Plant Gene Transfer and Expression Protocols

ISBN-13: 9781489940599 / Angielski / Miękka / 2013 / 466 str.

Heddwyn Jones

Opis

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 under- standing 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 differentia- tion 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 bio- technological products of industrial importance.

Recenzje

The chapters were written by outstanding scientists and [the book] can be recommended to any laboratory working with plants, providing tips and alternatives in case of problems. -Journal of Plant Physiology

Spis treści

Part I: Agrobacterium-Mediated Transformation. Tools for Expressing Foreign Genes in Plants. Introduction of Cloning Plasmids into Agrobacterium tumefaciens. Leaf Disk Transformation Using Agrobacterium tumefaciens-Expression of Heterologous Genes in Tobacco. Agrobacterium rhizogenes as a Vector for Transforming Higher Plants: Application in Lotus corniculatus Transformation. Agrobacterium-Mediated Transformation of Arabidopsis thaliana: Application in T-DNA Tagging. Agrobacterium-Mediated Transfer of Geminiviruses to Plant Tissues. Part II: Direct Gene Transfer. Stable Transformation of Barley via Direct DNA Uptake: Electroporation- and PEG-Mediated Protoplast Transformation. Gene Transfer into Plant Protoplasts by Electroporation. Transformation of Cereals by Microprojectile Bombardment of Immature Inflorescence and Scutellum Tissues. Part III: Use of Reporter Genes. The b-Glucoronidase (gus) Reporter Gene System: Gene Fusions, Spectrophotometric, Fluorometric, and Histochemical Detection. Chloramphenicol Acetyl Transferase Assay. NPTII Assays for Measuring Gene Expression and Enzyme Activity in Transgenic Plants. Part IV: Study of Gene Organization by Southern Blotting and Inverse PCR. Gene Characterization by Southern Analysis. Isolation and Characterization of Plant Genomic DNA Sequences via (Inverse) PCR Amplification. Part V: RNA Techniques for Studying Gene Expression. Isolation of Whole Cell (Total) RNA. Poly(A)+ RNA Isolation. In Vitro Translation. Northern Analysis and Nucleic Acid Probes. Nuclear 'Run-On' Transcription Assays. RNase A/T1 Protection Assay. Primer Extension Assay. Applications of RT-PCR. In Vitro Transcription of Class II Promoters in Higher Plants. Analysis of Plant Gene Expression by Reverse Transcription-PCR. In Situ Hybridization to Plant Tissue Sections. Xenopus Oocytes as a Heterologous Expression System. Heterologous Expression in Yeast. Part VI: Techniques for Studying Chloroplast Gene Expression. The Isolation of Intact Chloroplasts. In Vitro Protein Import by Isolated Chloroplasts. Targeting of Foreign Proteins to the Chloroplast. Part VII: Techniques for Studying Mitochondrial Gene Expression. Isolation of Mitochondria. Mitochondrial Nucleic Acid Purification and Analysis. In organello Protein Synthesis. Part VIII: Immunological Detection of Proteins. Separation of Plant Proteins by Electrophoresis. Western Blotting Analysis. ELISA Detection of Foreign Proteins. Immunocytochemical Localization of Proteins. Index.