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Glufosinate tolerance in T14 maize is the result of introducing a
gene encoding the enzyme phosphinothricin-N-acetyltransferase (PAT)
isolated from the common aerobic soil actinomycete, Streptomyces
viridochromogenes, the same organism from which glufosinate was
originally isolated. The PAT enzyme catalyzes the acetylation of
phosphinothricin, detoxifying it into an inactive compound. The PAT
enzyme is not known to have any toxic properties.
The term Recipient organism refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas Parental organisms refers to those that were involved in cross breeding or cell fusion.
pUC origin of replication
Phosphinothricin N-acetyltransferase gene
CaMV 35S terminator
The pat gene introduced is a was synthetic version which
was modified to optimize its expression in plants without altering
the amino acid sequence of the PAT enzyme.
Inserted DNA sequences
The pat gene is derived from the microorganism Streptomyces
viridochromogenes strain Tu494, and encodes for the enzyme
phosphinothricin acetyl transferase (PAT). This modifies and
inactivates the herbicide glufosinate ammonium, and its presence
thus confers to the plant tolerance to this herbicide. The pat gene
was modified at the DNA sequence level to increase its level of
expression in the plant. The modification to the DNA sequence of
the gene did not result in any changes to the amino acid sequence
of the PAT protein. Corn T25 contains the 35S promoter and
terminator sequences derived from cauliflower mosaic virus
(CaMV). The relevant promoter sequences are form pos. 6808
-7437 and the relevant terminator sequences are from pos. 7429
-7632 of the CaMV genome sequence (Franck et al., Cell21. (1980).
pp. 285-294). The 35S promoter directs high level constitutive
expression and is widely used as a promoter for high expression of
bla (0.86) | Ori-pUC (2.63) | P-35S (0.52) >> space
(0.029) >> pat (0.53) >> space (0.019)
>> T-35S( 0.2)
Note: the symbols >> and << indicate the sense and
anti-sense direction of transcription, respectively, between
different elements (typically, promoter - coding sequence -
terminator). The symbol | indicates elements that may be cloned
adjacently in a cassette but that presumably are not transcribed
together. The numbers in parenthesis indicate the approximate
length in Kb of each genetic element.
Molecular analyses of shows that event T14 contains 3 disrupted
copies of the vector. All of these copies appear to contain an
intact P-35S - pat - T-35s cassette and ori-pUC. None of
these copies have an intact bla gene. In one of these
copies bla gene appears to contain an insert and in two
others it is truncated.
Information on the vector
The vector used for the production of the recombinant maize T25 is
plasmid pUC/AC. To construct the plasmid, the synthetic pat gene
was cloned into the Sal1, between the CaMV derived 35S gene
promoter and terminator sequences of the pUC derived plasmid. The
chimeric pat gene cassette (35S promoter: pat:.35S terminator) can
be isolated as a 1.3 kb EcoR1 fragment. The construct
contains no other plant expressible genes. The pUC sequences
include an ampicillin resistance (ampR) gene and a bacterial origin
of replication. The ampR gene has regulatory signals
recognized in bacteria but not functional in transgenic corn cells.
- Resistance to antibiotics
- Resistance to herbicides
The maize line T14 was genetically engineered to express tolerance
to glufosinate ammonium, the active ingredient in phosphinothricin
herbicides (Basta®, Rely®, Finale®, and Liberty®). Glufosinate
chemically resembles the amino acid glutamate and acts to inhibit
an enzyme, called glutamine synthetase, which is involved in the
synthesis of glutamine. Essentially, glufosinate acts enough like
glutamate, the molecule used by glutamine synthetase to make
glutamine, that it blocks the enzyme's usual activity. Glutamine
synthetase is also involved in ammonia detoxification. The action
of glufosinate results in reduced glutamine levels and a
corresponding increase in concentrations of ammonia in plant
tissues, leading to cell membrane disruption and cessation of
photosynthesis resulting in plant withering and death.
Cultured protoplasts obtained from a yellow dent corn line were
transformed using a chemically mediated direct DNA introduction
method. Transformed cell colonies were selected for the presence of
the pat gene by regeneration on medium containing glufosinate
ammonium. The primary transformant T14 was then backcrossed with
parental lines of the yellow dent corn type. The resulting line
displayed field tolerance to phosphinothricin-containing
herbicides, thereby permitting farmers to use this herbicide for
weed control in maize cultivation.