龙葵阿特拉津抗性生物型的鉴定和阿特拉津抗性基因的克隆

通过直接施用除草剂、荧光诱导和正反杂交试验等鉴定出4种抗阿特拉津的龙葵生物型。从抗性最强的B_(13)株制备叶绿体DNA,构建限制性内切酶Bam HI片段的基因文库,并从中筛选出1个5kb Bam HI片段的克隆pSB 135,通过与探针的分子杂交,证明在pSB 135克隆的Bam HI片段中含有编码叶绿体32k蛋白质的阿特拉津抗性基因。     

龙葵叶绿体ATP合酶α-亚单位基因的克隆

本研究以菠菜叶绿体DNA 2.45kb的SalI片段(含有ATP合酶α-亚单位基因)为探针,从龙英叶绿体DNA BamHI片段文库中筛选出含龙葵叶绿体atpA基因的克隆。通过Southrcn吸印与探针杂交,证明了重组质粒pSB 132的插入片段含有atpA基因。同时将atpA基因定位在龙葵叶绿体DNA SalI、BglI、XhoI和BamHI 4种酶切图谱的限制性片段上。     

含蚕豆叶绿体rRNA基因重组质粒物理图谱的构建

以 pBR322 DNA 为载体,Escherichia coli HB101为受体菌,克隆了含蚕豆叶绿体 rRNA基因的二个 BamHI 片段。应用几种限制性内切酶酶切以及 Southern 印迹法构建了这二个特异片段的物理图谱。重组质粒 pVFB16含有一个4.70kb 的 BamHI 片段,其上含有完整的16S rRNA 基因;重组质粒 pVFB32含有一个5.65kb 的 BamHI 片段,其上含有23S rRNA基因,23S—4.5S/5S rRNA 基因的间隔区及4.5S/5S rRNA 基因。     

油菜叶绿体基因组雄性不育特异DNA片段的定位

实验选用油菜湘矮不育系及同核保持系叶绿体DNA为材料,用4种限制性内切酶EcoRⅠ、BamHⅠ、PstⅠ和SmaⅠ完全酶解。在EcoRⅠ酶解的保持系ctDNA图式中,观察到唯一的差异片段E3.2kb。将此片段连接于载体pUC9进行克隆,经克隆杂交及电泳分析鉴定,获得重组子。以rRNA基因为探针,与EcoRⅠ酶解的保持系ctDNA杂交,其中一条阳性带显示在差异片段E3.2。进一步以E3.2片段为探针,与经过限制酶BamHⅠ、EcoRⅠ、salⅠ、BgⅢ、HindⅢ和PstⅠ酶解后的rRNA基因反杂交。根据rRNA基因图谱,这一与不育性有关的E3.2kb片段被定位于距16S rRNA基因5'端+2.0至+5.4kb的前导序列中。此结果表明,这段可能与花粉育性形成有关的片段定位于叶绿体基因组反向重复区。     

菠菜叶绿体DNA 4.1 kb的SalⅠ片段的克隆

利用pBR 322衍生质粒DNA为载体,将菠菜叶绿体DNA的SalⅠ限制性片段插入质粒的Sal Ⅰ位点,获得52个含重组质粒的菌落(Ap~rTc~5)。并对每个克隆的质粒进行限制性内切酶分析,通过Southern吸印与探针杂交,证明了重组质粒pSS104含有的插入DNA是菠菜叶绿体DNA 4.1kb的SalⅠ片段。迄今在该片段尚未定位任何已知的叶绿体基因,用大肠杆菌的活体系统也未能发现这段DNA的转录产物,本文对此进行了讨论。     

水稻叶绿体基因文库的构建和精细限制图谱的制作

水稻幼叶在加有高浓度抗坏血酸的缓冲液中匀浆,以获得完整的叶绿体,从中分离到ctDNA得率高达100μg/100g叶,纯度足以用于限制性核酸内切酶分析。ctDNA经Mbo I部分酶解得到的片段克隆到载体pcos 2 EMBL的Bam HI位点,重组DNA经体外包装后感染宿主菌,筛选表型Tc~5Km~R的重组子,通过计数克隆有效率达5×10~4重组菌落/1微克插入DNA。用λ-末端酶对重组环状双链DNA在cos位点切成线性分子,产生两个(ON-L及ON-R)可供标记和杂交的末端,线性Cosmid DNA经限制酶部分消化,凝胶电泳分离,干燥凝胶放射自显影,得到了6种限制性核酸内切酶的限制图谱。水稻ctDNA全长为129.5kb,在ctDNA上Pvu Ⅱ、Sal Ⅰ、Pst Ⅰ、Hind Ⅲ、Eco RI及Bam HI的切点分别为11、12、17、37、67和44个,1R A和B为21.7kb,LSC为73.7kb,SSC为12.4kb。     

