Update on PKU transgenic soybean project (Spring 2008)
Over the last year we have been busy evaluating transgenic lines of soybean and
creating new transgenic lines containing phenylalanine (Phe)-free protein. Our first
soybean lines we have produced are in their third and fourth generations (defined and T3
and T4 generations). We have been busy screening plants of these generations identifying
which ones contain the PHE-free gene on both pairs of chromosome. (For those who are
not familiar with genetics, soybean plants (like most organisms) have two sets of
chromosomes; one pair is from the maternal side and one from the paternal side. When
we engineer soybean, the transgene is introduced onto only one of the chromosome pairs.
In order to get the transgene on both sets of the chromosomes (called a homozygous line),
we need to plant seeds from an engineered plant, select the seedlings that contain the Phefree gene, and then collect and grow seeds from these positive plants. Repeating this
process in successive generations we can identify specific soybean lines that are
homozygous for the Phe-free gene.) Using this method we have identified four lines
which are homozygous. Seeds from these plants have been grown in the greenhouse to
increase the number of seeds we have to extract protein. We will be planting these lines
in the field in 2008 to further increase the amount of soybean seeds we have for protein
extraction.
We have also generated 72 new events of putative transgenic soybean tissues
using co-bombardment with pMDZ200 (-conglycinin promoter::PHE-free -zein). The
reason for these new transgenic lines is to try to get additional lines that my have higher
expression profiles of the gene. Twenty-one of 72 events were confirmed to contain our
Phe-free gene. These lines are being regenerated into plants.
In addition to performing genetic analysis, selecting homozygous lines containing
the Phe-free protein and producing new transgenic events, we have been modifying the
protein extraction protocols. Antibodies made to our Phe-free protein are demonstrating
that our extraction protocols are working and we are obtaining mostly our transgenic
protein. However we are also detecting a small amount of a containment soybean protein
that is being co-isolated with our Phe-free protein. This additional protein is a small
amount and may be negligible but we need to send our samples out for amino acid
determination to see how much phenylalanine is actually in our purified sample. We
have been working primarily on the lines which were not homozygous as we needed
those for seed increase. Once we obtain enough homozygous seeds for protein extraction
we anticipate this contaminate protein will significantly be reduced. We will be able to
verify the amount of phenylalanine present in a partially purified sample through amino
acid analysis and take additional steps to clean up the product.
For the coming year we will continue to increase the amount of seed we have
available and will put forth most of our effort on modification of our extraction
techniques. I have a student who will be graduating this June (2008) who has expressed
interest in continuing with this project. Depending upon funding available he will work
on this project modifying our extraction techniques.
Last, Kansas State University has approved my research sabbatical leave for the
Spring of 2009. I will be working with a local biotech company that has experience with
extractions of transgenic proteins from plant tissue on a large scale. In addition to
working with their scientists to learn how to adopt our extraction protocols to large scale
methods, this company has experience in getting nutraceutical proteins though the FDA
regulations, a must for this project. Their expressed interest in the project and
willingness to host my sabbatical leave is a great way to push this project forward.
For this year’s funding request, I will be requesting approximately $30,000 from
multiple sources as in the past year. Last year we obtained a total of $14,070 from
various sources (Indiana PKU and Allied Disorders, Tennessee PKU Foundation, and
USMD) for our research project. The total amount requested will provide funding for a
research associate to work half-time on the project plus supplies necessary for modifying
our extraction procedures and continued analysis of the transgenic seeds.
Harold N. Trick
Department of Plant Pathology
Kansas State University