Subnucleolar distribution and organization of Vicia faba L. rDNA in situ

The distribution and organization of nucleolar DNA in Vicia faba L. was analyzed by specific cytochemical staining using NAMA-Ur. The results showed that nucleolar DNA was distributed in the FCs and at the FC/DFC junctions. Statistical analysis showed that the rRNA genes occupied about one-third of the total dense fibrillar component region. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, suggesting a possible origin for nucleolar DNA.     

Repeated DNA sequences found in the large spacer of Vicia faba rDNA

In the large spacer of the rDNA of Vicia faba, multiples of a 0.32 kilobasepair (kb) sequence reiterate to various degrees. We sequenced the repetitious region consisting of the repeating sequences and its flanking regions using two cloned plasmids, which contain V. faba rDNA segments encompassing the whole region of the large spacer. The repetitious region was found to consist of multiple complete copies and one truncated copy of a 325 bp repeat unit and to be flanked by direct repeat sequences of about 150 bp. The set of direct repeats located at either side of the repetitious region differed from each other with about 10% sequence heterogeneity. However, nucleotide sequences of the direct repeats were well conserved between the two clones examined. Southern blot hybridization indicated a widespread distribution within the whole V. faba genome of some related sequences with high homologies to the 325 bp repeat unit and to the direct repeats.     

Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase

Three cDNA clones were isolated which code for the ubiquitous chloroplast enzyme, polyphenol oxidase (PPO), from Vicia faba. Analysis of the cloned DNA reveals that PPO is synthesized with an N-terminal extension of 92 amino acid residues, presumed to be a transit peptide. The mature protein is predicted to have a molecular mass of 58 kDa which is in close agreement to the molecular mass estimated for the in vivo protein upon SDS-PAGE. Differences in the DNA sequence of two full-length and one partial cDNA clones indicate that PPO is encoded by a gene family. Analysis of the deduced amino acid sequence shows that the chloroplast PPO shares homology with the 59 kDa PPOs in glandular trichomes of solanaceous species. A high degree of sequence conservation was found with the copper-binding domains of the 59 kDa tomato PPO as well as hemocyanins and tyrosinases from a wide diversity of taxa.     

Molecular heterogeneity and genetics of Vicia faba seed storage proteins

Summary Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different -major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.Contribution no. 55 from the Center of Vegetable Breeding, Portici, Italy     

犬Ⅰ型腺病毒DNA的酶切分析及分子克隆

犬Ⅰ型腺病毒(CAV-1)弱毒用限制性内切酶EcoR Ⅰ,BamH Ⅰ,Pst Ⅰ,Sph Ⅰ和Hind Ⅲ消化分析后其图谱与强毒株相比没有差异。将弱毒DNA用Pst Ⅰ完全消化后以鸟枪法克隆到载体质粒pBluescrip'SK中,经用光生物素标记的CAV-1 DNA杂交筛选以及Pst Ⅰ分析重组质粒证明已将分子量为5.5,3.5,2.85,1.2,0.32和0.28Kb的CAV-1DNA片段克隆到质粒中。克隆到的这些片段将可考虑进一步研究作为探针检测犬及狐狸等野生动物的腺病毒感染。     

蚕豆豆类胰岛素基因的cDNA克隆及序列分析

为探明豆科植物中豆类胰岛素基因的结构特征与进化关系,在已获得大豆豆类胰岛素基因的基础上,以蚕豆种子胚根mRNA为材料,采用RT-PCR技术,克隆了蚕豆豆类胰岛素基因的cDNA序列,编码的前体多肽包括信号肽、成熟型豆类胰岛素及另一多肽的45个氨基酸残基。DNA序列分析表明,克隆片段与大豆和豌豆的同源性分别为62.5%和58.7%。在氨基酸水平上分别具有44.2%和43.6%的同源性,其中存在着高度保守的半胱氨酸位点,它们在维持豆类胰岛素的空间结构与生理功能方面,可能具有重要的作用。 Abstract:In order to elucidate the relationship between the structural features of leginsulin gene in legume plants and their phylogenetic significance,we have cloned the cDNA sequence of leginsulin gene from radicles of broad bean (Vicia faba) via RT-PCR techniques according to the leginsulin gene sequence we previously obtained from soybean (Glycine max).The cloned cDNA encoded for a precursor protein consisting of the signal peptide,mature leginsulin and an additional 45 amino acids of another polypeptide.A sequence search for homology comparison revealed the cloned leginsulin cDNA fragment shares 62.5% and 58.7% similarity to soybean and pea,respectively.The results also shown that leginsulin cDNA from broad bean presents 44.2% and 43.6% amino acid sequence homology with soybean and pea (Pisum sativum),respectively,and that there exists highly conserved cysteine sites among the leginsulin cDNAs,which may play a crucial role in maintaining the three-dimensional structure and the physiological functions of leginsulin.     

