Identification of yeast genes involved in Sauvignon Blanc aroma ...

More documents

Recommendations

Info

3074 bp2816 bp3302 bp2392 bp2263 bp1930 bp3302 bp3302 bp2972 bp2849 bp2326 bp2477 bp2263 bp2849 bp2477 bp3455 bp2641 bp2344 bp3455 bp2263 bp2789 bp3455 bpA.4 Deleting CYS3, CYS4, GLN3, IRC7, MET17, NPR1, SHM2 and URE2 in wine yeast 185A1kb+Irc7-Test1Met17-Int.Irc7-Test1Met17-Int.Ure2-Test1Met17-Int.Ure2-Test1Met17-Int.Shm2-Test1Met17-Int.Shm2-Test1Met17-Int.1kb+F15-irc7-IF15-wtF15-irc7-IF15-irc7-IIF15-wtF15-irc7-IIF15-ure2-IF15-wtF15-ure2-IF15-ure2-IIF15-wtF15-ure2-IIF15-shm2-IF15-wtF15-shm2-IF15-shm2-IIF15-wtF15-shm2-IIB1kb+Gln3-Test1Irc7-Int.Gln3-Test1Irc7-Int.Met17-Test1Irc7-Int.Met17-Test1Npr1-Test1Irc7-Int.Npr1-Test1Irc7-Int.Npr1-Test1Irc7-Int.1kb+F15-gln3-IF15-wtF15-gln3-IF15-gln3-IIF15-wtF15-gln3-IIF15-met17-IF15-wtF15-met17-IF15-met17-IIF15-wtF15-npr1-IF15-met17-IIF15-wtF15-npr1-IF15-npr1-IIF15-wtF15-npr2-IIFigure A.5: DNA band pattern of haploid wine yeast F15 (h, aux) knocked out in six candidategenes after PCR with corresponding Test1 primer pair. A: IRC7, URE2, SHM2; B:GLN3, MET17, NPR1; Met17 (A) and Irc7 (B) integration primers were used for PCRtemplate control. F15-gln3-II and F15-npr1-I both showed wild type bands.
186 APPENDICESA.5 Overexpression of CYS3, CYS4, GLN3, IRC7 andSHM2Five genes were overexpressed by replacing the native promoter of each target gene withthe UKP cassette containing the constitutive promoter PGK1. The procedure applied isdescribed in the materials and methods Chapter 2.10.6.Figure A.6 A shows the UKP cassettes containing sequence homology to each geneof interest after PCR amplification, purification and electrophoresis. This gene-specificUKP cassette was then transformed into haploid wine yeast F15 (h, aux) and diploidlaboratory yeast BY4743. Table A.3 displays the number of G418-resistant and uracilprototrophic colonies after transformation of the gene-specific UKP cassette. The transformationefficiency of the UKP amplicon in the wine yeast was 4 to 24 times lower thanin the laboratory yeast BY4743, although the control plasmid differed only ≈ 2-fold.The first four lanes in Figure A.6 B confirm that the PGK promoter had integrateddirectly in front of the CYS3 gene in all four BY4743 transformants tested. This can beconcluded, because the PCR with the primer pair employed (3’kanI-F/UKP-CYS3-test-R)amplifies the PGK promoter through to the beginning of the CYS3 gene (see Figure 2.3).All gene-specific overexpression cassettes were verified in BY4743 and F15 (h, aux) withthe corresponding primer pair (Figure A.6 C - E). However, it was not possible to amplifythe full length UKP-construct across the integration junctions with the appropriate testprimer pairs in the laboratory strain. BY4743 is diploid and was expected still to containone functional copy of the wild-type allele. The PCR most likely favoured the amplificationof the short wild-type construct (≈ 300 bp) over the much longer overexpressioncassette (≈ 3400 bp). In the haploid wine yeast F15 (h, aux) this long construct could beamplified for all five overexpressed genes as shown for genes CYS4 and GLN3 in A.6 (D+ E). PCR tests for the overexpression of genes CYS3, IRC7 and SHM2 were successfullyperformed, but are not shown in Figure A.6.These results suggested that the PGK promoter was successfully integrated upstreamof all five target genes in both F15 (h, aux) and BY4743. The success of this strategyin the wine yeast F15 (h, aux), despite the short (45 bp) regions of flanking homologyused, suggests that the recombination capability of this strain is similar to that of thelaboratory strain, and that standard methods of gene targeting developed in the laboratorystrain should be applicable to F15 (h, aux).
Page 1 and 2:
http://researchspace.auckland.ac.nz
Page 4 and 5:
IIIAbstractThe grape variety Sauvig
Page 6:
VIn my Blakean yearsI was so dispos
Page 9 and 10:
VIII• Kien Ly for letting me “b
Page 11 and 12:
XTABLE OF CONTENTS1.8.2 Winemaking
Page 13 and 14:
XIITABLE OF CONTENTS3.1 Introductio
Page 15 and 16:
XIVTABLE OF CONTENTS5.4 S-ethyl-L-c
Page 18 and 19:
XVIIList of figures1.1 Model of NCR
