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The Flavr Savr Tomato
November 25, 2014Lu Tian
Flavr Savr Tomato
• The first FDA approved genetically modified food • Licensed in 1994• Ripening causes production of an enzyme Polygalacturonase
in a gradual increasing level, which is responsible for softening of the tomato and which becomes the cause of rottening
• Calgene introduced a gene in plant which synthesize a complementary mRNA to PG gene and inhibiting the synthesis of PG enzyme.
Antisense RNA
Antisense RNA (asRNA) is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell.
Put PG gene sequences in the reverse orientation into the vector
Transformation
Isolate the nuclei used for in vitro transcription
Isolate the RNA
Hybridize RNA
Measure PG activity
Lycopene determination
Result
PG gene suppression is the result of RNAi
Promoter antisense cDNA poly A addition signal
antisense oligonucleotidemRNA
-antisenseRNA complex
dsRNA
Binding of dsRNA-specific nuclease
cleavage
mRNA is cleaved!
Nuclease-ssRNA complexHybridizes to mRNA
sense
antisense
Southern blot Two linked T-DNAs
Left T-DNA, lanes 1 and 2 (primers 1 and 4);Right T-DNA, lanes 3 and 4 (primers 2 and 3); full insert, lanes 5 and 6 (primers 1 and 2); unique junction region, lanes 7 and 8 (primers 5 and 6).
Diagram of the sequenced 13,621-bp T-DNA insertion and flanking genomic regions. DNA probes (bars C and D) and primers (arrows 1–6) used for confirmation PCRs are indicated.
Northern blot
Result
• Flavr Savr locus contains two linked T-DNAs• siRNAs from transgenes accumulate in Flavr
Savr tomatoes.