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Chromosomal Arrangement of Leghemoglobin Genes in Soybean

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Chromosomal Arrangement of Leghemoglobin Genes in Soybean Chromosomal Arrangement of Leghemoglobin Genes in Soybean and Kidney Bean by Jong Seob Lee A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of c Doctor of Philosophy Department of Biology MoGill University Montreal, Quebec Canada July 1984 Copyright @ Jong Seob Lee, 1984 0 To my wife Jung Sun 0 i ABSTRACT Leghemoglobin (Lb) genes are induced only following infection of the leguroo plant by Rhizobium. In soybean, there are four major leghemoglobins which are encoded by four separate genes. A chroroosoma.l walk has been carried out to investigate the arrangement of these Lb genes on the soybean chromosome by screening Alui-Haeiii and EcoRI genomic libraries. A cluster of four different Lb genes was isolated from the libraries in a set of overlapping clones which together include 45 kilobases (kb) of contiguous OOA. These four Lb genes, including a pseudogene, are present in the same transcriptional orientation and are arranged in the order: 5'-Lba.-Lbc -Lb 1/J -Lbc -3'. The intergenic regions average 2.5 kb. 1 1 3 In adddion to the main locus, there are other Lb genes in three other loci which do not appear to be contiguous to this locus. The second locus contains another leghemoglobin (Lbc2) gene and a second pseudo (truncated) gene. The two other loci only contain truncated sequences. A sequence which appears to be comnon to the 3' regions of all the Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is pr~rily expressed in root and leaf was found 5 1 to two Lb loci. In order to investigate the possible mechanism by which four Lb loci were generated, the structure and chromosomal arrangement of leghemoglobin genes in kidney bean have been determined. The nucleotide sequence of a kidney bean Lb gene showed the same intron/exon arrangement as that of soybean Lb genes, indicating their close evolutionary relationship. The presence in the kidney bean genome of four leghanoglobin genes with two 0 sequences, each specific to the 5' or 3' region of the soybean Lb loci ii suggests that tandom duplication of a single prilnordial plant globin gene had occurred to generate a four leghemoglobin gene locus before Glycine and Phaseolus species diverged. A large deletion in one of the 2 four-gene loci in soybean resulted in the generation of the Lbc2 locus containing two leghemoglobin genes. The truncated gene appears to have been generated in the genome of Glycine species, prior to the chromosome duplication by tetraploidization before the divergence of G. ~ and G. soja. iii .. ; RESUME Les genes de la leghanoglobine (Lb) sont induits seulement apres 1' infection de la legumineuse par le Rhizobium. Il y a quatre legbemoglobines majeures chez la feve soya qui sont codees par quatre genes separes. En parcourant le chromosome par hybridations successives dans des librairies genomiques Alui-Haeiii et EcoRI 1 1 arrangement des genes de la leghemoglobine sur le chromosome de la feve soya a ete analyse. Un groupe de quatre differents genes de Lb ont ete isole des librairies dans un ensemble de cl6nes se chevauchant lesquels totalisent 45 kilobases (kb) d'ADN contigu. Ces quatre genes Lb, incluant un pseudogene, sont presents dans la meme orientation transcriptionnelle et 1 1 sont places dans 1' ordre: 5 -Lba-Lbc -Lb 1jJ -Lbc -3 • Les regions 1 1 3 intergeniques sont en m:>yenne de 2.5 kb. En plus du locus principal, on trouve d' autres genes de la Lb dans trois autres loci qui ne senblent pas lies a ce locus. Le deuxiene locus contient un autre gene Lb (Lbc2) et un second pseudogene (tronque). Les deux autres loci ne contiennent que des sequences tronquees. Une sequence qui senble ~tre comnune aux regions 3 I de tous les loci Lb a ete trouvee pres du gene Lbc3. La region avoisinante de l 1 extremite 3 1 du locus principal contient aussi une s€quence qui semble ~tre exprimee plus frequernment dans le tissu racinaire. Une autre sequence qui est expri.Inee principalement dans les racines et les feuilles a ete trouvee a l'extremite 5 1 de deux loci de la Lb. Dans le but d 1 etudier les mecanismes possibles par lesquels les quatre loci de Lb ont ete generes, la structure et 1' arrangement chramosaniques des genes de la Lb dans la feve rouge ont ete determines. La sequence des nucleotides des genes de la Lb de la feve rouge montre le ~ arrangement iv intron/exon que les genes de la IJ:> chez la feve soya, indiquant leur proche parente evolutionaire. La presence dans le genome de la feve rouge de quatre genes de leghEmoglobine en plus des deux sequences, chacune specifique aux regions 5' ou 3' du