Progress 09/15/02 to 09/14/05
Outputs
The major objectives of this grant was to: 1) Construct expression vectors for soybean seed-specific expression of the native and recombinant phenylalanine-free gamma zein proteins under control of a lectin promoter (pGL-3); 2) Perform particle bombardment of soybean embryogenic cultures with the zein gene vector constructs and isolate fertile plants; and 3) Perform genetic and protein analyses of transgenic plants. All objectives were met during this funding cycle. To modify the gamma zein gene, the two phenylalanine codons (TTC) were replaced with codons for Tyrosine (TAT) and the codon usage was modified for optimal gene expression in soybean. To make this synthetic gene, several large overlapping primers were created along the gene and Klenow polymerase was used to elongate the fragment. These DNA segments, along with a shuttle vector, were then cut with the appropriate restriction enzymes and ligated together. To minimize the complexity of the ligation reaction,
we divided the gene into three subdivisions, making each one separately. After each was constructed and correctly sequenced, they were all ligated together into a vector containing either the Beta-conglycinin promoter/terminator cassette or soybean lectin promoters. The resulting plasmids were individually introduced into soybean somatic embryogenic cultures along with a plasmid carrying a hygromycin resistance gene. Seven independent events, three under control of the Beta-conglycinin promoter and four under control of the lectin promoter, were recovered and regenerated into fertile plants. Additionally, two transgenic lines were produced expressing the native zein gene. The native and MDZ proteins were isolated from the transgenic lines and Western blot analyses confirmed expression of the transgene. SDS-PAGE confirmed the recovery of an alcohol-soluble fraction contained predominantly the Gamma zein protein. Transgenic lines (both the native and the MDZ gene expressing lines) had a
significant increase in alcohol soluble protein with a 15- to 20-fold increase. Amino acid analysis of seed flour (total seed protein) indicated that amino acid profile was changed. Amino acid analysis of the purified zein was not possible at this time because the micro extraction techniques used (using only a half seed) could not yield enough protein to run an analysis.
Impacts
The current diet of PKU patients consists of a malodorous and distasteful medical formulation of dietary amino acids supplemented with foods low in protein and phenylalanine. The use of soybean-derived PHE-free protein would replace the malodorous and distasteful medical formulas and help patient to have strict low phenylalanine diet. Aside from the medical and nutritional benefits, Kansas agriculture would benefit from our proposal by creating a new market for soybean commodity.
Publications
- Li, Z. and Trick, H.N. 2005. Rapid method for high-quality RNA isolation from seed endosperm containing high levels of starch. Biotechniques 38 (6): 872-876.
- Li, Z., Meyer S., Essig J.S., Liu, Y., Schapaugh, M.A., Muthukrishnan, S., Hainline, B.E., Trick, H.N. 2005. High-level expression of maize gamma-zein protein in transgenic soybean (Glycine max). Molecular Breeding 16:11-20.
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Progress 01/01/04 to 12/31/04
Outputs
Two independent transgenic lines were studied at T1 generations. Seeds containing modified zein gene of these two lines were performed by DNA, RNA and protein analysis. Preliminary results suggested that transgenic seeds (T2 genotype) of these two lines continue expressed mRNA of modified zein gene and its protein. The modified zein protein is being analyzed for amino acid components. At present, these two lines are propagated to T2 plants (or T3 seeds) in the greenhouse for further analysis. More transgenic seeds (T2 and T3) are being collected for later bioassay on PKU mouse. Agrobacterium-mediated transformation was used to produce transgenic soybean seed expressing modified zein, as the Agrobacterium method has some advantages over biolistic method. Currently, several putative transgenic soybean shoots were obtained on hygromycin selection(hygomycin resistant gene was linked to GOI). Further analyses on these plants are being performed.
Impacts
The current diet of PKU patients consists of a malodorous and distasteful medical formulation of dietary amino acids supplemented with foods low in protein and phenylalanine. The use of soybean-derived PHE-free protein would replace the malodorous and distasteful medical formulas and help patient to have strict low phenylalanine diet. Aside from the medical and nutritional benefits, Kansas agriculture would benefit from our proposal by creating a new market for soybean commodity.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Three separate Phenylalanine (PHE)-free zein gene constructions have been designed. Two different promoters are being used to express this gene in seeds. One of the constructions also contains a molecular tag to facilitate the identification and extraction of the transgenic protein once the plants have been produced. Clones from our bombardments from FY2003 have yielded over 19 positive clones for the Zein gene and are currently in the green house setting seeds. Plants from all the PCR positive lines are being done DNA analysis by Southern blots. Twelve independent plants were tested by Southern analysis, and three transgenic plants were confirmed to contain the modified zein genes. Protein expression profiles are underway with immature seeds from these plants. The soybean plants regenerated from experiments with the native zein gene are in the second generation and seeds from one of these plants test strongly for the zein protein. Western-blot analysis indicates good
protein expression from these clones.
Impacts
The current diet of PKU patients consists of a malodorous and distasteful medical formulation of dietary amino acids supplemented with foods low in protein and phenylalanine. The use of soybean-derived PHE-free protein would replace the malodorous and distasteful medical formulas and help the patient have a strict low phenylalanine diet. Aside from the medical and nutritional benefits, Kansas agriculture would benefit from our proposal by creating a new market for soybean commodity.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The PHE-free zein gene has been codon optimized and synthesized by using the annealing of long oligonucleotides. Restriction sites have been designed to facilitate subcloning into the appropriate expression vectors. Sequencing of synthetic gene was performed for confirmation of modified sites. The gene was subcloned behind the beta-conglycinin promoter for seed-specific expression. Two separate bombardment experiments were performed to introduce this gene into soybean cultures. Currently these cultures are under selection. We are also in the process of placing this PHE-free zein gene behind a second seed-specific promoter. After this is accomplished, we will introduce this construct into soybean cultures as well.
Impacts
The current diet of PKU patients consist of a malodorous and distasteful medical formulation of dietary amino acids supplemented with foods low in protein and phenylalanine. The use of soybean-derived PHE-free protein would replace the malodorous and distasteful medical formulas and help patient to have strict low phenylalanine diet. Aside from the medical and nutritional benefits, Kansas agriculture would benefit from our proposal by creating a new market for soybean commodity.
Publications
- No publications reported this period
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