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 T-DNA

• 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