SMART技术构建栀子cDNA文库

目的:构建栀子叶片cDNA文库。方法:提取栀子叶片总RNA。利用SMART技术合成双链cDNA。双链cDNA经限制酶Sfil酶切后与pDNR-LIB质粒连接。利用电刺激转化法将重组质粒导入E.coli DH5α而获得文库。利用PCR法检测文库的重组率。结果:原始文库滴度为2.63×105cfu/ml。随机检测文库中的15个克隆,表明重组率约为86.7%。选择14个插入片段的长度在400bp以上的克隆进行测序和生物信息学分析,结果预测的全长基因占所检测序列的64.3%。结论:成功构建了栀子叶片的cDNA文库,为栀子基因的结构和功能的研究提供了基础。     

野生大豆未成熟种子总mRNA的分离及其cDNA的分子克隆

用氯化锂沉淀法从野生大豆(G.soja)未成熟种子中制备总RNA,经oligo(dT)-纤维素柱亲和层析,获得总mRNA,在兔网织红细胞体系中表现出一定翻译活性。以总mRNA为模板,oligo(dT)_(12)(?)为引物,反转录酶催化合成第一链cDNA,RNase H-DNA聚合酶Ⅰ协同合成第二链cDNA。双链cDNA的长度大约为200—5000 bp,且不存在发夹结构。将双链cDNA修补后钝端连接到pUC 19质粒的Sma 1位点,转化E.coli JM107,获得800多个白色重组子克隆。快速电泳检测及酶切分析表明,多数重组子带有插入片段,其中3个重组子的插入片段长度大致为1700 bp、2600 bp和1400 bp。     

烤烟品种南江3号均一化全长cDNA文库构建

目的:为了获得功能基因的信息,构建烤烟品种南江3号的均一化cDNA文库.方法:用RNeasy Plant Mini Kit提取烤烟南江3号叶片和花的RNA,用反转录酶逆转录合成第一链eDNA.以LD-PCR扩增获得的双链cDNA为模板,采用基于双链特异性核酸酶(Duplex-Specific Nuclease,DSN)的均一化cDNA文库技术,构建南江3号盛花期的均一化cDNA文库.结果:构建了南江3号盛花期的均一化cDNA文库,文库重组率大为97.47%,库容量约为1.26×10<'6>,插入片段平均长度大于1.2kb.从文库中随机48个克隆进行PCR检测,挑取20个克隆进行测序,序列通过BLAST比对结果显示文库可能包含大量基因和ESTs序列.结论:该文库为研究南江3号的基因功能和资源提供材料来源.     

PCR介导HaNPV罹病幼虫潜伏期cDNA文库的构建

以罹病棉铃虫幼虫为材料提取总RNA,反转录合成cDNA第一链, 加oligo(dG)同聚尾,PCR扩增合成双链cDNA,克隆到pGEM-T质粒载体中.随机筛选文库中阳性克隆,经酶切分析,cDNA插入片段大小在0.3～1.1kb之间.文库中原代重组子数为1. 66×105,重组百分比为87.8%.重组质粒的杂交分析表明,文库中HaNPV基因的cDNA克隆所占比例超过50%.     

中华大蟾蜍精巢组织全长cDNA文库的构建及泛素延伸蛋白基因Bg-ubi52的获得

运用SMART技术构建了中华大蟾蜍(Bufo bufo gargarizans)精巢全长cDNA文库。提取中华大蟾蜍精巢总RNA,用Clontech公司SMARTTM cDNA文库构建试剂盒反转录合成第一链cDNA,LD-PCR扩增获得全长cDNA双链;经SfiⅠ酶切、层析柱分离后,500bp以上的片段与λTriplEx2载体连接并包装,建成原始文库。经鉴定,原始文库滴度为2.21×106pfu/ml,重组率为91%;文库扩增后的滴度为2.94×109pfu/ml,重组率为93.7%。插入片段大小分布于0.4~2.0kb之间,平均长度约为1.0kb,说明已构建文库质量较高,为进一步筛选、克隆精巢特异表达基因奠定了基础。从该文库中克隆到了泛素延伸蛋白基因,全长561bp,包含完整的5′和3′非编码区,编码128个氨基酸,即泛素的76个氨基酸后融合了52个氨基酸的核糖体L40蛋白。     

甜菜夜蛾触角全长cDNA文库的构建与分析

目的:构建甜菜夜蛾触角全长cDNA文库。方法:利用TRIzol试剂提取甜菜夜蛾触角总RNA,以此为模板,通过SMAR-TScribeTM反转录酶反转录合成第一链cDNA,引物扩增获得双链cDNA,经Proteinase K消化、SfiⅠ酶切和CHROMA SPIN-400Column分级分离后,收集400～2 000 bp之间的片段重组于改造的pUC19载体并转化至大肠杆菌Escherichia coli DH5α,最终构建获得甜菜夜蛾触角全长cDNA文库。结果:对文库进行滴度测定和重组率分析,结果表明构建的cDNA初级文库滴度为1.6×107pfu/ml,重组率为94%,插入片段大小为0.5～3.0 kb,平均长度在1 kb以上,表明构建获得的文库是一个高质量的文库。结论:该文库的构建为今后克隆甜菜夜蛾嗅觉相关基因奠定了基础。     

PCR介导HaNPV罹病幼虫潜伏期cDNA文库的构建

以罹病棉铃虫幼虫为材料提取总RNA ,反转录合成cDNA第一链 ,加oligo(dG)同聚尾 ,PCR扩增合成双链cDNA ,克隆到 pGEM T质粒载体中。随机筛选文库中阳性克隆 ,经酶切分析 ,cDNA插入片段大小在 0 .3～ 1.1kb之间。文库中原代重组子数为 1.66× 10 5,重组百分比为 87.8%。重组质粒的杂交分析表明 ,文库中HaNPV基因的cDNA克隆所占比例超过 50 %。     

东亚三角涡虫cDNA文库的构建及EST初步分析

为了快速高效地分离鉴定东亚三角涡虫的发育和再生相关基因,以总RNA为模板,借助CreatorTM SMARTTM cDNA Library Construction Kit和Advantage 2 PCR Kit成功构建了东亚三角涡虫的cDNA文库.经测定,cDNA文库原始库容为1.22×105个独立克隆,重组率超过98 %,插入片段平均大小为900 bp.从文库中随机挑选重组克隆测序,并在NCBI数据库中比对,结果获得208个rRNA基因,148个编码蛋白基因,78个染色体片段基因.该研究为我国涡虫发育和再生的深入研究奠定了坚实的分子基础.     

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.     

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.     

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.     

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.     

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.