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Γενετικά Τροποποιημένα Φυτά
I. Γενετική τροποποίηση, Γενικές ΑρχέςΑγροβακτηρίδιοΒαλλιστικήΠαροδική ΈκφρασηΆλλες μέθοδοιΑναγέννηση σε ιστοκαλλιέργεια
II. Eφαρμογές ΓΤ, III. Έλεγχος-Ανίχνευση ΓΤ, Εκτίμηση Επικινδυνότητας
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Τα επόμενα ~50-70 χρόνια το Χερσαίο/θαλάσιο παραγωγικόσύστημα θα πρέπει να παράξει για την ανθρώπινη διατροφή
περίπου όσα τρόφιμα χρειάστηκαν από τότε που τοανθρώπινο γένος υπάρχει στη Γη !
http://www.biosci.ohio-state.edu/pcmb/osu_pcmb/courses/pb300_files/lamb_wi06/plant_biotech_breeding(8Mar06).ppt#259
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Green Revolution
• GR varieties - dwarfed & survive storms, some disease resistance, year round planting
• Dwarfism due to defects in gibberellic acid (GA)
http://www.biosci.ohio-state.edu/pcmb/osu_pcmb/courses/pb300_files/lamb_wi06/plant_biotech_breeding(8Mar06).ppt#259,4,Slide 4
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Green Revolution - undertaken in the 1960s
• Extensive effort to increase yield of wheat and other grains
• Included improvements in breeding, fertilization, and irrigationBreeding improvement example: hybrid corn (uniform,
therefore easier to harvest and much higher yields) HOWEVER, FARMERS MUST BUY THE HYBRID SEED EVERY YEAR, CAN’T PROPAGATE THEMSELVES
• Norman Borlaug won Nobel Peace Prize in 1970 for his part in the Green Revolution
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http://eeb.bio.utk.edu/EdSchilling/lectures/lecture9slides.pdf
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http://eeb.bio.utk.edu/EdSchilling/lectures/lecture9slides.pdf
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http://eeb.bio.utk.edu/EdSchilling/lectures/lecture9slides.pdf
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Cauliflower and broccoli are derived from the same genetic ancestor, Brassicaoleracea, but were developed over many years into individual and verydifferent vegetables through selection and breeding. Biotechnology can makethis process more precise and less time-consuming.
(California Agriculture June 2004, EDITORIAL OVERVIEWChallenges and opportunities for horticultural biotechnology )
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PLANT BIOTECHNOLOGYIN WAVES
Martina Newell=McGloughlin, Director, UC SystemwideBiotech Researchand Education Program http://ucbrep.ucdavis.edu 2003
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∆ύο Στρατηγικές εκτοπικής έκφρασης:
• Α. Παροδική έκφραση (transient expression)• Αγροεμποτισμός (Agroinfiltration)• Ιικοί φορείς
• Β. Σταθερή έκφραση• Γενετική τροποποίηση
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∆ύο Στρατηγικές εκτοπικής έκφρασης:
• Α. Παροδική έκφραση (transient expression)• Αγροεμποτισμός (Agroinfiltration)• Ιικοί φορείς
• Β. Σταθερή έκφραση• Γενετική τροποποίηση
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Παρατήρηση:
Kορονωτός κάλλος προκαλείται από το A.tumefaciens
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Agrobacterium tumefaciens
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To Aγροβακτηρίδιο έχει γενωμικό DNAαποτελούμενο από 4 μόρια και ένα μεγάλο
πλασμίδιο
