Weissbach Methods for Plant Molecular Biology

1. Auflage 2013
ISBN: 978-1-4832-6972-6
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 564 Seiten, Web PDF

Methods for Plant Molecular Biology is a collection of articles that focuses on the techniques used in plant molecular biology and genetics. The book discusses the isolation and characterization of nuclear, chloroplast, and mitochondrial nucleic acids and the factors and systems involved in transcription and gene expression. Procedures for the isolation of cell walls, chloroplast membranes, membrane proteins; techniques to carry out plant cell culture and protoplast formation; and methods for gene and organelle transfer are covered as well. Biologists, molecular biologists, botanists, and students will find the book very useful.

Autoren/Hrsg.

Weissbach, Arthur

Weissbach, Herbert

Inhaltsverzeichnis

1;Front Cover;1
2;Methods for Plant Molecular Biology;4
3;Copyright Page;5
4;Table of Contents;6
5;List of Contributors;10
6;Preface;14
7;Contents of Methods in Enzymology Volume 118;16
8;Section I: Cell Wall and Membrane;22
8.1;Chapter 1. Isolation and Characterization of Plant Cell Walls and Cell Wall Components;24
8.1.1;Sources of Cell Walls;25
8.1.2;Extraction and Purification of Cell Wall Components;29
8.1.3;Methods for Analysis of Cell Wall Polysaccharides;46
8.1.4;Acknowledgments;61
8.2;Chapter 2. Isolation of the Plasma Membrane;62
8.2.1;Purification Procedures;63
8.2.2;Membrane Markers;67
8.2.3;Critique of the Sucrose Gradient and Phase Separation Procedures;68
8.2.4;Acknowledgments;75
9;Section II: Nucleus;76
9.1;Chapter 3. Purification and Restriction Endonuclease Analysis of Plant Nuclear DNA;78
9.1.1;Isolation of DNA from Purified Nuclei;78
9.1.2;Restriction and Hybridization Analysis;86
9.1.3;Acknowledgments;96
9.2;Chapter 4. Analyzing Genome Variation in Plants;98
9.2.1;Measurement of Quantitative Variation in Repeated Sequences;98
9.2.2;Using Restriction Site Polymorphisms as Genetic Markers;105
9.2.3;Acknowledgment;108
10;Section III: Cytoplasmic Protein Synthesis;110
10.1;Chapter 5. The Synthesis of High-Molecular-Weight Proteins in the Wheat Germ Translation System;112
10.1.1;Preparation of the Wheat Germ Extract;114
10.1.2;Discussion of Experimental Conditions;119
10.1.3;Conclusions;122
10.1.4;Acknowledgments;122
11;Section IV: The Chloroplast;124
11.1;Chapter 6. Isolation and Structural Analysis of Chloroplast DNA;126
11.1.1;Analysis of Chloroplast Genome Structure by Restriction Site Mapping;133
11.1.2;Acknowledgments;145
11.2;Chapter 7. In Vitro Transcription of Chloroplast Protein Genes;146
11.2.1;Principle of the Method;147
11.2.2;Isolation of Transcriptionally Active Extracts;148
11.2.3;Analysis of In Vitro Transcription Products;153
11.2.4;Acknowledgments;167
11.3;Chapter 8. Coupled Transcription–Translation in Chloroplast
Lysates;168
11.3.1;Introduction;168
11.3.2;Methods;169
11.3.3;Some Applications of the Coupled in Vitro Transcription–Translation
System;176
11.3.4;Acknowledgments;179
11.4;Chapter 9. Translation by Isolated Pea Chloroplasts;180
11.4.1;Preparation of Chloroplasts;181
11.4.2;Translation by Isolated Chloroplasts;184
11.4.3;Measurement of Amino Acid Incorporation;190
11.4.4;Analysis of Products;191
11.4.5;Acknowledgments;193
11.5;Chapter 10. Isolation and Characterization of Chloroplast Envelope Membranes;194
