Arabidopsis Molecular Genetics HORT 301 – Plant Physiology October 31, 2007 Reading 1 (Alonso and Ecker (2006) Nature Reviews Genetics Reading 2 (Page and Grossniklaus (2002) Nature Reviews Genetics Function of all plant genes Assumption – majority of genes in plants have conserved function, including those in crops
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October 31, 2007Reading 1 (Alonso and Ecker (2006) Nature Reviews Genetics
Reading 2 (Page and Grossniklaus (2002) Nature Reviews Genetics
Function of all plant genes
Assumption – majority of genes in plants have conserved function, including those in crops
Lecture outline:
Genetic terminology
Arabidopsis thaliana, the current plant genetic model system
Resources for gene function determination
Phenotypic selection or screening of a T-DNA mutant population – forward genetics, identification of the mutated gene
Candidate gene based on predictions of function (phenotype) – reverse genetics
Terminology
Genetics – study of heredity/inheritance and function of genetic material
Gene – individual functional unit of the DNA that includes the promoter and encodes the messenger mRNA that is translated into a protein, i.e. locus
Locus – usually two alleles at each, contributed by two homologous chromosomes in a diploid (2n) organism
Molecular genetics – application of molecular biology to genetics, i.e. gene identification by DNA structure, genetic engineering
Genotype (genes) to phenotype (appearance and function)
Inheritance – single gene dominant and recessive phenotypes
Griffiths AF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM. 1996. An Introduction to Genetic Analysis. 6th ed. W.H. Freeman & Company
Yellow – 416 (3)Green – 130 (1)
Y (dominant) and y (recessive) are alleles
Allele – alternative state of a gene that may be different in each of the two chromosomes of the pair, dominant (Y) or recessive (y) allele
Mutation – change in DNA structure of a gene, alteration in gene function
WT siz1-2 siz1-3
Plant breeders improve crops by transferring “mutations” that confer better phenotypes/traits!!!!
See the maize example
Arabidopsis thaliana (L.) Heynh, plant genetic model – (Brassicaceae, mustard or crucifer family) common name Arabidopsis, rosette-type plant
25.1 (A) The shoot apical meristem in Arabidopsis thaliana; (B) An Arabidopsis plant
Morphological, developmental and molecular genetic attributes of Arabidopsis that make this plant a genetic model:
Small size – numerous plants can be grown in a small area
Short life cycle – six to eight weeks, 6 to 8 generations per year
Produces numerous seed – several thousand seeds per plant
Diploid (2n) – n = 5 (chromosomes)
Self-fertile – amenable to classic genetic manipulation by self or cross pollination
Natural variation – better alleles for certain traits in other accessions or ecotypes
Transformable using Agrobacterium tumefaciens – foreign DNA is easily transferred into the genome (complete set of genes and accompanying DNA)
Genome size is relatively small - ~120 x 106 bp, 26,819 genes encode proteins, 31,762 total including genes that encode miRNA, pseudogenes, and transposable elements, etc. http://www.arabidopsis.org/portals/genAnnotation/genome_snapshot.jsp
Genome sequence information is available, also cDNA sequence, http://signal.salk.edu/cgi-bin/tdnaexpress
Genetic resources for gene function identification - T-DNA insertional tagging mutagenesis - Agrobacterium-mediated transformation
Agrobacterium tumefaciens – crown gall disease, bacterial pathogen of plants that transfers DNA (T-DNA) into the plant genome during the infection process
Tumor cells produce carbon and nitrogen sources for use by the bacteria
Agrobacterium infection and tumor development
“Disarmed” Agrobacterium strains are used for genetic manipulation (engineering) of plants – bacteria are no longer pathogenic but are still capable of T-DNA transfer
T-DNA is inherited as a single dominant gene (locus)
T-DNA binary vector (plasmid) composition, e.g., pSKI015 - right border (RB) and left border (LB)
DNA between the borders is inserted into the plant genome (DNA), selectable marker gene
Selectable marker gene – e.g. herbicide resistance gene to “select” transformed plants
Activation sequence – e.g., 4X 35S that can activate expression of a native gene depending on the insertion position
Primary interest is to alter the function (cause a mutation) of every gene by T-DNA insertional mutatgenesis
It is estimated that ~300,000 random insertions will “saturate the genome”, a mutation in each gene, a population of 300,000 plants
Process is referred to as “tagging”
“Tag” – insertion of the T-DNA “tags” the region in the genome because the T-DNA sequence can be located in the genome because of sequence
Identification of the flanking sequence
Floral transformation of Arabidopsis
Each seed that is transformed has a unique mutation
Transformation procedure - inflorescences are dipped into a solution containing Agrobacterium
Plants are grown in the greenhouse and seeds are collected
Selection of transformed plants – typically based on resistance to a toxic agent (transgene expression results in detoxification), in this instance the herbicide bialaphos
Population is maintained through seed, i.e. inherited
Generation of T-DNA tagged population
Herbicide selection of transformantsPropagation and collection of
seed
Phenotypic screening of the T-DNA mutant population – forward genetics
Identify Salt Responsive Mutants in a T-DNA Insertion Population