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Γονιδιακή μεταφορά με Agrobacteriumtumefaciens
• H συχνότερη μέθοδος μεταφοράς• Χρησιμοποιεί το Agrobacterium tumefaciens,
‘φυσική’ γενετική μηχανική• Agrobacteria
– Βακτήρια εδάφους, gram-negative, συγγενικό τουRhizobium
– 4 ‘είδη’:tumefaciens- προκαλεί κορωνοτό κάλλοrubi- προκαλεί μικρούς κάλλους σε κάποια
δικότυλαrhizogenes- η ασθένεια των ‘μαλλιαρών ριζών’radiobacter- μη τοξικό
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Γονιδιακή μεταφορά με Agrobacteriumtumefaciens
• είναι η μέθοδος που επιλέγεται για τηντροποποίηση δικότυλων
• ∆εν είναι το ίδιο αποτελεσματική σε όλα ταδικότυλα (υπάρχει γενετική προδιάθεσηγι’αυτό)
• Περίπλοκο βακτηρίδιο –το γονιδίωμα είναιγνωστό; Αποτ. Από 4 χρωμοσώματα; ~ 5500
γονίδια
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Επιμόλυνση και δημιουργία όγκου
• Η μόλυνση συμβαίνει μόνο σε τραυματισμένουςιστούς
• Περιλαμβάνει αναγνώριση και χημειοταξίαπρος το τραύμα
• Οι κάλλοι είναι πραγματικοί όγκοι• Ο φαινότυπος είναι αποτέλεσμα μεταφοράς
DNA στο φυτό
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Συγκρότηση Ti πλασμιδίου
• Γονίδια τοξικότητας (vir) operons Α-H (24 genes)• Γονίδια σύζευξης (con)• ori V• Γονίδια καταβολισμού οπινών
• Γονίδια φυτο-ορμονών• Γονίδια σύνθεσης οπινών• Αλληλουχίες ορίων
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Ti Plasmid
• Μεγάλο (>200-kb)• Συζευκτικό• ~10% του πλασμιδίου μεταφέρεται στο φυτό
μετά τη σύζευξη• Το DNA που μεταφέρεται (ονομ. T-DNA)
ενσωματώνεται σχεδόν τυχαία στοχρωμοσωμικό DNA
• Το πλασμίδιο Ti επίσης κωδικοποιεί:1. Ένζυμα που σχετίζονται με το μεταβολισμό των
οπινών2. Πρωτεΐνες που σχετίζονται με την τοξικότητα
(Vir genes)
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Τα γονίδια του T-DNA και η ογκογέννεση
Το T-DNA των Αγροβακτηριδίων αγρίου τύπου κωδικοποιούνδύο τύπων λειτουργίες – παραγωγή φυτοορμονών και σύνθεσηοπινών
Τα γονίδια του T-DNA έχουν κατάλληλους υποκινητές(φυτικούς) και τέτοια χρήση κωδικονίων που τους επιτρέπουννα εκφράζονται στα φυτικά κύτταρα αλλά όχι στα βακτηριδιακά.
Για την παραγωγή φυτο-ορμονών υπεύθυνα είναι τα γονίδιαTms1 και Tms2 που μετατρέπουν την τρυπτοφάνη σεΙνδολ-3-οξικό οξύ (IAA), και το γονίδιοTmr/Ipt που μετατρέπει την αδενίνη σε ισοπεντυλ-αδενίνη.
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L-tryptophan indole-2-acetamide
indole-3-acetic acid (αuxin)
CO2NH3
Tms1
Tms2
adenosine - 5’-PO4
2-isopentenyl-PP
isopentenyladenine-5-PO4(a cytokinin)
PPiTmr1
O2
Το Αγροβακτήριο κωδικοποιεί και ένζυμα πουκαταλύουν τη βιοσύνθεση ορμονών και οδηγούν
στη δημιουργία όγκου
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Το A. rhizogenes περιέχει διαφορετικό μεγαπλασμίδιο(Ri) αλλά φέρει κατά το μεγαλύτερο μέρος τις ίδιεςλειτουργίες
Το πλασμίδιο Ri T-DNA φαίνεται ότι δεν κωδικοποιεί γιασύνθεση κυτοκινίνης και άρα παράγει υπερβολικά μεγάλοαριθμό ριζών αλλά όχι κάλλο.
∆εν έχουν χαρακτηριστεί όλα τα γονίδια του T-DNA
Το ORF 6b, μόλις πρόσφατα βρέθηκε ότι κωδικοποιεί μιαπρωτεΐνη που έχει χαρακτηριστικά ευκαρυοτικού παράγονταμεταγραφής (DNA-binding domain).
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Εύρος Ξενιστή
Το A. tumefaciens έχει εντυπωσιακά μεγάλο εύροςξενιστών που περιλαμβάνει σχεδόν όλες τις οικογένειεςδικότυλων. ∆εν είναι καλοί ξενιστές τα μονοκότυλα και ταγυμνόσπερμα.
