Introduction

Arabidopsis thaliana is a model plant because of its small size, short generation time and small genome.  Its genome (130 Mbp) was sequenced in 2000.  Several WWW databases (USA, Germany and Japan) are maintained to make all of this genomic information available to the plant community.  The present goal of the community is to understand the function of each of the +/-30,000 genes in Arabidopsis.  Traditional genetic approaches have been useful in revealing the function of a subset of these genes; those that when mutated lead to predictable phenotypes.  Such mutants can be analyzed to uncover the mutated gene that can then be cloned and and sequenced.  However, this approach won't work for many genes.  The completed sequence of the genome now allows researchers to identify genes based on sequence comparisons to known genes, and has also revealed many genes whose function is not predictable based on its sequence alone.  Using tools of reverse genetics it is now possible to identify the function of a gene starting from its sequence.

In this semester-long laboratory project you and your lab partners will investigate an Arabidopsis gene using reverse genetics.  Like any research project, we don’t know where the project will take us.  It is likely that some or even most of you will fail to learn the function of your gene.  However, by reading the literature, doing careful experiments and using your head it will likely lead somewhere!  You will be graded on how you carry out the project, not on whether you learn the function of your gene.

T-DNA Knockout Mutants

T-DNA is a fragment of DNA from a bacterial Ti-plasmid (tumor-inducing) that is inserted into a host plant’s genome by Agrobacterium tumefaciens during its pathogenic life cycle.  (Read more at http://helios.bto.ed.ac.uk/bto/microbes/crown.htm)  Encoded within the wild type T-DNA are genes encoding enzymes for auxin and cytokinin synthesis and for opine synthesis.  The auxin and cytokinin produced in transformed cells cause cell growth and division resulting in a tumor in which the bacteria live.  Opines are amino acids that provide the bacteria with a source of carbon and nitrogen that is unavailable to the plant.  A. tumefaciens is a soil bacterium capable of infecting many species of plants.  In the 1980s molecular biologists removed the genes for auxin, cytokinin and opine synthesis from a Ti plasmid, while retaining T-DNA transfer functions.  These plasmids are commonly used to transfer foreign DNA into plants and are the main workhorses of the plant biotechnology industry.

When foreign DNA is transferred into a plant there is a good chance that the T-DNA will land in a functional gene thus making it non-functional or "knocking it out".  It was then recognized that T-DNA could be a useful tool to knockout wild type genes because, unlike single base mutations which are difficult to locate at the molecular level, T-DNA tags are much larger and can be located more easily using molecular techniques.  In the late 1990s several groups including one at the Salk Institute initiated projects to attempt to saturate the Arabidopsis genome with T-DNA insertions and to locate them all on the genome map.  Many thousands of independent Arabidopsis transformants were grown, genomic DNA was isolated, PCR was performed to amplify DNA adjacent to the T-DNA insert, and the PCR products were sequenced.  This sequence information was then used to identify the location in the genome of the T-DNA insert.  This location information is now available to the community through a searchable web site.  Once a putative mutant in one's favorite gene is identified, seeds can be ordered at no charge.

The Project

Phase 1 - Working as a group, identify a Salk knockout mutant in each of two genes in a gene family using the Arabidopsis links page.  Identify all of the genes in your particular gene family and learn all you can using web resources about the structure and location of the proteins they encode.

Phase 2 - Begin reading the primary literature related to your gene.  Write three, 2-page reports to be turned in (individually) for grades in the first 4 weeks of the semester (see syllabus).

Phase 3 - Sow mutant and wild type seeds in week one, then isolate lead DNA and verify the insert using PCR (lab #7).  Sow seeds from a homozygote to begin Phase 4.

Phase 4 - Conduct experiments on the mutant to attempt to understand the function of the wild type gene (lab #8, weeks 9-14).

1/22/03 Copyright (C) 2003, Jonathan Monroe, monroejd@jmu.edu. All rights reserved.
URL: http://csm.jmu.edu/biology/courses/bio455_555/knockout.html