度、转化时间及冰浴处理对转化率的影响.采用1%PEG能有效促进细胞吸收DNA,获得10.5‰的转化率.实验初步证明,带有螺旋藻启动子的报告基因gfp能够在钝顶螺旋藻中顺利表达.     

螺旋藻的纯化

观察了螺旋藻生长过程中藻丝和杂菌的生长规律,发现中性细菌和碱性细菌的数量始终是藻丝的10^5～10^6倍。采用常规的稀释平板法、毛细管法和挑单藻法均无法可靠地获得无菌纯藻。设计用低速离心法洗涤下沉性藻丝,用过滤法洗涤上浮性藻丝,对藻丝进行预处理洗去大量杂菌;对迁移性和非迁移性藻株分别采用夹层法和平板法纯化藻株,使得单根藻丝在平板上形成藻落,获得无菌纯藻。     

螺旋藻的纯化*

观察了螺旋藻生长过程中藻丝和杂菌的生长规律,发现中性细菌和碱性细菌的数量始终是藻丝的10⁵～10⁶倍。采用常规的稀释平板法、毛细管法和挑单藻法均无法可靠地获得无菌纯藻。设计用低速离心法洗涤下沉性藻丝,用过滤法洗涤上浮性藻丝,对藻丝进行预处理洗去大量杂菌;对迁移性和非迁移性藻株分别采用夹层法和平板法纯化藻株,使得单根藻丝在平板上形成藻落,获得无菌纯藻。     

螺旋藻耐盐相关基因启动子区功能分析

文章利用绿色荧光蛋白基因作为报告基因,研究2个螺旋藻耐盐相关基因启动子区域的功能。通过启动子预测软件预测螺旋藻耐盐相关基因5′端非翻译区的启动子结构,用Primer3.0程序在线设计引物,以pMD18-T载体和pUC18载体克隆螺旋藻启动子序列、gfp和卡那霉素抗性基因,将螺旋藻启动子-GFP基因-卡那霉素抗性基因(pro-gfp-kanr)三联DNA片段克隆至pKW1188载体,并将该重组质粒pKW1188::pro::gfp::kanr转化至受体菌集胞藻6803,激光共聚焦显微镜观察不同盐浓度培养条件下、不同时间段集胞藻表达GFP的情况。结果显示,通过不同盐浓度和不同时间的诱导,2个螺旋藻启动子在0.4~0.6 mol/L NaCl条件下,培养6~8 h表达的绿色荧光蛋白最多。文章成功构建了以绿色荧光蛋白为报告基因、卡那霉素抗性基因为选择标记、集胞藻6803作为外源基因表达受体,进行螺旋藻耐盐相关基因功能研究的平台;另外,从螺旋藻启动子能被盐诱导大量表达GFP的结果看,与启动子相关的螺旋藻基因很可能与螺旋藻的耐盐性相关。     

利用荧光蛋白研究产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因CgGPD启动子

[目的]克隆产甘油假丝酵母(Candida glycerinogenes)胞浆3-磷酸甘油脱氢酶基因CgGPD的启动子(PCggpd),并通过报告基因gfp的差异表达来研究葡萄糖浓度对PCggpd在酿酒酵母(Saccharomyces cerevisiae)中的诱导特性.[方法]采用PCR扩增的方法分别从产甘油假丝酵母基因组和pCAMBIA1302载体中克隆出CgGPD的启动序列PCggpd和绿色荧光蛋白基因gfp.将两个基因同时构建到酿酒酵母表达载体pYX212-zeocin中,构建时将绿色荧光蛋白基因gfp置于CgGPD的启动序列下游,获得重组质粒pYX212-zeocin-PCggpd-gfp.通过电击转化酿酒酵母W303-lA.将重组酿酒酵母S.cerevisiae W303-1A-GFP置于不同葡萄糖浓度培养基中进行培养,利用荧光显微技术对其进行荧光检测.[结果]重组酿酒酵母能产生稳定的荧光,当葡萄糖浓度为2%时,重组酿酒酵母在YEPD培养基中产生较弱的荧光,随着葡萄糖浓度的升高,荧光强度有明显的增强.[结论]PCggpd属于环境胁迫诱导型启动子,高浓度的葡萄糖能诱导PCggpd启动绿色荧光蛋白的高水平表达,这对完善产甘油假丝酵母的遗传背景研究,阐明其高产甘油的机理具有重要意义.     