Σε κάποιες περιπτώσεις αυτό μοιάζει να είναι αποτέλεσματης έλλειψης συγκεκριμένου φαινολικού επαγωγέα
Σε άλλες περιπτώσεις φαίνεται ότι δεν υπάρχουνσυγκεκριμένα γονίδια «συνεργασίας» από τον ξενιστή
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Τα πρώτα στάδια της «επίθεσης»:
• Επαγωγή: Ακετοσυριγκόνη, σιναπικό οξύκ.α. φαινολικές ουσίες + σάκχαρα (VirAενεργοποιεί την VirG-μ.π. για άλλα Vir)+ χαμηλό pH (5-6)
• Πρόσδεση: Βιτρονεκτίνη + άλλες επιφανειακέςπρωτεΐνες + υδατάνθρακες
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Απλουστευμένη τοπολογία της ογκογόνου περιοχής (T-DNA) και συνοριακές αλληλουχίες ενός πλασμιδίου Ti
(pTiC58)
LB (left border): TGgCAGGATATATATTGtgGtGTAAAC| | | | | | | | | | | | | | | | | | | | | | | |
RB (right border): TGaCAGGATATATATTGgcGgGTAAAC
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Αλληλουχίες Συνοριακών άκρων διαφόρωνπλασμιδίων Τι και “consensus sequence”.
*pTiC58 (nopaline Ti) LB:TGgCAGGATATATATTGtgGtGTAAAC
RB:TGaCAGGATATATATTGgcGgGTAAAC
nopaline Ti LB:ggTGGCAGGATATATtgtggTGTAAACRB:ttTGGCAGGATATATtggcggGTAAAC
pTiAch5(Octopine Ti)TL-LB:ggcGGCAGGATATATtcaatTGTAAAcTL-RB:acTGGCAGGATATATaccgtTGTAATtTR-LB:ggtGGCAGGATATATcgaggTGTAAAaTR-RB:gaTGGCAGGATATATcgaggTGTAATa
Ri plasmid TL-LB:ggTGGCAGGATATATtgtgaTGTAAAaTL-RB:acTGaCAGGATATATgttccTGTcAtg
CONSENSUS xxTGGCAGGATATATxxxxxTGTAAA/T
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Η γονιδιακή περιοχή Vir του πλασμιδίου Τι εν δράσει: Τα οπερόνια VirA,B,C,D,E,F,Gεπάγονται από φαινολυκά συστατικά του φυτού, μονόκλωνες αλυσίδες του Τ-DNA
παράγονται με συνεργασία των πρωτενών πρωτεϊνη virC, VirD1 & 2, και«συσκευάζονται για μετακόμιση» από τη πρωτεϊνη VirE2 και μεταφέρονται στο φυτικό
κύτταρο μέσω της συζευκτικής γέφυρας που δομούν οι πρωτεϊνες VirB1-11
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Χαρακτηριστικά ενσωμάτωσης T-DNA
• Αντίθετα με τα μεταθετά στοιχεία το T-DNA δεν κωδικοποιεί για ενζυμικές δραστικότητεςικανές για εισαγωγή και ένθεση
• H ενσωμάτωση πρέπει να γίνετε από ένζυμα τουξενιστή (επιδιορθωτικοί μηχανισμοί DNA?)
•Στη ζύμη η ενσωμάτωση γίνεται με ομόλογοανασυνδυασμό
•Η ενσωμάτωση γίνεται πριν την σύνθεση τηςσυμπληρωματικής αλυσίδας ?
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H ενσωμάτωση του T-DNA στο γονιδίωμα του ξενιστή γίνετεσε –σχετικά- τυχαίες θέσεις με βάση αναλύσεις σε χιλιάδεςφυτά.
Η ενσωμάτωση πιστεύετε ότι συμβαίνει με ‘μη κανονικό’ ανα-συνδυασμό (illegitimate recombination).
