拟南芥实验手册 英文2025|PDF|Epub|mobi|kindle电子书版本百度云盘下载

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CHAPTER 1 How to Grow Arabidopsis,1

Morphology and Anatomy1

Seed Collection and Storage7

Cultivation of Arabidopsis7

General Considerations7

Growth Environment8

Cultivation of Plants in Soil10

Use of Solid Media12

Other Growth Conditions13

Pest Management14

Supplier's Web Sites17

References18

CHAPTER 2 Obtaining Mutants,19

Obtaining Previously Identified Mutants from the Stock Centers19

Forward Genetics:Finding Mutations That Cause Particular Phenotypes20

EMS Mutagenesis of Seed24

T-DNA Mutagenesis26

Special T-DNA Vectors26

Reverse Genetics:Finding Mutations in Particular Genes30

Screening DNA Pools for T-DNA Insertions31

Considerations for Phenotypic Characterization34

Beyond Mutants:Using Natural Variation to Identify Interesting Genes35

References37

CHAPTER 3 Genetic Analysis of Mutants,41

Setting up Ctosses41

Choosing Parent Plants41

Choosing Flowers41

Segregation Analysis43

Self-progeny43

F1 Progeny44

F2 Progeny44

Backcrosses and Cosegregation46

Complementation Testing48

Strategies for Identifying Double Mutants50

Finding Double Mutants in the F2 Generation50

Finding Double Mutants in the F3 Generation51

The Brute Force Approach52

References53

CHAPTER 4 How to Analyze a Mutant Phenotypically,55

Growth Parameters56

Quantitative Analysis of Root Growth56

Hypocotyl Length59

Flowering Time60

Germination Rate62

Fresh Weight Gain62

Water Loss62

Hormone Response63

Gibberellin/Abscisic Acid/Paclobutrazol63

Auxin64

Ethylene66

Brassinosteroids69

Response to the Abiotic Environment71

Root Elongation Under Salt/Hormone-induced Stress71

Germination Rate72

Electrolyte Leakage73

Fresh Weight Gain74

Water Loss75

Others:Proline and Sugar Content75

Heavy Metal Stress75

Bacterial Pathogens77

Preparation of Bacterial Cultures and Inoculation of Plants77

Testing the Hypersensitive Response78

Assessing Bacterial Growth79

Oomycete Pathogens:Peronospora parasitica81

Reviving Frozen Stocks and Inoculating Plants81

Rating Peronospora Infections83

Preparation of Frozen Tissue Sections83

Maintaining Infections84

Diaminobenzidine Stain for Hydrogen Peroxide85

Trypan Blue Stain for Fungi,Oomycetes,and Dead Plant Cells86

Histology87

Preparation of Tissue Sections of Fixed Material87

Specialized Staining Techniques93

The PAS Reaction for Staining Cell Walls93

Alcian Blue-PAS Reaction95

Phloroglucinol Stain for Lignin96

Vital Stain for Cytoplasm97

Neutral Red Staining for Vacuoles98

Whole-mount DAPI Staining and Measurement of DNA Content98

Nuclear Staining for Confocal Microscopy100

Cleared Tissue for Observation of Vascular Strands104

Agarose Imprints of Surfaces105

Scanning Electron Microscopy106

Standard SEM Protocol106

Dental Wax Impressions to be Viewed in SEM109

SEM"Quick-and-Easy"Fixation109

Imaging of Fresh Arabidopsis Tissues in the SEM110

Transmission Electron Microscopy112

Standard TEM Protocol112

TEM Freeze Substitution114

References115

CHAPTER 5 How to Transform Arabidopsis,119

Vectors and Agrobacterium Hosts119

Agrobacterium strains122

Transformation of Agrobacterium122

Transformation of Agrobacterium Using Electroporation123

Transformation of Agrobacterium Using the Freeze-Thaw Method125

PCR Analysis of Agrobacterium127

In Planta Transformation of Arabidopsis128

Plant Growth128

Floral Dip of Arabidopsis129

Alternative Protocol 1:Vacuum Infiltration130

Alternative Protocol 2:Spraying131

Selecting Transformed Plants131

Kanamycin Selection131

Basta Selection on Soil133

Root Transformation134

References140

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

Isolating a Gene Defined by an Insertion Mutation143

TAIL-PCR144

Isolating a Gene Known Only by the Phenotypes of Mutant Alleles:Positional Cloning155

STEP 1:Determining the Approximate Map Position for yfg155

STEP 2:Define the Map of yfg as Narrowly as Possible161

STEP 3:Finding YFG within the Mapped Interval162

Special Cases162

Serious Errors164

Purifying DNA from Arabidopsis165

CTAB DNA Miniprep165

Dellaporta DNA Miniprep166

Quick DNA Prep for PCR168

References169

CHAPTER 7 How to Study Gene Expression,171

RNA Expression171

RNA Extraction for Northern Blots and RT-PCR172

Semiquantitative Reverse Transcription followed by PCR173

In Situ Hybridization to Tissue Sections181

Radioactive In Situ Hybridization182

Nonradioactive In Situ Hybridization195

Whole-mount In Situ Hybridization212

Protein Expression215

Extraction of Total Protein for Western Blots215

Organelle Preparations216

FPLC Gel Filtration226

Nondenaturing Gel Electrophoresis of Proteins228

Protein Coimmunoprecipitation233

In Situ Localization of Proteins237

Reporter Genes241

Whole-mount GUS Staining243

Quantitative GUS Activity Assays249

Subcellular Localization of GUS-and GFP-Tagged Proteins in Onion Epidermal Cells252

Live-cell Imaging of GFP262

Liquid LUC Activity Assays267

LUC Imaging of Whole Plants269

References276

CHAPTER 8 How to Study Gene Function,281

Reducing Gene Expression282

Antisense RNA282

Cosuppression/RNAi284

Tissue-or Stage-specific Gene Knock-outs286

Misexpression287

Constitutive/Tissue-specific Promoter Fusions287

Two-component Systems for Tissue-specific Misexpression287

The Alc Gene Ethanol-inducible Switch system291

Glucocorticoid Inducible Control of Gene Expression293

Heat Shock Induction295

Glucocorticoid Fusions for Transcription Factors296

Transient Expression300

Transient Expression in Protoplasts300

Transgene Expression in Regenerated Roots304

Other Gain-of-Function Strategies308

Mosaic Analysis309

References313

APPENDIX 1 Where to Find Information on Arabidopsis317

APPENDIX 2 Critical x2 Values321

APPENDIX 3 Cautions323

APPENDIX 4 Suppliers341

Index,343