一株水稻促生长内生真菌的绿色荧光蛋白基因标记与示踪

将绿色荧光蛋白基因(gfp)转入到碱蓬内生真菌JP4-1中并检测菌株在水稻幼苗中的定殖情况。采用PEG-Ca Cl2介导的原生质体转化方法将携带gfp基因的p CT74质粒与菌株基因组整合获得转化子,用转化子侵染水稻幼苗,荧光显微镜下示踪JP4-1菌株及其侵染特性。转化子经连续传代6次仍能发出绿色荧光且荧光强度良好,能够稳定遗传;经PCR验证gfp基因已成功转入JP4-1菌株和水稻幼苗植株内并表达。转化可获得稳定表达GFP的JP4-1转化子,JP4-1菌株可定殖于水稻幼苗的根、茎、叶,定殖位置为细胞间隙,其促生作用与野生型菌株无明显差别。     

抑制螺旋藻胞内核酸酶酶活的研究

有效抑制胞内核酸酶的活性对提高外源DNA导入和稳定表达具有重要作用,本文通过对钝顶螺旋藻A9、抗刀豆氨酸（CS）突变株A9c、藻体长直型A9L这3种藻株胞内核酸酶活性的研究发现,在培养液中添加EDTA或不同温度不同时间处理藻丝体,都能明显抑制3种藻株胞内核酸酶的活性,并对合适的EDTA浓度、合适的处理温度和时间进行了探讨。     

根癌农杆菌介导的灰葡萄孢菌遗传转化研究

以pCAMBIA1300-N载体为骨架, 成功构建了以绿色荧光蛋白(gfp)为报告基因, 潮霉素(hph)为抗性筛选标记的载体pKPG, 并利用根癌农杆菌介导转化系统, 成功获得了能表达绿色荧光蛋白的重组灰葡萄孢菌。通过PCR检测转化子的绿色荧光蛋白基因和潮霉素抗性表达框, 观察菌丝和分生孢子的荧光表型, 以及gfp基因的Southern杂交验证, 结果表明：被测转化子基因组中均成功整合了目的基因片段。     

生防放线菌Ahn75的荧光标记及其在水稻中的定殖

【背景】目前gfp标记基因已成为研究靶标微生物与宿主之间互作的一种重要工具。利用gfp基因标记生防菌株,可以对生防菌株的生存及定殖能力进行有效追踪。【目的】对生防放线菌Ahn75进行荧光标记,探讨其在水稻中的定殖规律,为研究Ahn75的稻瘟病防治机制奠定基础。【方法】首先通过电激转化将含绿色荧光标记基因(gfp)的质粒pIJ8655导入大肠杆菌ET12567中,然后采用接合转移的方法将gfp整合到Ahn75基因组上;通过平板对峙试验检验Ahn75-GFP在标记绿色荧光后对稻瘟病病原菌的抑菌活性;采用喷施孢子液的方式将带荧光标记的Ahn75-GFP定殖水稻,并利用荧光显微镜观察生防菌在水稻中的定殖情况;对定殖水稻中的内生菌进行重分离,探究菌株在水稻组织中的分布规律。【结果】PCR扩增和荧光观察表明,绿色荧光标记基因成功整合到生防放线菌Ahn75中。通过平板对峙试验,发现Ahn75-GFP对稻瘟病病原菌抑菌活性与原始菌株没有显著差别。在荧光显微镜下,可以观察到Ahn75-GFP能稳定定殖于水稻的根、茎、叶等组织中,而水稻内生菌重分离试验表明该菌株在茎中的定殖力最强。【结论】获得一株绿色荧光标记生防菌株Ahn75-GFP,结果显示该菌株定殖水稻效果良好,这对于研究Ahn75的稻瘟病防治具有重要意义。     

板栗疫病菌绿色荧光与红色荧光蛋白共定位载体的构建

低毒病毒-板栗疫病菌组合是研究病毒与宿主相互作用的一个优秀的模式系统.我们构建了含绿色荧光蛋白基因gfp的载体pCPXHY2GFP与含红色荧光蛋白基因rfp的载体pCPXG418RFP,并用于转化野生型菌株EP155,获得了以潮霉素为筛选标记、表达绿色荧光蛋白的转化株pCPXHY2GFP/EP155和以G418为筛选标记、表达红色荧光蛋白的转化株pCPXG418RFP/EP155.将载体pCPXG418RFP转化pCPXHY2GFP/EP155,获得的转化株能观察到绿色荧光蛋白与红色荧光蛋白共定位的现象.板栗疫病菌绿色荧光与红色荧光共定位载体pCPXHY2GFP与pCPXG418RFP的构建,为深入研究病毒与宿主相互作用的分子机制提供了强有力的研究材料.     

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。     

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.     

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.     

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.     

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。