Φαίνεται να υπάρχει συσχέτιση μεταξύ συχνότηταςενσωμάτωσης και κυτταρική διαίρεση
Τα γενετικά τροποποιημένα από Αγροβακτηρίδιο κύτταραφέρουν συνήθως ένα αντίγραφο του T-DNA στο γονιδίωματους αν και έχουν παρατηρηθεί και πολλαπλά αντίγραφαδιάσπαρτα στο γονιδίωμα.
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Alonso et al Science 301:653 (2003)
Distribution of T-DNA insertions across the Arabidopsis genome.
Analysis of 225,000 independent events
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Φυτικά γονίδια που εμπλέκονται στην ενσωμάτωσητου T-DNA
Υπάρχουν αρκετά φυτικά γονίδια που φαίνεται ότι είναιαπαραίτητα για την μόλυνση από το Αγροβακτηρίδιο.
Προφανώς ο ρόλος τους σο κύτταρο είναι άλλος καιαπλώς χρησιμοποιούνται από τον εισβολέα
‘Έχουν βρεθεί μεταλλάγματα στο Arabidopsis σε διάφορουςγενετικούς τόπους που επηρεάζουν, περισσότερο ή λιγότερο τηνμόλυνση από το Αγροβακτήριο (Reduced efficiency Agrobacterium transformation mutants (rat mutants) (Gelvin 2001)
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From Covey & Grierson
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Τα γονίδια τοξικότητας (vir)
• Vir A αισθητήρας σινιάλου ενεργοποιεί τηνπρωτεΐνη G
• VirG επάγει την τοξικότητα (ενεργ. Vir γονιδίων)
• Vir D1,2, C1 αποκοπή μονόκλωνου T-DNA• Vir D2, Σύνδεση με το 5’ άκρο (RB) του T-DNA• VirE2, Συνδέεται με το ssDNA (προστασία)• Άλλες VirD προωθούν το DNA στο φυτό
8 οπερόνια Α-Η:
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Τα γονίδια τοξικότητας (vir), συνέχεια
• VirB- 11 πρωτεΐνες σχετιζόμενες με τονσυζευκτικό πόρο
• VirD2 VirE2 –Σχετίζονται και με τηνμεταφορά στον πυρήνα
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Hypothetical model for virB membrane channel
From P. Zambryski
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Άλλοι καθοριστικοί ως προς την ικανότηταμόλυνσης παράγοντες μπορεί να είναι:
i) Η ευαισθησία ή εξειδίκευση των VirA πρωτεϊνών
ii) Άλλα vir γονίδια
iii) Τα ογκογονίδια μπορεί να μην είναι συμβατάμε το ξενιστή
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Γενετική Μηχανική Φυτών και Αγροβακτήριο
Το T-DNA ενσωματώνεται μέσα στο γονιδίωμα
Κανένα από τα γονίδια που βρίσκονται μέσα στο T-DNA δενείναι απαραίτητο για την μεταφορά και ενσωμάτωσηΜόνο τα ‘όρια’ του T-DNA και οι λειτουργίες vir είναιαπαραίτητες
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Τa Τεχνικά Προβλήματα
Πώς μπορούν να διατηρηθούν οι λειτουργίες vir χωρίς ναείναι το πλασμίδιο τεράστιο;
Εάν απαλειφθούν τα γονίδια του όγκου πως θα ξέρουμε ποιακύτταρα τροποποιήθηκαν;
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Σύστημα δυαδικών φορέων
Στρατηγική1. Μεταφορά του T-DNA σε ένα ξεχωριστό μικρό
πλασμίδιο2. Αφαιρούνται τα γονίδια aux (Τms) και cyt(Tmr)3. Εισάγεται κατάλληλο γονίδιο επιλογής4. Τα γονίδια Vir διατηρούνται αλλά σε διαφορετικό
πλασμίδιο μόνιμα μέσα στο στέλεχος
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Σύστημα δυαδικών φορέων (συν.)
