gene functional analysis.ppt
DESCRIPTION
gene functional analysisTRANSCRIPT
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Some useful statistics:
Small genome (114.5 Mb/125 Mb total)
has been sequenced in the year 2000
(SequenceViewer, AGI).
Extensive genetic and physical maps
of all 5 chromosomes (MapViewer).
A rapid life cycle (about 5-8 weeks from
germination to mature seed).
Prolific seed production and easy
cultivation in restricted space.
Efficient transformation methods
utilizing Agrobacterium tumefaciens.
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SIGnAL "T-DNA Express" Arabidopsis Gene Mapping Tool
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Moving forward in reverse: genetic technologies to
enable genome-wide phenomic screens in Arabidopsis
Jose M. Alonso* and Joseph R. Ecker (2006) Nature
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Work PlanScreening of Arabidopsis mutants on MS plates
Planting mutant lines on Soil
DNA isolation
Amplify the flanking sequences of each insertion
(Using gene specific Primers)
Homozygosity and Heterozygosity analysis for T-DNA
through PCR
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Identification of interesting phenotype linked with
homozygous T-DNA line
Amplification of Gene from wild type
Cloning and Sequencing of gene
Construct designing into pBINAR expression vector
Transformation into heterozygous mutant plant
Complementation of mutant phenotype
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Germination of the different plant lines on MS medium
The resistant plants of mutant 165B07
are germinated on MS having
sulfadiazine
The resistant plants of mutant 060B06 are
germinated on MS having sulfadiazine
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Description of the Mutant
Phenotype of GABI mutant
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Disrupted gene:
The disrupted gene was At5g16980
genomic sequence length: 1601 nucleotides
coding sequence length: 701 nucleotides.
T-DNA Sequence
AATCTGTGCGCGGGAATTATCGCATAAATTGGCTTTGGGATCCTCCC
TATGGGAGCAGAAGACTCAACGCGATAGAGATAGCGAAAAGAAAAA
CCCGAAGAGACACAATTTCTTCTCAGAGTCTTCAAGAACACCAAAT
TGTCATGTAGTTTCTACATCAATAAGAAGCATCTA
Gene AnnotationThe gene At5g16980 codes for a putative NADP-dependent
oxidoreductase like protein Protein propertiesLength : 311 amino acids
molecular weight : 34528.9 Da
Isoelectric point is 6.32
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Disrupted gene
At4g29060 which has 4312 nucleotides for genomic DNA
sequences and 2862 nucleotides for coding sequences.
T-DNA Sequence
GGGTTCATACTCATTGCTGGTGTCTCTCCCCTGTGCTATTTTTATTTCGTTTT
CCCTCTCTTGGTGGATAGAGGAGATAAGAGTCTTCAAAGATTACTACAGGTGA
TTGTCTTTTTCTCTCTCTCCTGCGCCATCAAGACTCGAGCTTCAATATGGGCT
TACGGGCTTCATACTAAGGCCCATCAATAGCCTATATCGATGTGAAACTCCCC
CCCAGACATTACATAGTAGCAATGCCTCCTTTTCATGCTGGTCAATTTCTTAA
TAAATCAATAAGAGGCGTCGAACAAGACTTATTCAACATTTTATTACTAAGAGT
CTAAAAGACTTTAGTTTTTGACCCTCCAAGTCATACAGATAAAGCCTATTTAGA
TCTTTTTCATATTATAATCATATGGTTTTGACCAAAAAAAGAAAAACTTATGGT
AAGATCATTCATGAGAGGAAAAAAGAATTGCCAAACGATGA
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TCTACCACTCTCCTCGTTTTCGCTTCGTGCTTATTTGTCAAACGCGAACATTTCA
CCGAAACTTAGCGACTTTTCTCGCTTTACTCTTTCTGGGAGCAATTTCTCAGAG
CATAGGCGAGCTTAAAGGTAGCTCCTTTTCTTAGCTTTGCTCAAATGGGTTTCT
TAGTTACTCTTCTTATGTTTCCGTGATAAGATGGTTCGAAATTTACTATGGGAT
CCTCCGTATAGTGAGGCGTTNTTACTAANNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNAAGAAAAAGAGTGGTCCTTTTCTTCATATGCTTTCAGATTAG
TCTGTGTTGTTGTCTGAGGAAAGCTTGTAATTTTGACAGTGTGTAATGGTTT
TCTATGTGTAGGTGACAGTTCTTAGCTATGGCTACGATTACACCTTCTTCTAT
TAGCAATGCCTGGCTCATTCCTGGAGCTTCTTTTACTGTAAAGAAGAGTGAT
TGTTCCATCAAATGTA
Gene AnnotationAt4g29060 codes for a putative Quinone oxidoreductase like protein which
functions in chain elongation during polypeptide synthesis at the ribosome
Protein Properties
length : 953 amino acids
Molecular weight 103782 Da
Isoelectric point : 4.95
localized in chloroplast and Ribosome
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PCR amplification of small pale green plants with gene specific forward and GK-
LB primer.
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Amplification and cloning of the At5g59650 gene from Col-0
Genevestigator database (https://www.genevestigator.ethz.ch)
3.7Kb
3.7Kb
Restriction digestion of the cloned At5g59650 and pGEMT
vector 3.7kb, with Sal-1 and Xba-1 restriction enzymes.