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was to delete 75% of the T-DNA including those sequences responsible for
oncogenicity (Monsanto).
In addition to the engineered recipient T-DNA, an intermediate vector needed to
be designed for integration of the genes to be transferred into the T-DNA.
Intermediate vectors have three requirements:
selectable markers
o kanamycin resistance
o hygromycin resistance
o bleomycin resistance
scorable markers
o NPT II activity
o opine production
o beta-glucuronidase activity
o chloramphenicol acyl transferase activity
o luciferase activity
cloning site for the gene of interest
o most vectors have only a few sites for integration or a gene
sequences; these sites can be situated next to a promoter (such as
NOS or CaMV 35S) and 3' polyadenylation signals for constitutive
expression or next to a marker gene such as NPT II or CAT for
promoter analysis
sequences for homologous recombination
o T-DNA sequences
o pBR 322 sequences
Scorable markers can also be used as reporter genes. These genes can be
placed under the control of a specific promoter. If the necessary cell machinery is
present, then the promoter will be activated, RNA polymerase will make the
mRNA of the reporter gene and it will be translated. To determine if the gene was
activated, plant tissue is treated with the appropriate substrate and expression
can be monitored. If expression of the reporter gene is detected then the
expression pattern of the promoter can be determined.
Binary vectors offer several advantages to the cointegrate vectors. The primary
advantage is that they are much easier to construct since they do not require
sequences for cointegration into the Ti-plasmid. Cloning sequences into them is
generally easier since you can transform the engineered plasmid into standard E.
coli strains. Finally, specialized sequences can be added to them such as cos
sites so theoretically large fragments of plant DNA can be cloned into them and
the fragment can then be introduced into the recipient A. tumefaciens strain using
packaged phage particles.