locus de la IJ:> de la feve soya, suggeke qu' i1 y a eu une duplication en tandem d 'un seul gene primi tif de globine vegetale avant que les esp€ces Glycine et Phaseolus ne divergent. Une large deletion dans un des deux loci de quatre genes dans la feve soya a generl§ le locus IJ:>c2 contenant deux genes de la leghemoglobine. Le gene tronque semble avoir ete genere dans le genome de l'espece Glycine avant la duplication des chromosomes par tetraploidisation survenue avant la divergence de G. max de G. soja. Traduit par Diane Longtin V ACKN<MLEDGEMENTS 0 I wish to thank my supervisor, Dr. Desh Pal s. Verma, for his support, guidance and discussions throughout my work in his laboratory. I wish also to thank the rcenbers of my supervisory comni tee, Drs. Yutaka Nishioka and Gorden Shore, for their guidance and discussions in the course of my study at McGill universi~. My thanks are extended to the rcenbers of the laboratory, particularly Forrest Fuller, Norman Brisson, Eric Olson, Michael Sadowsky, Claude Lalande, Vincent Mauro, Shri Purohit, Indra Verma, Truyen Nguyen, Yvette Mark, Diane Longtin, Rose-Marie Riha, Victoria Foster and Joel Delisle for their various help. I also thank Drs. Robert Goldberg of the Universi~ of california, Los Angeles and Jerry Slightam of the Agrigenetic Inc., Madison, Wisconsin for providing soybean or kidney bean genomic libraries and Mr. Guy L'Heureux and Robert Lamache for their photographic work. 0 vi TABLE OF CONTENTS Page ABSTRACI' i RESUME... "" iii ACKN<MELEDGMENTS V TABLE OF CONTENTS vi LIST OF FIGURES xi ABBREVIATIONS xiii I. INTRODUCTION AND LITERATURE REVIEW 1 A. Deve1op:oont of the Root Nodule 2 (1). Invasion of root hair cells 2 (2). Infection of cortical cells 4 B. Nodule Maturation 6 (1). Plant factors 6 (2). Bacterial factors 7 c. AssDni1ation of Symbiotically Fixed Nitrogen 8 (1). Asparagine synthesis 10 (2). Ureide synthesis 10 D. Plant Genes Expressed in the Root Nodule 12 (1}. Nodulins 12 (a) • Uricase 13 (b). Glutamine syntheta~::;e 14 (c). Other nodul ins 14 vii Table of contents {cont'd) Page (2}. Leghemoglobin 16 (a) • Location 16 (b). Structure 17 (c) • Function 21 (d) • Biosynthesis 22 (e). Leghemoglobin genes 24 II. MATERIAL AND METHODS 27 A. Materials 27 (1). Enzymes and chanicals 27 (2}. Biological materials 28 (3). Growth media for bacteria and bacteriophage 29 B. Methods 32 (1). Growth of plant tissues 32 (2) • Preparation of DNA 32 (a). DNA from plant tissues 32 {b) • Plasmid OOA 33 {c) • Phage DNA 35 (3). Isolation of poly(A)+ RNA 36 (4). DNA digestion and electrophoresis 37 (a). Restriction enzyme digestions 37 (b). Gel electrophoresis 37 1. Polyacrylamide gel electrophoresis 37 2. Agarose gel electrophoresis 38 viii Table of contents (cont'd) Page (5). Southern hybridization 38 (a). Transfer of DNA from gels 38 (b). Hybridization 39 1. Hybridization to DNA bound to GeneScreen 39 2. Colony hybridization 40 (6). Screening of genomic libraries 40 (7). Molecular cloning 42 (a) • Synthesis and cloning of cONA 42 (b). Subcloning of genomic clones 43 (8) • DNA sequencing 44 (a). Cloning precedures 44 1. Isolation of DNA fragments 44 2. Ligation 45 3. Transformation 45 4. Preparation of single-stranded template 46 (b) • Sequencing procedures 46 1. Annealing primer to template 46 2. Sequencing reaction 47 3. Gel electrophoresis 47 4. Autoradiography 47 5. Canparison of DNA sequences 48 0 ix Table of contents (cont' d) page III. RESULTS 49 A. Chromosanal Arrangement of Leghanoglobin Genes in Soybean 49 (1). Isolation of genomic fragments containing leghemoglobin sequences 49 {2) • Chramosanal walk on a leghemoglobin locus 59 (3). Other regions containing leghemoglobin sequences 56 (4). Analysis of leghemoglobin sequences on clone 2 58 {5). Structure of a leghemoglobin sequence on clone 43 63 (6). A sequence flanking the leghemoglobin loci 63 (7). Other genes flanking leghemoglobin loci 67 B. Structure and Chramosanal Arrangement of Leghemoglobin Genes in Kidney Bean 72 (1). Construction of a partial cDNA library 72 (2). Isolation of a kidney bean leghemoglobin gene 73 (3) • Structure of a kidney bean leghanoglobin gene 73 (4). Hybridization of kidney bean genomic INA with pJSLbl5 89 (5) • Search for the 3' repeat sequence in kidney bean and Glycine soja genon:e> 89 (6). Search for the 5' sequence expressed in root and leaf 82 IV. DISCUSSION 86 A. Chromosanal Arrangement of Leghemoglobin Genes in Soybean 86 (1). Multiple loci of leghemoglobin genes 86 (2). Flanking regions of leghemoglobin loci 88 X Table of contents (cont'd) page c B. Chramosanal Arrangements of Legha:noglobin Genes in Kidney Bean and Wild Soybean (G. soja) 89 (1) • Structure of a legha:noglobin gene in kidney bean 89 (2).
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