11.5.1;General Considerations;195
11.5.2;Preparation of Intact Chloroplasts;197
11.5.3;Preparation of Envelope Membranes;197
11.5.4;Separation of Inner and Outer Envelope Membranes;198
11.5.5;Characterization of Envelope Membranes;201
11.6;Chapter 11. Characterization of the Thylakoid Membrane by Subfractionation Analyses;204
11.6.1;Thylakoid Membrane Subfractionation by Mechanical Disruption and Phase Partition;205
11.6.2;Thylakoid Membrane Subfractionation by Detergent Treatment
and Centrifugation;213
11.6.3;Applicability of the Various Thylakoid Subfractionation Procedures;215
11.6.4;Acknowledgments;216
11.7;Chapter 12. Subunit Structure and Biogenesis of ATP Synthase and Photosystem I Reaction Center;218
11.7.1;Preparation of NaBr-Treated Chloroplasts;219
11.7.2;Purification of the Chloroplast Photosystem I Reaction Center;220
11.7.3;Properties of the Purified Reaction Center;223
11.7.4;Isolation of Individual Subunits from Gels and Prepartions of Antibodies;224
11.7.5;Use of Electrotransfer and Immunodecoration for Quantitative Estimation of Proteins;226
11.7.6;Extraction of Polypeptides from Leaves;230
11.7.7;Following the Relative Amounts of Individual Polypeptides during
Chloroplast Development by Immunodecoration;233
11.8;Chapter 13. Cloning and Expression of Genes for the Small Subunit of Ribulosebisphosphate Carboxylase;236
11.8.1;Introduction;236
11.8.2;Screening of Genomic Clones;239
11.8.3;Rapid Gel Analysis of Positive Clones;244
11.8.4;Expression of Individual SSU Genes;246
11.8.5;Acknowledgments;249
11.9;Chapter 14. Separation and Reassembly of the Subunits of Ribulosebisphosphate Carboxylase;250
11.9.1;Separation of Small Subunits from Large Subunit Octamer;251
11.9.2;Properties of the Isolated Subunits;253
11.9.3;Measurement of RuBisCo Subunits18;253
11.9.4;Reassembly of Functional Holoenzyme;256
11.9.5;Properties of the Reassembly Process;257
11.9.6;Acknowledgment;258
11.10;Chapter 15. The Cloning and Expression in Escherichia coli of RuBP Carboxylase/Oxygenase Large Subunit Genes;260
11.10.1;Introduction;260
11.10.2;Expression of R. rubrum RuBisCo in E. coli;261
11.10.3;Expression of Z. mays RuBisCo LS-Gene in E. coli;270
11.10.4;Acknowledgments;274
12;Section V: Mitochondria;276
12.1;Chapter 16. The Isolation of Mitochondria and Mitochondrial DNA;278
12.1.1;Suspension Culture Mitochondria and mtDNA
Isolation;280
12.1.2;mtDNA Isolation from Green Tissues;287
12.1.3;Examination for Plastid DNA Contamination of mtDNA Preparations;290
12.1.4;Suggestions for Alternative Procedures;292
12.1.5;Acknowledgments;294
12.2;Chapter 17. Analysis of the Genome Structure of Plant Mitochondria;296
12.2.1;Materials and Methods;298
12.3;Chapter 18. Strategies for the Identification and Analysis of Higher Plant Mitochondrial Genes;314
12.3.1;Preparation of Material;315
12.3.2;Identification of Gene-Containing Restriction Fragments in Plant mtDNA;316
12.3.3;Cloning and Sequencing Strategies;320
12.3.4;Identification of Protein-Coding Open Reading Frames in Higher Plant mtDNA Sequences;322
12.3.5;Confirmation of Gene Expression;325
12.3.6;Alternative Approaches for the Identification of Higher Plant
Mitochondrial Genes;326
12.4;Chapter 19. Isolation of Plant Mitochondrial RNA1;330
12.4.1;Principle;331
12.4.2;Plant Material;331
12.4.3;Isolation of Mitochondria;332
12.4.4;Gradient Purification of Mitochondria;332
