Plant Gene Transfer and Expression Protocols

Methods in Molecular Biology John M. Walker, SERIES EDITOR

35. Plant Cell Electroporation and Electrofusion Protocols, eihei by Jac A. Nickoloff. 1995

54. YAC Protocols, edited by David Markie, 1995 53. Yeast Protocols: Methods in Cell and Molecular Biology,

edited by Ivor H. Evans, 1996 52. Capillary Electrophoresis: Principles, Instrumentation,

and Applications, edited by Kevin D. Altria, 1996 51. Antibody Engineering Protocols, edited by Sudhir Paul,

1995-50. Species Diagnostics Protocols: PCR and Other Nucleic

Acid Methods, edited by Justin P. Clapp, 1996 49. Plant Gene Transfer and Expression Protocols,

edited by Heddwyn Jones, 1995 48. Animal Cell Electroporation and Electrofusion Proto­

cols, edited by Jac A. Nickoloff, 1995 47. Electroporation Protocols for Microorganisms, edited by

Jac A. Nickoloff 1995 46. Diagnostic Bacteriology Protocols, edited by Jenny

Howard and David M. Whitcombe, 1995

45. Monoclonal Antibody Protocols, edited by William C.

Davis, 1995

44. Agrobttcterium Protocols, edited by Kevan M. A. Gartland

and Michael R. Davey, 1995

43. In Vitro Toiicity Testing Protocols, edited by Sheila

O'Hare and Chris K. Atterwill, 1995

42. ELISA: Theory and Practice, by John R. Crowther, 1995

4 i. Signal Transduction Protocols, edited by David A. Kendall

and Stephen J. Hill, 1995

40. Protein Stability and Folding: Theory and Practice,

edited by Bret A. Shirley, 1995

39. Baculovirus Expression Protocols, edited by Christopher

D. Richardson, 1995

3$. Cryopreservation and Freeze-Drying Protocols, edited by John G. Day and Mark R. McLellan, 1995

37. In Vitro Transcription and Translation Protocols, edited by Martin J. Tymms, 1995

36. Peptide Analysis Protocols, edited by Ben M. Dunn and Michael W. Pennington, 1994

35. Peptide Synthesis Protocols, edited by Michael W. Pennington and Ben M. Dunn, 1994

34. Immunocytochemical Methods and Protocols, edited by Lorette C. Javois, 1994

33. In Situ Hybridization Protocols, edited by K. H. Andy

Choo. 1994

32. Basic Protein and Peptide Protocols, edited by John M.

Walker, 1994

31. Protocols for Gene Analysis, edited by Adrian J. Harwood,

1994

30. DNA-Protein Interactions, edited by G. Geoff Kneale, 1994

29. Chromosome Analysis Protocols, edited by John R. Gosden, 1994

28. Protocols for Nucleic Acid Analysis by Nonradioactive Probes, edited by Peter G. Isaac, 1994

27. Biomembrane Protocols: //. Architecture and Function, edited by John M. Graham and Joan A. Higgins, 1994

26. Protocols for Oligonucleotide Conjugates: Synthesis and Analytical Techniques, edited by Sudhir Agrawal, 1994

25. Computer Analysis of Sequence Data: Part II. edited by Annette M. Griffin and Hugh G. Griffin, 1994

24. Computer Analysis of Sequence Data; Part I, edited by Annette M. Griffin and Hugh G. Griffin. 1994

23. DNA Sequencing Protocols, edited by Hugh G. Griffin and Annette M. Griffin, 1993

22. Microscopy, Optical Spectroscopy, and Macroscopic Techniques, edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, 1993

21. Protocols in Molecular Parasitology, edited by John E. Hyde, 1993

20. Protocols for Oligonucleotides and Analogs: Synthesis and Properties, eiilti by Sudhir Agrawal, 1993

19. Biomembrane Protocols: /. Isolation and Analysis, eiileibyJohn M. Graham and Joan A. Higgins, 1993

18. Transgcncsis Techniques: Principles and Protocols, eiitei by David Murphy and David A. Carter, 1993

17. Spectroscopic Methods and Analyses: NMR, Mass Spec­trometry, and Metalloprotein Techniques, edited by Chris­topher Jones, Barbara Mulloy, and Adrian H. Thomas, 1993