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

前沟藻18S rDNA序列克隆和分子鉴定分析

旨在扩增一株未知微藻18S rDNA,并进行序列分析和物种鉴定分析.提取该微藻总DNA,用PCR技术扩增其18S rDNA,对序列进行测序,在GenBank进行序列比对分析,用ClustalX软件构建进化树;用光学显微镜进行形态学分析.经测序其序列长度为1 736 bp,G+C为47％;同源性分析表明,与GenBank中多个强壮前沟藻种的18S rDNA基因序列同源性迭99％;与前沟藻属的其它藻种同源性均迭95％以上.而与裸甲藻属和环藻属的藻株同源性为85％左右.经光学显微镜观察,该藻具有典型的强壮前沟藻形态.结合该藻18S rDNA序列分析和粗略的形态学分析,推断该藻可能为前沟藻属的强壮前沟藻.微藻形体微小,结构简单,需借助电子显微镜才可准确地从形态学上签定其种属,鉴定工作繁琐费时;而利用分子技术,则可能使鉴定工作变得简单快捷.     

Cloning and Characterization of New Repetitive Sequences in Field Bean (Vicia faba L.)

Annals of Botany83: 535–541, 1999 It is regretted that Figure 1 of this paper was incorrect; itshould have appeared as follows: Fig. 1. Genomic organization of new repetitive sequences. Southernblots of field bean genomic DNA digested using various restrictionenzymes (H, Hind III; Hc, Hinc II; T,Taq I; A, Alu I; R, RsaI; M, Mbo I; F, Fok I) were probed with the individual repeats.The bands from which the sequences were isolated are markedby arrows. The bands from which full length sequence of T133and TIII17 repeats were isolated are marked by asterisks.     

Isotachophoretic Analysis of Ions in Guard Cells of Vicia faba

Isotachophoretic analysis of ions was performed on guard cells of Vicia faba cv. Ryosai Issun with either open or closed stomata. In guard cells of open stomata, K⁺ and malic acid concentrations were 5–7 and 5–10 times higher, respectively, than in guard cells of closed stomata. The content of citric acid (plus isocitric acid) also increased during stomatal opening, but the increment was smaller than that of malic acid. Sodium ions, phosphoric and glyceric acids were present in low concentrations but did not increase during the opening. Other cations and anions could not be measured because of low concentrations. Malic acid provided 68–79% of the counter anions for the potassium taken up by guard cells during stomatal opening.     

合成生物学中不依赖限制性内切酶的分子克隆策略

随着合成生物学的兴起和发展,基因克隆和DNA大片段组装成为了常规操作。利用人工智能和液体操作机器人进行高通量的DNA组装和功能筛选已被广泛应用。传统的依赖于限制性内切酶识别位点的克隆技术对序列有选择性、步骤繁琐、实验人员的培训周期长,不利于以流水线形式进行工程化使用,已经逐步在生物工程领域内被淘汰。文中论述了一系列适于机械化操作的新一代分子克隆技术,即不依赖基因序列和连接反应克隆方法、Gibson组装、聚合酶环形延伸克隆、细胞裂解物体外无痕连接和细胞体内组装克隆。对这些方法的建立、基本原理及应用前景等方面进行了总结,并对其优缺点进行了比较。     