12.4.5;Lysis of Mitochondria and Isolation of Nucleic Acids;333
12.4.6;Denaturation of RNA;336
12.4.7;Gel Elpctrophoresis;337
12.4.8;Hybridization Conditions;337
13;Section VI: Nitrogen Metabolism;340
13.1;Chapter 20. Isolation and Characterization of Nitrogenase from Klebsiella pneumoniae;342
13.1.1;Methods;343
13.1.2;Acknowledgments;350
13.2;Chapter 21. Tn5 Mapping of Rhizobium Nitrogen Fixation Genes;352
13.2.1;Types of Tn5 Mutagenesis;353
13.2.2;Tn5 Marker Exchange in Rhizobium;355
13.2.3;DNA Mini-Prep Procedures;359
14;Section VII: Cell Culture and Transformation;362
14.1;Chapter 22. Establishment of Calli and Suspension Cultures1;364
14.1.1;Expiant Procedure;365
14.1.2;Laboratory Protocol: Expiant Preparation: Aseptic Germination of Seeds;365
14.1.3;Laboratory Protocol: Medium Preparation;368
14.1.4;Growth Curve;374
14.2;Chapter 23. Protoplasts: Isolation, Culture, Plant Regeneration;376
14.2.1;An Idealized Example;376
14.2.2;Parameters Affecting Protoplast Isolation and Proliferation and Plant Regeneration;378
14.2.3;Protocols for the Preparation and Culture of Protoplasts and Regeneration of Plants;396
14.3;Chapter 24. Fusion and Transformation of Plant Protoplasts;406
14.3.1;Plant Protoplast Fusion;406
14.3.2;Plant Protoplast Transformation;409
14.4;Chapter 25. Organelle Transfer;424
14.4.1;Transfer of Organelles and Organelle-Controlled
Traits;424
14.4.2;Identification of Organelle-Controlled Traits;432
14.4.3;Acknowledgment;440
15;Section VIII: Gene Transfer;442
15.1;Chapter 26. Gene Transfer in Plants: Production of Transformed Plants Using Ti Plasmid Vectors;444
15.1.1;Introduction of Foreign Genes into Modified A. tumefaciens Strains;445
15.1.2;Cocultivation of A. tumefaciens Strains with Plant Tissue;451
15.1.3;Cocultivation with A. tumefaciens Strains;452
15.1.4;Selection and Regeneration of Transformants;452
15.1.5;Analysis and Verification of Gene Expression in Transgenic Plants;454
15.2;Chapter 27. Plant Virus Vectors: Cauliflower Mosaic Virus;458
15.2.1;Methods;458
15.2.2;Comments;466
15.2.3;Acknowledgments;467
15.3;Chapter 28. Direct Gene Transfer to Plants;468
15.3.1;Principle;468
15.3.2;Methods;469
15.3.3;Evaluation of Results;475
15.3.4;General Comments;483
15.3.5;Acknowledgments;484
16;Section IX: Virology;486
16.1;Chapter 29. In Vitro Transcription of Infectious Viral RNA from Cloned cDNA;488
16.1.1;Introduction;488
16.1.2;cDNA Cloning;491
16.1.3;Production and Analysis of Transcripts;495
16.1.4;Plant Infection with in Vitro Viral Transcripts;499
16.1.5;Acknowledgments;500
16.2;Chapter 30. Use of RNA Probes to Detect Plant RNA Viruses;502
16.2.1;Principle of the Method;502
16.2.2;Methods;503
16.2.3;Discussion;506
16.3;Chapter 31. Preparation and Use of cDNA Probes for Detection of Viral Genomes;508
16.3.1;Preparation of cDNA Probes;509
16.3.2;Hybridization Techniques with cDNA Probes;517
16.4;Chapter 32. ELISA Techniques;528
16.4.1;Types of ELISA;529
16.4.2;Sample Preparation;531
16.4.3;Antiserum Production;532
16.4.4;Purification of Immunoglobulins from Antisera;533
16.4.5;Conjugate Preparation;536
16.4.6;Substrates;538
16.4.7;Preparation of Substrates;539
16.4.8;Solid Phases;540
16.4.9;ELISA Procedures;540
16.4.10;Adsorptive Membranes;545
16.4.11;Reagent Evaluation and General Considerations;547
16.4.12;Quantitative Tests;549
17;Appendix: Buffer Formulations;550
18;Subject Index;552