16. Enzymes of Molecular Biology, edited by Michael M. Burrell, 1993

15. PCR Protocols: Current Methods and Applications, edited by Bruce A. White, 1993

14. Glycoprotein Analysis in Biomedicine, edited by Eliza­beth F. Hounsell, 1993

13. Protocols in Molecular Neurobiology, edited by Alan Longstaffand Patricia Revest, 1992

12. Pulsed-Field Gel Electrophoresis; Protocols, Methods, and Theories, edited by Margit Burmeister and Levy Ulanovsky, 1992

II. Practical Protein Chromatography, edited by Andrew Kenney and Susan Fowell, 1992

10. Immunochemical Protocols, edited by Margaret M. Manson, 1992

9. Protocols in Human Molecular Genetics, edited by Chris­topher G. Mathew, 1991

8. Practical Molecular Virology: Viral Vectors for Gene Expression, edited by Mary K. L Collins, 1991

1. Gene Transfer and Expression Protocols, edited by Edward J. Murray, 1991

6. Plant Cell and Tissue Culture, edited by Jeffrey W. Pollard and John M. Walker, 1990

5. Animal Cell Culture, edited by Jeffrey W. Pollard and John M. Walker, 1990

Methods in Molecular Biology™ • 49

Plant Gene Transfer

and Expression Protocols

Edited by

Heddwyn Jones University of Hertfordshire, Hatfield, UK

H u m a n a P r e s s ^ 1 ^ Totowa, New Jersey

© 1995 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

All rights reserved.

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in molecular biology™ is a trademark of the Humana Press Inc.

All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher.

This publication is printed on acid-free paper. <S> ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials.

Cover illustration: Fig. 2A from Chapter 25, "In Situ Hybridization to Plant Tissue Sections," by Shirley R. Burgess.

Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Himiana Press Inc., provided that the base fee of US $4.00 per copy, plus US $00.20 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Trans­actional Reporting Service is: [0-89603-32l-X/95 $4.00 + $00.20].

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Library of Congress Cataloging in Publication Data

Main entry under title: Methods in molecular biology™.

Plant gene transfer and expression protocols / edited by Heddwyn Jones, p. cm. — (Methods in molecular biology™ 49)

Includes index. ISBN 0-89603-321-X (alk. paper) 1. Plant genetic engineering—^Laboratory manuals. 2. Crops—

Genetic engineering—^Laboratory manuals. 3. Plant gene expression— Laboratory manuals. I. Jones, Heddwyn. II. Series: Methods in molecular biology™ (Totowa, NJ); 49. QK981.5.P567 1995 581.87'322'0724—dc20 95-23429

CIP

Preface

The development of recombinant DNA technology and methods for transferring recombinant genes into plants has brought about significant advances in plant science. First, it has allowed investigation, using reporter genes, into the transcriptional regulation of plant genes—a key to the under­standing of the biochemical basis of growth and development in plants. Second, gene transfer technology has facilitated the molecular cloning, by tagging genomic sequences, of important genes (e.g., homeotic genes) whose gene products control the normal pattern of growth and differentia­tion of plants. Third, overproducing foreign or endogenous proteins in plants can often lead to a better understanding of biochemical and physiological processes. Fourth, gene transfer technology has allowed the improvement of plant agricultural productivity. For example, plants have been engineered with improved viral resistance or the ability to withstand herbicide attack, therefore allowing a more effective use of herbicides to kill weeds. Fifth, there have been recent successes that demonstrate the potential use of plants as biotechnological chemical factories. For example, it is possible to use plants in the production of human antibodies and antigens of medical importance. It has been demonstrated recently that plants can be engineered to produce modified oils and even plastics! This paves the way to redirect agriculture from the production of surplus foods to the production of bio­technological products of industrial importance.

Plant Gene Transfer and Expression Protocols presents a range of protocols for introducing genes into plants, and the analysis of their expression at the mRNA and protein level. The book does not attempt to cover basic nucleic acid manipulation; there are already some excellent books in this area. Moreover, basic DNA technology over the last 10 years or so has been increasingly commercialized and made more amenable to researchers by several biotechnology companies; for example it is now possible to buy a ready-made genomic or cDNA library!