gamma-Guanidinobutyraldehyde Dehydrogenase of Vicia faba Leaves

γ-Guanidinobutyraldehyde dehydrogenase was purified 27-fold in 40% yield from extracts of Vicia faba leaves. High specificity exist only for γ-guanidinobutyraldehyde and γ-aminobutyraldehyde; the K_m value was 3.4 micromolar for γ-guanidinobutyraldehyde, 25 micromolar for γ-aminobutyraldehyde, and 84 micromolar (case of γ-guanidinobutyraldehyde) for NAD, respectively. The enzyme had a molecular weight of approximately 83,000. Optimal pH and temperature for activity were 9.5 and 45°C, respectively. The enzyme was inhibited strongly by p-chloromercuribenzoate, N-ethylmaleimide, and zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene).     

Analysis of growth of tetraploid nuclei in roots of Vicia faba

Growth of nuclei of a marked population of cells was determined from G1 to prophase in roots of Vicia faba. The cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G1 and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. Of the marked population of cells, about 65% had completed a cell cycle 14--15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.     

Sister chromatid exchanges in Vicia faba

The relationship between chromosomal aberrations and sister chromatid exchanges (SCE's) after treatment of Vicia faba root tips with thiotepa, caffeine and 8-ethoxycaffeine (EOC) was studied by using a modified fluorescent plus Giemsa (EPG) technique. At concentrations which had little effect on the frequency of chromosomal aberrations, thiotepa strongly increased the frequency of SCE's, provided that the chromosomes were allowed to replicate between treatment and fixation. Frequently, the size of the exchanged material was smaller than the diameter of the chromatid. Post-treatments with caffeine of roots previously exposed to thiotepa strongly increased the frequency of aberrations, but had little effect on the frequency of SCE's. In contrast to thiotepa, EOC caused only a slight increase in the frequency of SCE's even at concentrations which produced a high frequency of chromosomal aberrations. Thus, there was not a close correlation between SCE's and chromosomal aberrations. Single-strand exchanges between the DNA double helices in sister chromatids were not detected.     

Sister chromatid exchanges in Vicia faba

Evolutionary loss of the Y chromosome has occurred in Climacia areolaris (Hagen) of the neuropteran family Sisyridae. The diploid set comprises 6 pairs of autosomes, plus 2 X chromosomes in the female and 1 X in the male. The Y is retained in Sisyra vicaria (Walker) of the same family: its chromosome number is 14 in both sexes including 2X chromosomes in the female and 1X plus Y in the male. Two alternative pathways for the segregation of the sex chromosomes-distance segregation and sex bivalent formation-co-exist in the latter species in a ratio of approximately 1 to 6; the possible phylogenetic significance of this feature is discussed.     

Flow karyotyping and sorting of Vicia faba chromosomes

Summary Chromosome suspensions were prepared from formaldehyde-fixed, synchronized Vicia faba root tips. After staining with the DNA intercalating fluorochrome propidium iodide, the suspensions were analysed with a flow cytometer. The resulting histograms of integral fluorescence intensity contained peaks similar to those of theoretical V.faba flow karyotypes. From V. Faba cv Inovec (2n = 12) only one peak, corresponding to a single chromosome type (metacentric chromosome), could be discriminated. However, it was found that the peak also contained doublets of acrocentric chromosomes. Bivariate analysis of fluorescence pulse area (chromosome DNA content) and fluorescence pulse width (chromosome length) was necessary to distinguish the metacentric chromosome. To achieve a high degree of purity, a two-step sorting protocol was adopted. During a working day, more than 25 000 metacentric chromosomes (corresponding to 0.2 g DNA) were sorted with a purity of more than 90%. Such chromosomes are suitable for physical gene mapping by in situ hybridization or via the polymerase chain reaction (PCR) and allow the construction of chromosome-specific DNA libraries. While it was only possible to distinguish and sort one chromosome type from V. Faba cv Inovec with the standard karyotype, it was possible to sort with a high degree of purity five out of six chromosome types of the line EFK of V. Faba, which has six pairs of morphologically distinct chromosomes. This result confirmed the possibility of using reconstructed karyotypes to overcome existing problems with the discrimination and flow sorting of individual chromosome types in plants.