Page 20 and 21:
XIX5.3 Fermentation rate and volati
Page 22 and 23:
XXIList of tables1.1 Characteristic
Page 24 and 25:
XXIIIAbbreviationsAbbreviations of
Page 26:
XXVUUVUKPv/vVAV maxwtw/vw/wYAN2-ami
Page 29 and 30:
2 INTRODUCTIONfactor to enable this
Page 31 and 32:
4 INTRODUCTIONThis work is entirely
Page 33 and 34:
6 INTRODUCTION• Sulfur-containing
Page 35 and 36:
8 INTRODUCTIONnitrogen sources for
Page 37 and 38:
10 INTRODUCTION1.3 An overview of t
Page 39 and 40:
12 INTRODUCTIONH 2 S into organic s
Page 41 and 42:
14 INTRODUCTIONTable 1.1: Character
Page 43 and 44:
16 INTRODUCTIONThe current model of
Page 45 and 46:
18 INTRODUCTIONThe C6 compound (E)-
Page 47 and 48:
20 INTRODUCTIONThe only indication
Page 49 and 50:
22 INTRODUCTIONgrape-bunch pressing
Page 51 and 52:
24 INTRODUCTION3. The third goal wa
Page 53 and 54:
26 MATERIALS AND METHODSNameTable 2
Page 55 and 56:
28 MATERIALS AND METHODSSynthetic d
Page 57 and 58:
30 MATERIALS AND METHODSTable 2.4:
Page 59 and 60:
32 MATERIALS AND METHODSTo determin
Page 61 and 62:
34 MATERIALS AND METHODSx 10 6 cell
Page 63 and 64:
36 MATERIALS AND METHODSTable 2.6 -
Page 65 and 66:
Page 67 and 68:
40 MATERIALS AND METHODS2.9.3 Isola
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
46 MATERIALS AND METHODSPCR product
Page 75 and 76:
48 MATERIALS AND METHODS45 bpUKP-Ge
Page 77 and 78:
50 MATERIALS AND METHODS2.12.2 Tran
Page 79 and 80:
52 MATERIALS AND METHODSTable 2.8:
Page 81 and 82:
54 MATERIALS AND METHODSGeneral set
Page 83 and 84:
56 MATERIALS AND METHODSSD pooled =
Page 85 and 86:
58 OPTIMISED FERMENTATION IN GRAPE
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 110 and 111:
834 Identification of yeast genesin
Page 112 and 113:
4.2 Candidate genes for volatile th
Page 114 and 115:
Page 116 and 117:
Page 118 and 119:
4.3 Screening of single-gene deleti
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
4.4 Deletion of candidate genes in
Page 132 and 133:
4.5 Volatile thiol release in yeast
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
4.6 Supplementation of grape juice
Page 140 and 141:
Page 142 and 143:
Page 144 and 145:
4.7 Discussion and conclusion 1174.
Page 146 and 147:
4.7 Discussion and conclusion 1194.
Page 148 and 149:
4.7 Discussion and conclusion 121Th
Page 150 and 151:
4.7 Discussion and conclusion 123Ta
Page 152 and 153:
4.7 Discussion and conclusion 125to
Page 154 and 155:
4.7 Discussion and conclusion 127ca
Page 156 and 157:
4.7 Discussion and conclusion 129me
Page 158 and 159:
4.7 Discussion and conclusion 131an
Page 160:
4.7 Discussion and conclusion 133ce
Page 163 and 164: 136 GENETIC MAPPING APPROACHES TO A
Page 194 and 195: 1676 Concluding DiscussionMy resear
Page 196 and 197: 6.1 Laboratory yeast can be used in
Page 198 and 199: 6.2 Sulfur metabolism plays an impo
Page 200 and 201: 6.4 Volatile thiol synthesis of 3MH
Page 202 and 203: 6.5 S-ethyl-L-cysteine as a model c
Page 204: 6.6 Concluding thoughts 177from dif
Page 207 and 208: 180 APPENDICEScompounds. However, w
Page 209 and 210: 182 APPENDICESA.3 Variation of vola
Page 211: 2287 bp1765 bp2103 bp2286 bp184 APP
Page 215 and 216: 188 APPENDICESAUKP-GeneX-F/R3236 bp
Page 217 and 218: 190 APPENDICESTable A.4: S-ethyl-L-
Page 219 and 220: 192 APPENDICESTable A.5: SEC phenot
Page 221 and 222: 194 APPENDICESA.9 S-ethyl-L-cystein
Page 223 and 224: 196 APPENDICESA.10 Deleting DUG1 in
Page 225 and 226: 198 APPENDICESA.11 Differentiation
Page 227 and 228: 200 APPENDICESA.12 Overexpression o
Page 229 and 230: 202 APPENDICESA.12.2 Transformation
Page 231 and 232: 204 APPENDICESCumulative weight los
Page 234 and 235: 207ReferencesAllen, M. S. and Lacey
Page 236 and 237: 209Ciani, M., Beco, L., and Comitin
Page 238 and 239: 211cerevisiae.” Mol. Cell. Biol.,
Page 240 and 241: 213Kumar, C., Sharma, R., and Bachh
Page 242 and 243: 215Nakagawa, S. and Cuthill, I. C.
Page 244 and 245: 217Schlenk, F. and Zydek, C. R. (19
Page 246 and 247: 219thesis, University of Bordeaux 2
Page 248 and 249: 221expression of some stress induce
show all

Identification of yeast genes involved in Sauvignon Blanc aroma ...

Create successful ePaper yourself

Delete template?

Save as template?