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Genomanalyse Von Prodiamesa Olivacea

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Genomanalyse Von Prodiamesa Olivacea Genomanalyse von Prodiamesa olivacea Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften (Dr. rer. nat.) am Fachbereich Biologie der Johannes Gutenberg-Universität in Mainz Sarah Brunck geb. 08.08.1987 in Mainz Mainz, 2016 Dekan: 1. Berichterstatter: 2. Berichterstatter: Tag der mündlichen Prüfung: ii Inhaltsverzeichnis Inhaltsverzeichnis ................................................................................................................................ iii 1 Einleitung ........................................................................................................................................... 1 1.1 Die Familie der Chironomiden ................................................................................................. 1 1.1.1 Die Gattung Chironomus ..................................................................................................... 3 1.1.2 Die Gattung Prodiamesa ....................................................................................................... 6 1.2 Die Struktur von Insekten-Genomen am Beispiel der Chironomiden ............................... 9 1.2.1 Hochrepetitive DNA-Sequenzen ..................................................................................... 11 1.2.2 Mittelrepetitive DNA-Sequenzen bzw. Gen-Familien ................................................. 13 1.2.3 Gene und genregulatorische Sequenzen ........................................................................ 17 1.3 Zielsetzung ................................................................................................................................ 20 2 Material und Methoden .................................................................................................................. 22 2.1 Versuchstiere ............................................................................................................................. 22 2.2 Speziesbestimmung P. olivacea ............................................................................................... 22 2.3 Geschlechtsbestimmung bei Chironomiden-Larven ........................................................... 23 Molekularbiologische Methoden ................................................................................................. 24 2.4 Isolation von Nukleinsäuren .................................................................................................. 24 2.4.1 Isolation von genomischer DNA aus Chironomiden-Larven ..................................... 24 2.4.2 Isolation von Einzellarven- DNA .................................................................................... 24 2.4.3 Isolation von Plasmid- DNA ............................................................................................ 25 2.4.4 Präparation von Gesamt-RNA (Guanidinthiocyanat (GTC)- Methode) (Chomczynski und Sacchi 1987) ............................................................................................... 25 2.4.5 Präparation von Gesamt-RNA aus Einzellarven (GTC-Methode) ............................. 26 (Chomczynski und Sacchi 1987) ............................................................................................... 26 iii 2.5 Konzentrationsbestimmung von Nukleinsäuren ................................................................ 26 2.5.1 NanoDrop® ........................................................................................................................ 26 2.5.2 QuBit™ ................................................................................................................................ 26 2.5.3 Agilent 2100 Bioanalyzer .................................................................................................. 27 2.6 Aufreinigung von Nukleinsäuren .......................................................................................... 27 2.6.1 Phenol-Chloroform-Extraktion ........................................................................................ 27 2.6.2 Fällen von Nukleinsäuren ................................................................................................ 27 2.6.3 Entfernen von Verunreinigungen ................................................................................... 28 2.7 Elektrophoretische Auftrennung von DNA ......................................................................... 28 2.7.1 Testgele ............................................................................................................................... 29 2.7.2 Präparative bzw. analytische Gele .................................................................................. 29 2.8 Wiedergewinnung von DNA-Fragmenten aus Agarosegelen ........................................... 29 2.8.1 Elektroelution ..................................................................................................................... 29 2.8.2 Wiedergewinnung mittels Silica-Membran ................................................................... 30 2.9 Klonierung von DNA............................................................................................................... 30 2.9.1 Restriktion genomischer DNA ........................................................................................ 30 2.9.2 Ligation ............................................................................................................................... 30 2.9.3 Transformation mittels Elektroporation ........................................................................ 31 2.10 DNA-Markierungstechniken ................................................................................................ 31 2.10.1 Random Primed Oligo Labeling .................................................................................... 31 2.10.2 Nick Translation .............................................................................................................. 32 2.11 Hybridisierungstechniken ..................................................................................................... 32 2.11.1 Fluoreszenz-in-situ-Hybridisierung (FISH) ................................................................. 32 2.11.2 Southern-Hybridisierung bzw. „Pirrotta“ ................................................................... 34 2.11.3 Koloniefilter-Hybridisierung (Grunstein und Hogness 1975) .................................. 36 2.12 Polymerase Kettenreaktion (PCR) (Mullis et al. 1986) ...................................................... 37 iv 2.12.1 Standard-PCR ................................................................................................................... 37 2.12.2 cDNA-Synthese ................................................................................................................ 38 2.12.3 RT-PCR .............................................................................................................................. 38 2.13 Sequenzierung ........................................................................................................................ 38 2.13.1 Sanger Sequenzierung .................................................................................................... 38 2.13.2 Illumina Next-Generation Sequencing ............................................................................. 39 2.14 Bioinformatische Methoden .................................................................................................. 40 2.14.1 Konvertierung der Rohdaten ......................................................................................... 40 2.14.2 Importieren der Rohdaten .............................................................................................. 40 2.14.3 Trimmen der Rohdaten .................................................................................................. 40 2.14.4 De novo-Assembly der getrimmten Daten ................................................................... 40 2.14.5 Kartierung von genomischen bzw. transkriptomischen Daten ................................ 41 2.14.6 Blast ................................................................................................................................... 41 2.15 Puffer und Lösungen ............................................................................................................. 42 3 Ergebnisse ......................................................................................................................................... 44 3.1 Isolierung von Nukleinsäuren aus P. olivacea-Larven/–isolierten Gehirnen .................... 44 3.1.1 Isolierung von genomischer DNA aus unterschiedlichen Geweben ......................... 44 3.1.2 Präparation von Gesamt-RNA aus P. olivacea-Larven ................................................. 45 3.2 NGS-generierte Daten .............................................................................................................. 45 3.3 Analyse von hochrepetitiver DNA aus P. olivacea ............................................................... 47 3.3.1 Analyse von hochrepetitiver DNA mittels Restriktionsverdau genomischer DNA 47 3.3.2 Analyse von hochrepetitiver DNA durch Southern-Analyse ..................................... 48 3.3.3 Chromosomale Lokalisation von hochrepetitiver DNA mittels FISH ....................... 49 3.3.4 Sequenzanalyse von hochrepetitiver DNA aus P. olivacea .......................................... 50 3.4 Die Globin-Gene von P. olivacea ............................................................................................
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