拟南芥实验手册 英文PDF电子书下载

CHAPTER 1 How to Grow Arabidopsis, 1

Morphology and Anatomy 1

Seed Collection and Storage 7

Cultivation of Arabidopsis 7

General Considerations 7

Growth Environment 8

Cultivation of Plants in Soil 10

Use of Solid Media 12

Other Growth Conditions 13

Pest Management 14

Supplier's Web Sites 17

References 18

CHAPTER 2 Obtaining Mutants, 19

Obtaining Previously Identified Mutants from the Stock Centers 19

Forward Genetics:Finding Mutations That Cause Particular Phenotypes 20

EMS Mutagenesis of Seed 24

T-DNA Mutagenesis 26

Special T-DNA Vectors 26

Reverse Genetics:Finding Mutations in Particular Genes 30

Screening DNA Pools for T-DNA Insertions 31

Considerations for Phenotypic Characterization 34

Beyond Mutants:Using Natural Variation to Identify Interesting Genes 35

References 37

CHAPTER 3 Genetic Analysis of Mutants, 41

Setting up Ctosses 41

Choosing Parent Plants 41

Choosing Flowers 41

Segregation Analysis 43

Self-progeny 43

F1 Progeny 44

F2 Progeny 44

Backcrosses and Cosegregation 46

Complementation Testing 48

Strategies for Identifying Double Mutants 50

Finding Double Mutants in the F2 Generation 50

Finding Double Mutants in the F3 Generation 51

The Brute Force Approach 52

References 53

CHAPTER 4 How to Analyze a Mutant Phenotypically, 55

Growth Parameters 56

Quantitative Analysis of Root Growth 56

Hypocotyl Length 59

Flowering Time 60

Germination Rate 62

Fresh Weight Gain 62

Water Loss 62

Hormone Response 63

Gibberellin/Abscisic Acid/Paclobutrazol 63

Auxin 64

Ethylene 66

Brassinosteroids 69

Response to the Abiotic Environment 71

Root Elongation Under Salt/Hormone-induced Stress 71

Germination Rate 72

Electrolyte Leakage 73

Fresh Weight Gain 74

Water Loss 75

Others:Proline and Sugar Content 75

Heavy Metal Stress 75

Bacterial Pathogens 77

Preparation of Bacterial Cultures and Inoculation of Plants 77

Testing the Hypersensitive Response 78

Assessing Bacterial Growth 79

Oomycete Pathogens:Peronospora parasitica 81

Reviving Frozen Stocks and Inoculating Plants 81

Rating Peronospora Infections 83

Preparation of Frozen Tissue Sections 83

Maintaining Infections 84

Diaminobenzidine Stain for Hydrogen Peroxide 85

Trypan Blue Stain for Fungi,Oomycetes,and Dead Plant Cells 86

Histology 87

Preparation of Tissue Sections of Fixed Material 87

Specialized Staining Techniques 93

The PAS Reaction for Staining Cell Walls 93

Alcian Blue-PAS Reaction 95

Phloroglucinol Stain for Lignin 96

Vital Stain for Cytoplasm 97

Neutral Red Staining for Vacuoles 98

Whole-mount DAPI Staining and Measurement of DNA Content 98

Nuclear Staining for Confocal Microscopy 100

Cleared Tissue for Observation of Vascular Strands 104

Agarose Imprints of Surfaces 105

Scanning Electron Microscopy 106

Standard SEM Protocol 106

Dental Wax Impressions to be Viewed in SEM 109

SEM"Quick-and-Easy"Fixation 109

Imaging of Fresh Arabidopsis Tissues in the SEM 110

Transmission Electron Microscopy 112

Standard TEM Protocol 112

TEM Freeze Substitution 114

References 115

CHAPTER 5 How to Transform Arabidopsis, 119

Vectors and Agrobacterium Hosts 119

Agrobacterium strains 122

Transformation of Agrobacterium 122

Transformation of Agrobacterium Using Electroporation 123

Transformation of Agrobacterium Using the Freeze-Thaw Method 125

PCR Analysis of Agrobacterium 127

In Planta Transformation of Arabidopsis 128

Plant Growth 128

Floral Dip of Arabidopsis 129

Alternative Protocol 1:Vacuum Infiltration 130

Alternative Protocol 2:Spraying 131

Selecting Transformed Plants 131

Kanamycin Selection 131

Basta Selection on Soil 133

Root Transformation 134

References 140

CHAPTER 6 How to Isolate a Gene Defined by a Mutation, 143

Isolating a Gene Defined by an Insertion Mutation 143

TAIL-PCR 144

Isolating a Gene Known Only by the Phenotypes of Mutant Alleles:Positional Cloning 155

STEP 1:Determining the Approximate Map Position for yfg 155

STEP 2:Define the Map of yfg as Narrowly as Possible 161

STEP 3:Finding YFG within the Mapped Interval 162

Special Cases 162

Serious Errors 164

Purifying DNA from Arabidopsis 165

CTAB DNA Miniprep 165

Dellaporta DNA Miniprep 166

Quick DNA Prep for PCR 168

References 169

CHAPTER 7 How to Study Gene Expression, 171

RNA Expression 171

RNA Extraction for Northern Blots and RT-PCR 172

Semiquantitative Reverse Transcription followed by PCR 173

In Situ Hybridization to Tissue Sections 181

Radioactive In Situ Hybridization 182

Nonradioactive In Situ Hybridization 195

Whole-mount In Situ Hybridization 212

Protein Expression 215

Extraction of Total Protein for Western Blots 215

Organelle Preparations 216

FPLC Gel Filtration 226

Nondenaturing Gel Electrophoresis of Proteins 228

Protein Coimmunoprecipitation 233

In Situ Localization of Proteins 237

Reporter Genes 241

Whole-mount GUS Staining 243

Quantitative GUS Activity Assays 249

Subcellular Localization of GUS-and GFP-Tagged Proteins in Onion Epidermal Cells 252

Live-cell Imaging of GFP 262

Liquid LUC Activity Assays 267

LUC Imaging of Whole Plants 269

References 276

CHAPTER 8 How to Study Gene Function, 281

Reducing Gene Expression 282

Antisense RNA 282

Cosuppression/RNAi 284

Tissue-or Stage-specific Gene Knock-outs 286

Misexpression 287

Constitutive/Tissue-specific Promoter Fusions 287

Two-component Systems for Tissue-specific Misexpression 287

The Alc Gene Ethanol-inducible Switch system 291

Glucocorticoid Inducible Control of Gene Expression 293

Heat Shock Induction 295

Glucocorticoid Fusions for Transcription Factors 296

Transient Expression 300

Transient Expression in Protoplasts 300

Transgene Expression in Regenerated Roots 304

Other Gain-of-Function Strategies 308

Mosaic Analysis 309

References 313

APPENDIX 1 Where to Find Information on Arabidopsis 317

APPENDIX 2 Critical x2 Values 321

APPENDIX 3 Cautions 323

APPENDIX 4 Suppliers 341

Index, 343