5. Εισάγεται το ΕΓ ανάμεσα στα δεξιά καιαριστερά όρια
6. Και τα δύο πλασμίδια πρέπει να είναιταυτόχρονα παρόντα στοAγροβακτηρίδιο
7. Ο φυτικός ιστός συν-καλλιεργείται μετα Αγροβακτήρια
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Πώς μπορούν να διατηρηθούν οι λειτουργίες vir χωρίςνα είναι το πλασμίδιο τεράστιο;
Οι λειτουργίες vir δε είναι ανάγκηνα βρίσκονται in cis το Τ-DNA
virregion
‘disarmed’Ti plasmid
cloning vector plasmid
LB RB
ori
kanR
YFG
MCS
+
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Virplasmid
Binary vector
T-DNA
Separate vir and binary plasmids
Cointegratingvector
T-DNA
Cointegrating vector
Plant Cell
Agrobacterium
∆υαδικοί φορείς: τα γονίδια τοξικότηταςμεταφέρονται σε δεύτερο πλασμίδιο πουβρίσκεται στο ίδιο κύτταρο και δρουν in trans
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Transfering the binary vector to agrobacterium via “triparentalmating”. One E. coli strain carries a self-transmissible plasmid
(“helper”) which is transferred (by a process known as “conjugation”) to the other E. coli strain carrying the vector
plasmid on which the transgene cassette is already assembled. The helper plasmid provides the conjugation mechanism for the vector plasmid, promoting its “mobilization” into agrobacterium.
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Γονίδια επιλογής
Μόνο ένα μικρό μέρος από τακύτταρα μετασχηματίζεται
Πρέπει να χρησιμοποιηθούνγονίδια τα οποία θα εξασφαλίζουνότι τα μετασχηματισμένα ζουν ταάλλα πεθαίνουν.
= επιλογήcloning vector plasmid
LB RB
orι
kanR
YFG
MCS
Εάν απαλειφθούν τα γονίδια του όγκου πως θα ξέρουμε ποιακύτταρα τροποποιήθηκαν;
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Τα πρώτα γονίδια επιλογής ήταν γονίδια ανθεκτικότηταςσε αντιβιοτικά
Ορισμένα αντιβιοτικά που δρουν στα βακτηρίδιανεκρώνουν και φυτικά κύτταρα (οργανίδια)
♦ kanamycin♦ hygromycin
Υπάρχουν όμως και βακτηριδιακά γονίδια που μεταβολίζουντο αντιβιοτικόΠ.χ. neomycin phosphotransferase II (nptII; kanR)
Η έκφραση ενός τέτοιου γονιδίου στα φυτικά κύτταρα τακαθιστά ανθεκτικά στο αντιβιοτικό και συνεπώς το nptIIμπορεί να χρησιμοποιηθεί ως παράγοντας επιλογής
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Νεώτερα γονίδια επιλογής:
Γονίδια ανθεκτικότητας σε ζιζανιοκτόνα- glyphosate tolerance (mod. EPSP synthase)- phosphinothricin tolerance (converted)
Γονίδια μετατροπής του μεταβολισμού- phosphomannose isomerase- D-amino acid oxidase
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Τα γονίδια που θα εισαχθούν στον πυρήνα πρέπει ναεκφραστούν επαρκώς στον πυρήνα
Οι συχνότεροι υποκινητές που χρησιμοποιούνται σήμερα είναι οnos (γονίδιο της nopaline synthase) και ο CaMV35S (από τον ιόCaMV)
cloning vector plasmid
LB RB
ori
kanR
YFG+ kanR
MCS
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Εισαγωγή επιθυμητού γονιδίου (ΕΓ)
Κάθε γονίδιο που εισάγεται για έκφραση πρέπει να έχει:♦ κατάλληλο εκκινητή και καταληκτική αλληλουχία♦ κατάλληλη αλληλουχία έναρξης μετάφρασης♦ Χρήση κωδικονίων φυτικού τύπου.
