毛冠鹿大脑组织全长cDNA文库构建

运用SMART技术构建了毛冠鹿(Elaphodus cephalophus)大脑组织全长cDNA文库。提取大脑组织总RNA,Oligotex mRNA Kit纯化、获得poly(A) RNA,以CDSⅢ/3′PCR引物进行逆转录,LD-PCR扩增获得全长双链cDNA,经SfiⅠ酶切及柱层析分离后,500 bp以上的片段与载体λTripIEx2连接,体外包装得到cDNA文库。经鉴定原始文库滴度为5.1×105pfu/ml,扩增后文库滴度为1.5×109pfu/ml,重组率达到85%以上,插入片断平均长度约为1.0 kb,说明构建文库质量符合要求,可用于大脑特异表达基因的筛选。从该文库中克隆到了rig基因全长,包含5′和3′非编码区,从第43至477个核苷酸为一完整阅读框(ORF),此阅读框可编码一个145氨基酸的rig蛋白。     

星星草cDNA文库构建和金属硫蛋白(MT-1)基因的克隆

以受NaHCO3胁迫后的星星草叶片组织为材料构建了cDNA文库,文库的初始滴度为2.0×106pfu,重组率为95%,平均插入片段长度为0.8kb,扩增后文库的滴度为4.0×109pfu.mL-1.文库克隆随机测序获得了星星草的金属硫蛋白(MT-1)基因的全长cDNA序列,显示文库中含有一定量的全长基因.MT-1基因全长622bp,其中5'非翻译区57 bp,3'非翻译区343 bp,开放读码框长222 bp,编码73个氨基酸,氨基酸序列中具有植物MT-1蛋白特有的金属响应元件(MRE)序列,MT-1蛋白的分子量为7.814 kD,理论等电点为4.72.     

巨型艾美耳球虫孢子化卵囊cDNA文库的构建

为构建巨型艾美耳球虫(Eimeria maxima)孢子倾卵囊cDNA表灰文库,从E.maxima孢子化卵囊中提取总RNA,以总RNA为模板、λTriplex2TM为栽体,利用SMARTTM cDNA文库构建试剂盒构建全长cDNA表达文库.经测定构建的E.maxima cDNA表达文库的原始文库容量为1×106 pfu/ml,扩增后的文库滴度为5×1010pfu/ml,重组率为95%,插入片断主要集中在0.5～1kb之间.根据已知序列设计引物,能从文库中扩增出编码E.maxima免疫球蛋白重链结合蛋白的基因序列片段.结果 表明所构建的E.maxima cDNA表达文库质量良好,为克隆、筛选E.maxima的功能性基因奠定了基础.     

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。     

蒙古沙冬青冷冻胁迫SMART cDNA文库的构建及序列分析

以强抗逆植物蒙古沙冬青 (Ammopiptanthus mongolicus)为材料,用SMART技术构建了其在冷冻胁迫下的全长cDNA文库。原始文库滴度为9.44?06 pfu.mL-1,重组率为98.3%,插入片段长度在0.5~2.5kb,平均达到1kb。扩增文库滴度为1.98?010pfu.mL-1。从原始文库中随机挑取480个阳性克隆进行序列分析,获得348个Unigene,其中有些是逆境应答相关基因和新基因。此外,以扩增文库为模板,经PCR方法克隆到读码框为1083bp的沙冬青类RD22基因。这些结果表明所建文库质量较高,可以满足后续研究的要求。     

草豆蔻cDNA文库的构建及鉴定

以草豆蔻花序原基为材料,构建了cDNA文库。原始文库滴度为0.8×106pfu/mL,扩增后滴度为4.23×1011pfu/mL。插入片段大小在500bp～1.5kb之间,重组率为95.3%。以水稻RAP1A基因中包含MADS-box保守区段的序列为探针对该文库进行筛选,获得的阳性克隆经测序及序列比对分析,确认其中共有10个含MADS-box的阳性克隆。     

盐生植物海马齿盐胁迫全长cDNA文库的构建与分析

为了研究盐生植物耐盐基因表达调控,本实验以海水浇灌的海马齿植株为供试材料,构建了盐胁迫下的全长cDNA文库。构建方法如下:采用改良的CTAB法提取总RNA,SMART法反转录合成cDNA,LD-PCR方法合成双链cDNA。LD-PCR产物经蛋白酶K消化和SfiⅠ酶切后,经CHROMA SPIN+TE-1000分离柱子除去小片段DNA后,回收0.5kb以上的片段,按照适当的比例连接λTripIEX2载体。连接产物利用MaxPlaxTMLambda Packaging Extracts进行体外包装,得到海马齿初级cDNA文库。初始文库的独立克隆数为2.4×106pfu,初级文库滴度大于4.80×106pfu/mL,重组率为93.75%,插入片段为0.5～5kb,扩增文库的滴度为1.21×109pfu/mL,所得文库质量较高。本研究表明该cDNA文库适合于盐生植物海马齿相关基因的克隆和分析。     

红果人参叶中cDNA文库的构建

以四年生红果人参叶片为材料,提取叶中总RNA合成cDNA,连接到质粒载体pDNR-LIB上。采用电穿孔法将重组质粒转化到DH5α中。经文库质量鉴定表明:原始文库滴度为1.008×106pfu·mL-1,扩增后的文库滴度为2.968×109pfu·mL-1,重组率接近100%,插入片段大小在0.5~2kb之间,平均为0.85kb,表明已成功构建了红果人参叶中cDNA文库。     

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.     

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.     

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.     

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.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.     

Time of detection of recessive genes: effects of system of mating and number of examined individuals

Summary Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, Siete Cerros 66 and Penjamo 62, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of Penjamo 62 in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.     

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.