Some useful statistics:

Small genome (114.5 Mb/125 Mb total)

has been sequenced in the year 2000
(Sequenceie!er" #$%).
&'tensi(e genetic and )hysical ma)s
of all 5 chromosomes (Ma)ie!er).
# ra)id life cycle (about 5*+ !ee,s from
germination to mature seed).
-rolific seed )roduction and easy
culti(ation in restricted s)ace.
&fficient transformation methods
utili.ing Agrobacterium tumefaciens.
S%$n#/ 01*23# &')ress0 #rabido)sis $ene Ma))ing 1ool
Mo(ing for!ard in re(erse: genetic technologies to
enable genome*!ide )henomic screens in #rabido)sis
4ose M. #lonso5 and 4ose)h 6. &c,er (2007) 3ature
Work Plan
Screening of Arabidopsis mutants on MS plates

Planting mutant lines on Soil

DNA isolation

Amplify the flanking sequences of each insertion

(Using gene specific Primers)

omo!ygosity and etero!ygosity analysis for "#
DNA
through P$%
Identification of interesting phenotype linked &ith
homo!ygous "#DNA line

Amplification of 'ene from &ild type

$loning and Sequencing of gene

$onstruct designing into p(INA% e)pression *ector

"ransformation into hetero!ygous mutant plant

$omplementation of mutant phenotype

Germination of the different plant lines on MS medium
1he resistant )lants of mutant 175809
are germinated on MS ha(ing
sulfadia.ine
1he resistant )lants of mutant 070807 are
germinated on MS ha(ing sulfadia.ine
2escri)tion of the Mutant
Phenotype of GABI mutant
Disrupted gene:
1he disru)ted gene !as #t5g17:+0
genomic sequence length: 1701 nucleotides
coding sequence length: 901 nucleotides.
T-DNA Sequence
AATCTGTGCGCGGGAATTATCGCATAAATTGGCTTTGGGATCCTCCC
TATGGGAGC#$##$#;1;##;$;$#1#$#$#1#$;$####$#####
;;;$##$#$#;#;##111;11;1;#$#$1;11;##$##;#;;###1
1$1;#1$1#$111;1#;#1;##1##$##$;#1;1#
Gene Annotation
1he gene #t5g17:+0 codes for a )utati(e 3#2-*de)endent
o'idoreductase li,e )rotein
Protein properties
/ength : <11 amino acids
molecular !eight : <452+.: 2a
%soelectric )oint is 7.<2
Disrupted gene
At4g29060 which has 4312 nucleotides for genomic DNA
sequences and 262 nucleotides for coding sequences!
T-DNA Sequence
GGGTTCATACTCATTGCTGG1$1;1;1;;;;1$1$;1#11111#111;$1111
;;;1;1;11$$1$$#1#$#$$#$#1##$#$1;11;###$#11#;1#;#$$1$#
11$1;11111;1;1;1;1;;1$;$;;#1;##$#;1;$#$;11;##1#1$$$;1
1#;$$$;11;#1#;1##$$;;;#1;##1#$;;1#1#1;$#1$1$###;1;;;;
;;;#$#;#11#;#1#$1#$;##1$;;1;;1111;#1$;1$$1;##111;11##
1###1;##1##$#$$;$1;$##;##$#;11#11;##;#1111#11#;1##$#$1
;1####$#;111#$11111$#;;;1;;##$1;#1#;#$#1###$;;1#111#$#
1;11111;#1#11#1##1;#1#1$$1111$#;;AAAAAAAGAAAAACTTATGGT
AAGATCATTCATGAGAGGAAAAAAGAATTGCCAAACGATGA
TCTACCACTCTCCTCGTTTTCGCTTCGTGCTTATTTGTCAAACGCGAACATTTCA
CCGAAACTTAGCGACTTTTCTCGCTTTACTCTTTCTGGGAGCAATTTCTCAGAG
CATAGGCGAGCTTAAAGGTAGCTCCTTTTCTTAGCTTTGCTCAAATGGGTTTCT
TAGTTACTCTTCTTATGTTTCCGTGATAAGATGGTTCGAAATTTACTATGGGAT
CCTCCGTATAGTGAGGCGTTNTTACTAANNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNA#$#####$#$1$$1;;1111;11;#1#1$;111;#$#11#$
1;1$1$11$11$1;1$#$$###$;11$1##1111$#;#$1$1$1##1$$111
1;1#1$1$1#$$1$#;#$11;11#$;1#1$$;1#;$#11#;#;;11;11;1#1
1#$;##1$;;1$$;1;#11;;1$$#$;11;1111#;1$1###$##$#$1$#1
1$11;;#1;###1$1#
Gene Annotation
#t4g2:070 codes for a )utati(e =uinone o'idoreductase li,e )rotein !hich
functions in chain elongation during )oly)e)tide synthesis at the ribosome
Protein Properties
length : :5< amino acids
Molecular !eight 10<9+2 2a
%soelectric )oint : 4.:5
locali.ed in chloro)last and 6ibosome
-;6 am)lification of small )ale green )lants !ith gene s)ecific for!ard and $>*
/8 )rimer.
Amplification and cloning of the At5g5!5" gene from #ol-"
$ene(estigator database
(https:$$%%%&gene'estigator&eth(&ch)
<.9>b
<.9>b
6estriction digestion of the cloned #t5g5:750 and )$&M1
(ector <.9,b" !ith Sal*1 and ?ba*1 restriction en.ymes.