Μπορούν να εισαχθούν ταυτόχρονα πολλά γονίδια σε ένανμετασχηματισμό αλλά:
•Όσο πιο μεγάλο το T-DNA τόσο πιο δύσκολο η ενσωμάτωση
•Όσο πιο μεγάλος ο φορέας τόσο πιο δύσκολος ο χειρισμός
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‘Άμεση μεταφορά DNA
• Εισαγωγή ‘γυμνού’ DNA στα κύτταρα και μετάεπιλογή μετασχηματισμένων
• Εισαγωγή DNA :1. Ηλεκτροπόρωση2. Βομβαρδισμός σωματιδίων (Βιο-βαλιστική)3. Χημική εισαγωγή (PEG)
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Ηλεκτροπόρωση
• Χρησιμοποιούνται κύτταρα χωρίς τοιχώματα(πρωτοπλάστες)
• Εφαρμόζεται ηλεκτ. Πεδίο υψηλού voltage, το οποίο προκαλεί πρόσκαιρους πόρους στημεμβράνη επιτρέποντας στο DNA να εισέλθειστο κύτταρο
• Χρησιμοποιείται στα μονοκότυλα (καλαμπόκι, ρύζι)
• Μειονέκτημα: πολύ δύσκολη η αναγέννησηαπό πρωτοπλάστες
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Electroporation
Overview of method
Removal of cell walls not alwaysnecessary
1.5 kV
Ion concentrationKCl 125 mM
Plasmid size3-6 Kb
Plasmid concentration200μg/mL
Protoplast density2 x 106mL-1
Many variables need optimizingMany variables need optimizing
Examine cells for activity of transgene(e.g. GUS expression)
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Isolated protoplasts on a hemocytometer
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Χημικά επαγόμενος μετασχηματισμός
• Συνήθως εφαρμόζεται σε πρωτόπλαστες• ∆ιάφορα πρωτόκολλα:
1. Εισαγωγή του DNA σε τεχνητές μεμβράνες(λιποσώματα), που θα συντηχθούν με την κυτ. μεμβράνη
2. Πρόσδεση του DNA με πολύ-κατιόντα (PEG) γιατην εξουδετέρωση φορτίων, ορισμένα κύτταραθα εισάγουν με ενδοκυτ. το σύμπλοκο
3. Συνδυασμός των 1 και 2
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Βομβαρδισμός σωματιδίων• Λιγότερα εμπόδια από την ηλεκτροπόρωση• Μπορεί να χρησιμοποιηθεί σε οποιοδήποτε ιστό• Μπορεί να τροποποιήσει οργανίδια• Αρχές Μεθόδου:
1. Κατακρήμνιση DNA σε μικρά (0.6-1.6 μm)σφαιρίδια βολφραμίου ή χρυσού (ειδικόβάρος+ανενεργότητα)
2. Επιτάχυνση σωματιδίων σε ιστούς3. Επιλεκτική αναγέννηση όπως και για το
Αγροβακτηρίδιο
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Original biolistic gun.
DNA is bound to the microprojectiles, which impact the tissue or immobilized cells at high speeds.
From J. Sanford et al.
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Particle bombarment using helium powered “gene gun”
Helium gas
Rupturedisk
Macroprojectile
Target cellStops macroprojectile
Overview of method
DNA-coatedmicocarrers
1. Bombard cells with DNA-coated microbeads2. Detect activity of GUS-fusion proteins by adding X-Glc
- Dos not require cell culture or pre-treatment of recipient tissue- Technique currently favored for transformation of monocots
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Particle bombardmentBiolistic Gun
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Σύγκριση Αγροβακτηριδίου/Βαλλιστικής
• Λίγες ενθέσεις ανά μετασχηματισμό• Σχετικά εύκολη διαδικασία (σε κάποιαφυτά)
• Σχεδόν τυχαία ένθεση• Μίμηση «φυσικού» τρόπου ένθεσης
Μειονεκτήματα:• ∆εν τροποποιεί όλα τα φυτικά είδη
Ι. Αγροβακτήριο
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Σύγκριση Αγροβακτηριδίου/Βαλλιστικής
• Εφαρμόσιμη σε κάθε είδος ιστού• Μετασχηματίζει και οργανίδια• Εύκολη
Μειονεκτήματα:• Πολλαπλές ενθέσεις, συχνά πολύπλοκες• Σχετικά ακριβή
ΙΙ. Βαλλιστική
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∆ύο Στρατηγικές εκτοπικής έκφρασης:
• Α. Παροδική έκφραση (transient expression)• Αγροεμποτισμός (Agroinfiltration)• Ιικοί φορείς
• Β. Σταθερή έκφραση• Γενετική τροποποίηση
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Overexpression of GFP on a GFP expressing plant by Agroinfiltration
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Agroinfiltration of GFP-Virp1 fusionI. Whole leaf
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Agroinfiltration of GFP-Virp1 fusionII. protoplasts
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Αναγέννηση μετασχηματισμένων φυτών
• Αρχές:• Τα φυτικά κύτταρα είναι πολυδύναμικά
(polypotent)• H ανάπτυξή τους δεν είναι προκαθορισμένηκαι μπορεί να τροποποιηθεί ορμονικά
• Υπάρχει η δυνατότητα να οδηγηθούν προςαναγέννηση μόνο εκείνα τα κύτταρα που είναιμετασχηματισμένα
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Eξαίρεση: η γενετική τροποποίηση τουArabidopsis που γίνεται με εμβάπτιση τωνάνθεων.