Plant Gene Transfer and Expression Protocols is divided into eight sections, which roughly reflect the flow of biochemical information from DNA to proteins. The first section describes the various expression and reporter gene cassettes that are currently used for studying gene regulation

vi Preface

and for the production of recombinant proteins in plants. This is followed by methods for introducing genes into plants using Agrobacterium and, in the second section, direct gene transfer methods. The first section includes a chapter that deals with cloning plant genes by T-DNA tagging of Arabidopsis thaliana.

The third section of the book deal specifically with the use of reporter genes (e.g., gus), and the fourth section deals with determining gene orga­nization and copy number (including the analysis of gene families) by South-em blotting and inverse PCR technology. The fifth and largest section of the book provides protocols for isolating RNA and for the analysis of gene expression at the RNA level using such methods as Northern analysis, RNase protection, RT-PCR, and in situ hybridization analysis. Also included in this section are chapters that deal with Xenopus oocytes and yeast cells as heterologous hosts for the expression of plant mRNA and cDNA. These systems have recently facilitated the molecular cloning and characteriza­tion of various membrane transporters.

The sixth and seventh sections of the book are devoted to methods for studying and manipulating chloroplast and mitochondria, respectively. The final section provides protocols for analyzing recombinant proteins in plants. In this section, protocols are provided for extracting and separating plant proteins by various electrophoretic systems, and for their analysis by Western blotting and ELISA. There is also a chapter in this section on the detection of proteins at the cellular level using immunocytochemical techniques.

Although the emphasis throughout Plant Gene Transfer and Expres­sion Protocols is on the analysis of transgenes, the sections on gene orga­nization, RNA, organelles, and protein methodology should also be of value to any researcher who wishes to study an endogenous gene without neces­sarily resorting to gene transfer technology. Following the previous books of the Methods in Molecular Biology™ series, emphasis has been placed on generally applicable protocols, with each chapter ending with a Notes sec­tion highlighting potential problems and providing tips and alternatives. The protocols are detailed, and with the help of the Notes section, should allow the efficient transfer of these techniques to any laboratory, whether engaged in fundamental or applied aspects of plant science.

I would like to thank the series editor, John Walker, for his help and patience during the preparation of the book and also thank the contributing authors for the excellent material they have provided for this volume.

Heddwyn Jones

Contents

Preface v

Contributors xi

PART I. AGROBACTERIUM-MEDIATED TRANSFORMATION

CH. 1. Tools for Expressing Foreign Genes in Plants, Francois Guerineau 1

CH. 2. Introduction of Cloning Plasmids into Agrobacterium tumefaciens, Charles H. Shaw 33

CH. 3. Leaf Disk Tiansformation Using Agrobacterium tumefaciens-Expression of Heterologous Genes in Tobacco,

Patrick Gallois and Paulo Marinho 39 CH. 4. Agrobacterium rhizogenes as a Vector for Transforming Higher

Plants: Application in Lotus comiculatus Transformation, Jens Stougaard 49

CH. 5. Agrobacterium-Med-iaicd Transformation of Arabidopsis thaliana: Application in T-DNA Tagging,

Jennifer F. Topping, Wenbin Wei, Michael C. Clarke, PaulMuskett, and Keith Lindsey 63

CH. 6. y4groftacrenMffj-Mediated Transfer ofGeminiviruses to Plant Tissues, Margaret I. Boulton 77

PART II. DIRECT GENE TRANSFER

CH. 7. Stable Transformation of Barley via Direct DNA Uptake: Electroporation- and PEG-Mediated Protoplast Transformation,

Paul A. Lazzeri 95 CH. 8. Gene Transfer into Plant Protoplasts by Electroporation,

Heddwyn Jones 107 CH. 9. Transformation of Cereals by Microprojectile Bombardment

of Immature Inflorescence and Scutellum Tissues, Pilar Barcelo and Paul A. Lazzeri 113

PART III. USE OF REPORTER GENES

CH. 10. The P-Glucuronidase (gus) Reporter Gene System: Gene Fusions; Spectrophotometric, Fluorometric, and Histochemical Detection,

