Παρουσίαση πρακτικής άσκησης Ιουλίου – Αυγούστου 2012 Δημήτρης Κοκορέτσης Α.Μ.: Τμήμα Γεωπονικής Βιοτεχνολογίας, Εργαστήριο Φυσιολογίας και Μορφολογίας Φυτών Επιβλέπων: Σταμάτης Ρήγας Περιεχόμενα: 1.Έλεγχος μετασχηματισμού φυτών D-3 background (lon1- 1) με p35S:LON1:FLAG. 2.Αξιολόγηση επιπτώσεων οξειδωτικού stress σε φυτά D-3 και D-3 μετασχηματισμένα με p35S:LON1:FLAG. 3.Βιοπληροφορική ανάλυση των γονιδίων AT1G63740, AT2G41510 και AT4G Στρατηγική κλωνοποίησης του AT4G02530.

Έλεγχος μετασχηματισμού φυτών D-3 background με 35S:LON1:FLAG (Hyg 30 μg/ml)

Line 15Line 16 Line 19 Δοκιμή 2

Line 20 Δοκιμή 2 Line 23

Δοκιμή 2 (πιο αξιόπιστη) Βιώσιμα φυτά (8η ημέρα): Line 15: 11/16 (ή 68,75%) Line 16: 15/15 (ή 100%) Line 19: 3/15 (ή 20%) Line 20: 9/16 (ή 56,25%) Line 23: 10/15 (ή 66,67%)

Δοκιμή 3 Line 15Line 16 Line 19

Δοκιμή 3 Line 20 Line 23

Δοκιμή 3 Βιώσιμα φυτά (8η ημέρα): Line 15: 5/12 (ή 41,67%) Line 16: 10/12 (ή 83,33%) Line 19: 3/11 (ή 27,27%) Line 20: 6/12 (ή 50%) Line 23: 7/12 (ή 58,33%)

Methyl Viologen Αξιολόγηση επιπτώσεων οξειδωτικού stress σε φυτά D-3 και D-3 μετασχηματισμένα με p35S:LON1:FLAG

Γονότυποι φυτών: Col-0 (wild-type) D-3 (lon1-1) D-3 + p35S:LON1:FLAG (Lines 15, 16, 20, 23) Θρεπτικά υποστρώματα: ½ MS + agarose (control) ½ MS + agarose + 10 nM Methyl Viologen Φωτογράφηση και μέτρηση μήκους ρίζας (day 5 & day 7) Μέτρηση βιομάζας (day 13)

Col-0 D-3 Day 5 Line 15Line 16 Control Day 5

Line 20Line 23 Control

Day 5 10 nM MV Line 15Line 16 Col-0 D-3 Day 5 10 nM MV Col-0 D-3 Line 16 Col-0 D-3 Line 15Line 16 Col-0 D-3

Line 23Line 20 Day 5 10 nM MV Μήκος ρίζας (cm)Col-0D-3Line 15Line 16Line 20Line 23 Control1,6810,3660,9830,9941,6280, nM MV0,6640,3650,7740,8060,7100,557

Line 16Line 15 Col-0 D-3 Day 7 Control Line 16 D-3 Control Line 16 D-3 Col-0 Line 16 D-3 Line 15 Col-0 Line 16 D-3 Day 7 Line 15 Col-0 Line 16 D-3 Day 7

Control Line 23Line 20Line 23Line 20

Day 7 10 nM MV Line 15Line 16 Col-0D-3 10 nM MV Day 7

Line 23Line 20 Day 7 10 nM MV Μήκος ρίζας (cm)Col-0D-3Line 15Line 16Line 20Line 23 Control:1,9350,6291,4101,3331,9940, nM MV:0,8010,6711,0591,1610,8530,741

Βιοπληροφορική ανάλυση γονιδίων Dual translation & protein targeting AT1G63740 (group III) AT2G41510 (group I) AT4G02530 (group II)

AT1G63740 TIR-NBS-LRR disease resistance protein Group III

annotated start codon recommended start codon AT1G63740 (disease resistance protein) 1 row = 100 bp

Targeting predictions AT1G63740 (disease resistance protein)

