Transcript Plant Transformation

Lecture 7
Manipulation of foreign gene and
secretion of foreign protein
Manipulation of gene expression
• Isolation and Use of Different Promoters
• Targeted Alterations in Plant DNA
• Targeting foreign DNA into chloroplast
genome
Isolation and Use of Different
Promoters
• Use of specialized vectors, called promoter tagging
(labeling) vectors
• approach relies on the Agro bacterium mediated Ti plasmid
transformation system
• a promoter less reporter gene is placed next to the right
border of the Ti plasmid vector
• after transfer of the T-DNA into a plant chromosome the
reporter gene from the vector is situated adjacent to the
plant DNA
• if the T-DNA is inserted at the promoter region of a
functional gene, transcription of the reporter gene occurs
Isolation and Use of Different
Promoters
• For eg: neomycin phosphotransferase (npt)
gene as a reporter
• expression detected by selecting
kanamycin-resistant transformants (Fig A)
• difficult to identify (tag) a promoter that is
active only during a certain developmental
stage or that is induced by a specific
environmental factor
Isolation and Use of Different
Promoters
• To overcome this, two-gene selectable marker
system was devised (Fig B)
• In this case, a hygromycin resistance gene was
placed under the control of a constitutive promoter
next to a promoter less reporter gene within the TDNA
• After hygromycin-resistant transformants are
selected, the transformants can be checked by an
enzyme assay under different conditions for
expression of the reporter gene
Targeted Alterations in Plant DNA
• use of chimeric oligonucleotides
• delivery into a plant cell by microprojectile bombardment
• DNA repair enzymes recognize the mismatches between
the targeted gene and the chimeric oligonucleotide
• During the repair process, the altered DNA is incorporated
into the plant genome
• detected phenotypically
• similar to conventional mutagenesis and selection
procedures
An example of a chimeric oligonucleotide used to change the nucleotide
sequence of plant DNA. The 2’-O-methyl RNA residues are shown in
lower case letters, the DNA residues are shown in uppercase letters
Targeting foreign DNA into
chloroplast genome
• 2 ways for specific foreign protein to be
introduced into the chloroplast or mitochondrion
1.a fusion gene encoding the foreign protein
and additional amino acids that direct the transport
of the protein to the organelle inserted into the
chromosomal DNA, and, after synthesis, the
recombinant protein transported into the targeted
organelle
2.the gene for the foreign protein inserted
directly into chloroplast or mitochondrial DNA
(Fig C)
Fig C. Plasmid vector used for integrating tandem genes
into the chloroplast genome
Secretion of foreign protein
•
•
•
•
•
By rhizo secretion
Three different foreign proteins :
xylanase from Clostridium thermocellum
GFP from the jelly fish Aequorea victoria
human placental secreted alkaline
phosphatase
• directed to the root apoplast using three
different direction signal
Secretion of foreign protein
• genetic construct contained a DNA
fragment encoding a signal peptide
• placed upstream of the gene whose protein
was targeted for secretion
• both the 35S promoter expressed in all types
of plant cells
• mas2’ promoter, expressed in roots direct
the synthesis of target protein in root tissue