Gillian A. Hull and Martine Devic 125

vii

via Contents

CH. 11. Chloramphenicol Acetyl Transferase Assay, Michael R. Davey, Nigel W. Blackhall,

and J. Brian Power 143 CH. 12. NPTII Assays for Measuring Gene Expression and Enzyme Activity

in Transgenic Plants, Ian S. Curtis, J. Brian Power, and Michael R. Davey 149

PART IV. STUDY OF GENE ORGANIZATION BY SOUTHERN BLOTTING AND INVERSE P C R

CH. 13. Gene Characterization by Southern Analysis, Paolo A. Sabelli and Peter R. Shewry 161

CH. 14. Isolation and Characterization of Plant Genomic DNA Sequences via (Inverse) PCR Amplification,

Remko Offringa and Frederique van der Lee 181

PART V. RNA TECHNIQUES FOR STUDYING GENE EXPRESSION

CH. 15. Isolation ofWhole Cell (Total) RNA, Rod J. Scott 197

CH. 16. Poly(A)* RNA Isolation, Rod J. Scott 203

CH. 17. In Vitro Translation, Rod J. Scott 207

CH. 18. Northern Analysis and Nucleic Acid Probes, Paolo A. Sabelli and Peter R. Shewry 213

CH. 19. Nuclear "Run-On" Transcription Assays, John Gatehouse and Andrew J. Thompson 229

CH. 20. RNase AJT^ Protection Assay, Craig G. Simpson and John W. S. Brown 239

CH. 21. Primer Extension Assay, Craig G. Simpson and John W. S. Brown 249

CH. 22. Applications of RT-PCR, Craig G. Simpson and John W. S. Brown 257

CH. 23. In Vitro Transcription of Class II Promoters in Higher Plants, Richard Cooke and Paul Penon 271

CH. 24. Analysis of Plant Gene Expression by Reverse Transcription-PCR, Mark Bell 291

CH. 25. In Situ Hybridization to Plant Tissue Sections, Shirley R. Burgess 301

CH. 26. Xenopus Oocytes as a Heterologous Expression System, FredericaL, Theodoulou and Anthony J. Miller 317

CH. 27. Heterologous Expression in Yeast, Laurence J. Trueman 341

Contents ix

PART VI. TECHNIQUES FOR STUDYING CHLOROPLAST GENE EXPRESSION

CH. 28. The Isolation of Intact Chloroplasts, Johnathan A. Napier and Simon A. Barnes 355

CH. 29. In Vitro Protein Import by Isolated Chloroplasts, Johnathan A. Napier 361

CH. 30. Targeting of Foreign Proteins to the Chloroplast, Johnathan A. Napier S69

PART VII. TECHNIQUES FOR STUDYING MITOCHONDRIAL GENE EXPRESSION

CH . 31. Isolation of Mitochondria, Stefan Binder andLutz Grohmann 377

CH. 32. Mitochondrial Nucleic Acid Purification and Analysis, Stefan Binder 383

CH. 33. In organella Protein Synthesis, Lutz Grohmann 391

PART VIII. IMMUNOLOGICAL DETECTION OF PROTEINS

CH. 34. Separation of Plant Proteins by Electrophoresis, Peter R. Shewry, Arthur S. Tatham, and Roger J. Fido 399

CH. 35. Western Blotting Analysis, Roger J. Fido, Arthur S. Tatham, and Peter R. Shewry 423

CH. 36. ELISA Detection of Foreign Proteins, E. N. Clare Mills, Geoffrey W. Plumb,

and Michael R. A. Morgan 439 CH. 37. Immunocytochemical Localization of Proteins,

John Davies 453

Index 463

Contributors

PILAR BARCELO • Biochemistry and Physiology Department, Institute of Arable Crops Research, Harpenden, UK

SIMON A. BARNES • Department of Plant Sciences, University of Cambridge, UK; Current address: Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY

MARK BELL • Department of Agricultural Sciences, University of Bristol, UK STEFAN BINDER • Institutfiir Genbiologische Forschung, Berlin, Germany NIGEL W . BLACKHALL • Department of Life Science, University

of Nottingham, UK MARGARET I. BOULTON • Department of Virus Research, Norwich

Research Park, Colney, Norwich, UK JOHN W . S. BROWN • Cell and Molecular Genetics Department, Scottish

