Analyses of transcriptional alterations during the mutualistic interaction between the model legume Medicago truncatula and the arbuscular mycorrhiza fungus Glomus intraradices, and characterisation of mycorrhiza-specific lectins [Elektronische Ressource] / von André Frenzel

Andre Frenzel

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2006
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Analyses of transcriptional alterations during the mutualistic
interaction between the model legume Medicago truncatula and
the arbuscular mycorrhiza fungus Glomus intraradices, and
characterisation of mycorrhiza- specific lectins
Von der
Naturwissenschaftlichen Fakultät
der Universität Hannover
zur Erlangung des Grades
eines Doktors der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation
von
Dipl.-Bi ol. André Frenzel
geboren am 12 .01.1976
in Ankum
2 006Referentin: PD Dr. Franziska Krajinski
Korreferent: Prof. Dr. Edgar Maiß
Tag der Promotion: 13. März 2 006Contents
Contents
Abstra ct......................................................................................................................................1
Zusam m enfa ssung ........................................................... ............................................................2
1. Introduc tion.............................................................................................................................3
1.1 The mode l plant Medicago t runc atul a..............................................................................4
1.2 The arbus cul ar m yc orrhi za ..............................................................................................6
1.3 Tra nscriptiona l ana lyses to identify AM-related gene s....................................................9
1.4 Objective s of this w ork ..................................................................................................11
2 Material and M ethods ...........................................................................................................13
2.1 G ene ra l Methods ...........................................................................................................13
2.1.1 P lant grow th and inoc ul ations................................................................................13
2.1.2 S taini ng of funga l struc ture s...................................................................................13
2.1.3 P rotein e xtra ction for E MSA ana lysis..................................................................14
2.1.4 M easure m ent of prot ein conc entra tion a ccordi ng t o Bra dford ..............................14
2.1.5 D N A-i sol ation from Medicago t runc atul a.............................................................15
2.1.6 RN A isol ation.........................................................................................................15
2.1.7 Re ve rse tra nscription for c D N A synthesis..............................................................16
2.1.8 Pol ym era se chain re actions...................................................................................16
2.1.9 Re striction di gests...................................................................................................17
2.1.10 E thanol puri fication of D N A................................................................................17
2.1.1 1 Cloni ng of P CR fragments....................................................................................17
2.2 Microbi ol ogical Methods ...............................................................................................18
2.2.1 P re pa ra tion of c om pe tent Escherichia col i cells.....................................................18
2.2.2 Tra nsform ation of E scherichia col i.........................................................................19
iContents
2.2.3 Plasm id isol ation.....................................................................................................19
2.2.4 S eque nc ing ..............................................................................................................20
2.2.5 P re pa ra tion of c om pe tent Agroba cterium rhizoge ne s cells....................................20
2.2.6 Tra nsform ation of Agroba cterium rhizoge ne s........................................................20
2.3 Tissue cul turi ng m ethods ................................................................................................21
2.3.1 Agroba cterium rhizoge ne s-mediated tra nsform ation of M . trunc atul a..................21
2.3.2 Agroba cterium rhizoge ne s-mediated tra nsform ation of N icotiana tabacum ..........21
2.3.3 Histoc hem ical ana lysis of tra nsgeni c root s ...........................................................22
2.4 Microa rra y Ana lyses.......................................................................................................22
2.4.1 S cope and layout of the microa rra y........................................................................22
2.4.3 Ana lysis of microa rra y im age data.......................................................................23
2.5 In silico t ra nscriptiona l ana lyses...................................................................................23
2.5.1 c D N A libraries and s eque nc ing ..............................................................................23
2.5.2 S eque nc e proc essing, annotation a nd c lustering ...................................................24
2.5.3 In s ilico ana lysis of gene expre ssion ....................................................................24
2.5.4 Q ua ntitative real-tim e RT-PCR...............................................................................25
2.5.5 S eque nc e Ana lysis..................................................................................................26
2.6 Ana lyses of AM-spe cific lectin-like gene s.....................................................................26
2.6.1 Cl oni ng of w hol e cD N A seque nc es........................................................................26
2.6.1.1 Cl oni ng of genom ic seque nc es ......................................................................27
2.6.2 S eque nc e Ana lyses................................................................................................27
2.6.3 Cons truc tion of G FP fus ion prot eins.....................................................................28
2.6.4 G FP detection us ing confoc al laser s canni ng m icropscopy....................................28
2.7 Prom oter ana lysis of AM-spe cific lectin-like gene s.......................................................28
2.7.1 Is ol ation of l ectin prom oters from BAC s eque nc es and by inve rse PCR...............28
iiContents
2.7.2 P rom oter D eletion Ana lyses...................................................................................29
2.7.3 E lectrophore tic Mobility Shift Assay...................................................................30
2.7.3.1 3' end labelling of D N A.................................................................................30
2.7.3.2 D ot bl ot to c heck l abelling efficienc y.............................................................31
2.7.3.3 E lectrophore tic mobility s hift assay................................................................31
2.7.4 Computationa l ana lyses.........................................................................................32
2.8 Chem icals and s ol utions.................................................................................................33
3 Resul ts....................................................................................................................................34
3.1 Tra nscriptiona l ana lyses us ing Microa rra y hybri disation..............................................34
3.1.1 M . trunc atul a is 6 dpi w ith G. i ntra ra dices in an e arly-m yc orrhi zal pha se............34
3.1.2 M icroa rra y hybri disation........................................................................................34
3.1.3 M icroa rra y data are conc orda nt w ith form er s tudi es..............................................35
3.1.4 U p and dow n-re gulated gene s are distribut ed equa lly duri ng a com pl etely
deve lope d AM..................................................................................................................36
3.1.5 Six day early-m yc orrhi zal roots show tra nscriptiona l decre ase of defenc e-i nvol ve d
gene s...............................................................................................................................37
3.1.6 Thirty-s ix gene s show tra nscriptiona l altera tion 6 a nd 21 dpi................................38
3.2 Tra nscriptiona l ana lyses us ing electroni c Northern a pproa ch.......................................41
3.2.1 Gene ra tion of E ST-clusters from two M. trunc atul a-G. intra ra dices AM cD N A
libraries............................................................................................................................41
3.2.2 In silico s cre eni ng for nove l AM-spe cific tra nscripts............................................42
3.2.3 RN A accum ul ation s tudi es ....................................................................................47
3.2.4 A fam ily of M . trunc atul a lectin gene s is induc ed duri ng arbus cul ar m yc orrhi za..49
3.3 S eque nc e ana lyses of the AM-spe cific lectins...............................................................50
3.4 Targeting of AM-spe cific MtLE C5 prot ein...................................................................53
iiiContents
3.4.1 M tLE C5 is pre dicted to be targeted at the vacuol e.................................................53
3.4.2