Annotated EST analysis AT1G63740 (disease resistance protein) Out of 19 annotated ESTs for AT1G63740, 2 ESTs that cover area upstream of the reference mRNA were detected: GenBank accession numbers ES ES They were then aligned with the gene + 5’ UTR:

Annotated EST analysis AT1G63740 (disease resistance protein) Translated EST and gene + 5’ UTR sequence alignment:

AT1G63740 (disease resistance protein) EST search BLASTn: [Met #1…annotated Met] X ESTs (all organisms) DK & DK match start codon #3 (MASSSSS…)

AT1G63740 (disease resistance protein) DK505268: full-length kale cDNA library (capsules) Brassica oleracea var. acephala cDNA clone KALE-130A02 5-, mRNA sequence DK490237: full-length kale cDNA library (seedlings) Brassica oleracea var. acephala cDNA clone KALE-090L08 5-, mRNA sequence EST search

AT1G63740 (disease resistance protein) BLASTn: DK X nucleotide collection (Brassica)

EST search AT1G63740 (disease resistance protein) BLASTn: DK X nucleotide collection (Brassica)

AT1G63740 (disease resistance protein) Nucleotide alignment BrTNLs: annotated CDS AT1G63730: annotated CDS AT1G63740: long CDS, starting in Met #3 (MASSS…) AT1G63740 annotated Met AT1G63730 & BrTNLs annotated Met

AT1G63740 (disease resistance protein) Amino acid alignment BrTNLs: annotated proteins AT1G63730: annotated protein AT1G63740: long protein, starting in Met #3 (MASSSSS…)

Nucleotide alignment AT1G63740 (disease resistance protein) AT1G63740 annotated Met AT1G63730 annotated Met AT1G63740 X AT1G63730 (5’ end + UTR)

Amino acid alignment AT1G63740 (disease resistance protein) AT1G63740 X AT1G63730 (5’ end + UTR) translated AT1G63730 annotated Met AT1G63740 annotated Met

TIR-NBS-LRR disease resistance gene conservation

TIR-NBS-LRR disease resistance gene conservation (amino acid)

AT2G41510 Cytokinin oxidase/dehydrogenase 1 Group I

AT2G41510 (AtCKX1) recommended start codon annotated start codon 1 row = 100 bp

Targeting predictions AT2G41510 (AtCKX1)

Annotated EST analysis Out of 4 annotated ESTs for AT2G4510, no ESTs that cover area upstream of the reference mRNA were detected.

AT2G41510 (AtCKX1) EST search BLASTn: [Met #1…annotated Met] X ESTs (all organisms) NO results

Subcellular localization according to Werner et al. (2003)Werner et al. (2003) Vacuole

AT4G02530 Chloroplast thylakoid lumen protein Group II

1 row = 100 bp AT4G02530 (chloroplast thylakoid lumen protein) annotated start codon recommended start codon

Targeting predictions AT4G02530 (chloroplast thylakoid lumen protein)

EST search BLASTn: Met #1…Met # bp X ESTs (Arabidopsis thaliana)

Annotated EST analysis AT4G02530 (chloroplast thylakoid lumen protein) Out of 79 annotated ESTs for AT4G02530, 1 EST that covers area upstream of the reference mRNA was detected: BP (GenBank accession)

AT4G02530 (chloroplast thylakoid lumen protein) Detected in thylakoid lumen, according to proteomics analysis by Schubert et al. (2002)Schubert et al. (2002) Subcellular localization

p35S:AT4G02530:YFP cloning strategy

δεν κόβει μέσα στο γονίδιο AT4G02530 (genomic) restriction sites Restriction sites: XhoI (1), NcoI (1), SalI (1), PstI (1)

Primers For: 5’-AAAAGTAATGAAAAAAATATTGAGAG-3’ Rev: 5’-TTCAACGCGTCAGTCCCTTAAGCTGTTCCAAAAGA-3’ (ACGCGT: MluI restcitrion site) PCR gDNA Arabidopsis PCR product:

Insertion in SmaI site of PUC18 vector Orientation check with PstI sense: 1326 bp / antisense: 562 bp

YFP insertion in MluI site Orientation check with BamHI sense: 2574 bp / antisense: 1872 bp

XbaISacI Digestion with XbaI + SacI

Insertion in CaMV 35S/pGPTV-HPT cassete

Forward primer BLAST

Reverse primer BLAST