Crop Research Institute, Dundee, Scotland SHIRLEY R. BURGESS • Department of Agricultural Sciences, University

of Bristol, UK MICHAEL C. CLARKE • Department of Botany, University of Leicester, UK RICHARD COOKE • Laboratoire de Physiologic et Biologic Moleculaire

Vegetale, Universite de Perpignan, France IAN S. CURTIS • Department of Life Science, University of Nottingham, UK MICHAEL R. DAVEY • Department of Life Science, University

of Nottingham, UK JOHN DA VIES • Department of Molecular Pathology, Glaxo Research

and Development Ltd., Greenford, UK MARTINE DEVIC • Laboratoire de Physiologie et Biologic Moleculaire,

Universite de Perpignan, France ROGER J. FIDO • Department of Agricultural Sciences, University

of Bristol, UK PATRICK GALLOIS • Laboratoire de Physiologie et Biologic Moleculaire

Vegetale, Universite de Perpignan, France JOHN GATEHOUSE • Department of Biological Sciences, University

of Durham, UK LuTZ GROHMANN • Institutfiir Genbiologische Forschung, Berlin, Germany

xi

xii Contributors

FRANCOIS GUERINEAU • Department of Botany, University of Leicester, UK GILLIAN A. HULL • Laboratoire de Physiologie et Biologie Moleculaire,

Universite de Perpignan, France HEDDWYN JONES • Department ofBiosciences, University of Hertfordshire,

Hatfield, UK PAUL A. LAZZERI • Biochemistry and Physiology Department,

AFRC Institute of Arable Crops Research, Harpenden, UK KEITH LINDSEY • Department of Botany, University of Leicester, UK PAULO MARINHO • Laboratoire de Physiologie et Biologie Moleculaire

Vegetale, Universite de Perpignan, France ANTHONY J. MILLER • Biochemistry and Physiology Department,

AFRC Institute of Arable Crops Research, Harpenden, UK E. N. CLARE MILLS • Department of Food Molecular Biochemistry,

AFRC Institute of Food Research, Norwich, UK MICHAEL R. A. MORGAN • Department of Food Molecular Biochemistry,

AFRC Institute of Food Research, Norwich, UK PAUL MUSKETT • Department of Botany, University of Leicester, UK JOHNATHAN A. NAPIER • Department of Agricultural Sciences, University

of Bristol, UK REMKO OFFRINGA • Institute of Molecular Plant Sciences, Leiden

University, Leiden, The Netherlands PAUL PENON • Laboratoire de Physiologie et Biologie Moleculaire

Vegetale, Universite de Perpignan, France GEOFFREY W . PLUMB • Department of Food Molecular Biochemistry,

AFRC Institute of Food Research, Norwich, UK J. BRIAN POWER • Department of Life Science, University of Nottingham, UK PAOLO A. SABELLI • Department of Agricultural Sciences, University

of Bristol, UK ROD J. SCOTT • Department of Botany, University of Leicester, UK CHARLES H . SHAW • Department of Biological Sciences, University

of Durham, UK PETER R. SHEWRY • Department of Agricultural Sciences, University

of Bristol, UK CRAIG G. SIMPSON • Cell and Molecular Genetics Department, Scottish

Crop Research Institute, Dundee, Scotland JENS STOUGAARD • Department of Molecular Biology, University

ofAarhus, Denmark ARTHUR S. TATHAM • Department of Agricultural Sciences, University

of Bristol, UK

Contributors xiii

FREDERICA L. THEODOULOU • Biochemistry and Physiology Department, AFRC Institute of Arable Crops Research, Harpenden, UK

ANDREW J. THOMPSON • Department of Biological Sciences, University of Durham, UK

JENNIFER F . TOPPING • Department of Botany, University of Leicester, UK LAURENCE J. TRUEMAN • lACR—Rothamsted Experimental Station,

Harpenden, UK FREDERIQUE VAN DER LEE • Mogen International nv, Leiden, The Netherlands WENBIN WEI • Department of Botany, University of Leicester, UK;

Current address: Centre for Plant Biochemistry and Biotechnology, University of Leeds, UK