• Ακολουθείεπιλογή τωνσπόρων πουπεριέχουντουλάχιστον έναδιαγονίδιο
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∆ύο γενικές κατευθύνσεις:
• Α. Αναγέννηση μέσω de novoοργανογένεσης
• Β. Αναγέννηση μέσω εμβρυογένεσης
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“orange” cells are “transformable”
“blue” cells can be regenerated
“black” cells can regenerate transformants
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GERMINATION
The surface-sterilized tomato seeds are placed in a petri dish with nutrient medium for germination
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CUT EXPLANTS
After 7 days, the tomato seedings are at the stage where the cotyledons (explants) can be cut for use in transformation.
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CUT EXPLANTS
The cotyledon explants are cut from the seedlings in liquid MS. The wounded cells made by cutting will be the site of DNA transfer from the Agrobacterium.
EXPOSE TO AGROBACTERIUM
After overnight preconditioning on the feeder plates, the explants are competent for transformation and are exposed to the agrobacterium.
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BLOT EXCESS AGROBACTERIUM Excess agrobacterium is blotted from the explants on filter paper to avoid excess growth of the bacteria during co-cultivation.
DNA TRANSFER
The explants are co-cultivated with the agrobacterium for 48 hours to allow time for the agrobacterium to transfer the engineered DNA to the wounded plant cells.
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TOMATO PLANT REGENERATIONThe explants are placed on a medium containing antibiotics for selection and control of the Agrobacterium. The medium also contains the growth regulator, zeatin riboside. Here the formation of an initial callus can be seen on the explant. The callus occurs at the site of wounding and is a result of stimulated plant cell growth caused by the growth regulator, zeatin riboside.Time point: 3 weeks
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SHOOTS BEGINAfter 6 weeks the cotyledon explants are cut from the calli and discarded. Shoots are beginning to form and are transferred to fresh MSZ media.
ROOTING MEDIAThe tomato shoots have regenerated from the calli and are ready to be transferred to rooting media. This media lacks zeatin, but contains kanamycin for selection of transformants.
Time point: 9 weeks
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TRANSGENIC PLANTS
These are fully differentiated transgenic tomato plants rooting in MS media with kanamycin.
Time point: 11 weeks
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TRANSFER TO SOIL
Rooted plantlets are then transferred to boxes containing soil.
The plants must be acclimated to the air, or "hardened off".
The process takes 4-5 days.
Time point: 13 weeks
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TO THE GREENHOUSE
These transgenic plants are ready for transfer to the greenhouse. Shown are two cultivars of tomato; Moneymaker (left), and Motelle (right). Both contain engineered genes.
Time point: 15 weeks
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A GREENHOUSE OF TRANSGENIC PLANTS.
Plants and growth conditions are carefully monitored and controlled. The plants are used for analysis, seed production, or are transplanted to the field.
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Ubiquitin promoter which is monocot specific controlling a bacterial reporter gene, gusA, in transgenic rice.
Particle bombardmentBiolistic Gun
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a-Amylase promoter which is endosperm specific controlling a bacterial reporter gene, gusA, in transgenic rice. The blue stain, is expressed to peak levels at day 6 in the germinating transgenic seed.
Reporter gene under tissue specific promoter
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Expected and unexpected effects of plant transformation –Insertion locus effects
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Expected and unexpected effects in plant transformation-somaclonal variation
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http://mbclserver.rutgers.edu/~dooner/PGRPpage.html
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Expected and unexpected effects of plant transformation – Insertion locus
effects