蚕豆叶绿体DNA BamH I克隆库的构建及含16S-25S rRNA基因克隆的分析

蚕豆叶绿体DNA(ct—DNA)经BamH I酶切产生26个片段,最大的为14．00kb,最小的为0．42kb。本文以pBR322为载体,E．Coli HB101为受体菌,采用标准分子克隆法构建了蚕豆ct—DNA BamH I克隆库,并从库中分离得到含叶绿体rRNA基因的克隆。32P标记的E．Coil 16S、23S rRNA能和蚕豆ct—DNA BamH I第6(B₆,5．65kb)和第9(B₉,4．70kb)个片段杂交,含有这二个片段的克隆分别命名为pVFB32和pVFBl6。利用几种限制性内切酶酶切和Southern印迹法构建了pVFBl6的物理图谱。pVFBl6电镜下观察到有一变性环(A—T丰富区),经Hind I酶切,电镜观察定位此A—T丰富区位于16S和23S rRNA基因的间隔顺序内,推测该环可能与DNA复制有关。     

变铅青链霉菌启动子的克隆和表达

利用启动子探测质粒pIJ486(Tsr~(?) Neo~(?))将变铅青链霉菌(streptomyces lividans)TK24染色体DNA的BamHI酶切片段插入pIJ486的BamHI位点。获得4个硫链丝菌肽抗性、新霉素抗性的重组质粒。它们分别命名为pMGI(10.6kb)、pMG40(7.6kb)、pMG50(10.8kb)和pMG88(7.92kb)。BamHI酶切分析及再转化试验表明,新霉素抗性的恢复确实来自载体的外源插入序列。用BgⅢ酶切已将pMG40的插入序列缩小到0.78kb的pMG40-2,pMG50的插入序列缩小到2.2kb的pMG50-25,仍保留启动子活性。重组质粒pMG50-25的新霉素抗性水平高达90μg/ml(卡那霉素抗性水平为500μg/ml),表明这是一个活性很强的启动子。     

细胞质雄性不育高粱叶绿体 ndh D 基因的序列变异

片段SAAU－02 700特异地扩增自7种具可育细胞质的高粱材料的总DNA,含有叶绿体psa C(88bp)和ndh D(192bp)基因的部分序列。该片段与Eco Ri HindⅢ酶切的总DNA,线粒体DNA和叶绿体DNA杂交,在总DNA中获得了0.74kb的杂交带,而在叶绿体中获得0.74kb和0．45kb两条杂交带。与线粒体DNA无杂交;与经Hae Ⅲ酶切的总DNA杂交,在不育系中获得4．9kb的杂交带,而保持系的杂交带为4．45kb。参考GenBank中高粱的近缘物种玉米叶绿体基因组的序列,构建了ndh D基因区的酶切位点图谱,借此分析得出高粱不育系的叶绿体ndh D基因序列已发生改变。这种变异与高粱细胞质雄性不育反生的关系正在探讨中。     

苏云金芽胞杆菌cryⅡ基因的克隆和表达

分离的苏云金芽胞杆菌(Bacillus-thuringiensis)YBT-791对鳞翅目小菜蛾(Plutellaxylostella)有毒力,将其质粒DNA提纯,经HindⅢ酶切后与杀虫晶体蛋白cryⅡ基因探针杂交,显示出分子量分别为5kb和9．4kb两条DNA阳性片段。把5kb的DNA阳性片段克隆到pUCl8的HindⅢ位点上并转化大肠杆菌TGl,经酶切和杂交检测,证明斑点杂交阳性克隆子中含有5kb的cryI片段。把这个含cryⅡ基因的5kbHindⅢ片段进行亚克隆,将4kb的BamHI－Pstl酶切片段插入穿梭载体pXl61中,并用电脉冲法克隆于苏云金杆菌不产伴胞晶体的突变株中,得到产生单一cry Ⅱ基因编码的杀虫晶体蛋白的克隆菌株M一5。经电镜观察,该克隆菌株能形成出发菌YBT－791多种形态伴胞晶体中的一种方形伴胞晶体;经免疫双扩试验,它只能与Cry Ⅱ晶体蛋白抗血清形成沉淀线;经SDS—PAGE电泳,克隆菌的伴胞晶体只含有一种65kDa的晶体蛋白。生物测定结果表明它既对鳞翅目小菜蛾(Piutella xylostella)有毒性,又对双翅目致倦库蚊(Culex quinquefasciatus)有毒性。     

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.     

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.     

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.     

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.     

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。     

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.