James Bird and Dr. Craig E. Coleman, Botany and Range Sciences
Maize is one of the most abundantly produced grains on earth and is widely used in human and livestock diets. 90% of maize protein is found in the endosperm portion of the seed in the form of small spherical aggregates called protein bodies. These protein bodies consist of four structurally distinct alcohol soluble proteins called á-, â-, ã-, and ä-zeins.1 Initial stages of protein body formation involve â- and ãzeins forming spherical accretions which later enlarge by the accumulation of á-zeins inside the â- and ãzein spheres.2 á-Zeins alone constitute 70% of the protein body and therefore make up a very substantial fraction of maize storage proteins. á-Zeins are also completely void of lysine, an essential amino acid. Maize alone, therefore, lacks adequate levels of lysine to maintain good nutrition. Societies that depend on corn as the main staple of their diet often deprive themselves of sufficient lysine and can become malnourished. If á-zein genes could be modified to include lysine, without disrupting protein body formation and other endosperm properties, the nutritional value of maize would be significantly enhanced.
Our objective has been to modify á-zein genes with carefully placed lysine inserts and to observe the interaction between ã-zeins and á-zeins in protein body formation. Zein interactions are difficult to study within the maize plant itself due to the presence of non-modified proteins in the endosperm produced by other non-modified zein genes in the genome. Tobacco plants transformed with isolated zein genes, however, have proven to be very effective tools to observe zein interactions and protein body formation. Studying modified maize genes in tobacco allows us to observe the interactions between the modified proteins without the interference of similar non-modified proteins. Earlier studies have shown that á-zeins and ã-zeins interact to form stable protein aggregates in transgenic tobacco.3 Whether or not á-zeins with a modified primary structure will aggregate normally to form stable proteins bodies is not yet known. We hypothesize that modified á-zeins with lysine inserts will be stabilized by ã-zeins and form protein bodies in transgenic tobacco endosperm.
Before I began the project, four different point mutations designed to incorporate a lysine codon in the á-zein gene had already been generated. Each gene was then transformed into ten different tobacco plants using Agrobacterium tumafaciens. My first objective was to verify the presence of the modified gene in each of these plants using the polymerase chain reaction (PCR) procedure with primers complementary to the á-zein gene. Several attempts were made to verify the presence of the gene over a period of three months, September 1999 – November 1999, without any success. Considering the sensitivity of the PCR technique, problems with the tests may have been due to a variety of variables such as wrong concentrations of MgCl2, inactive Taq DNA polymerase, or poor DNA extractions from plant leaf tissue. After several attempts with no success we turned to a different protocol with new PCR reagents. Subsequently, many of the plants do contain the point mutated genes and are ready for protein analysis.
While I was working with the PCR to verify transformation, I was also busy performing cross pollenations between ã-zein tobacco plants and the transformed á-zein plants, and collecting seed from those crosses. Several crosses were performed on each of the forty plants and the seeds were stored at -80EC to be analyzed later. Cross pollenations and collections were preformed from September 1999 – February 2000.
In order to qualitatively verify presence of á-zein protein in the endosperm of the collected tobacco seed, we extracted the endosperm protein and preformed SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting with á-zein specific antibodies. The SDS-PAGE and blotting processes were very difficult and time consuming. Several variations had to be made to the protocols. Some success has been observed, indicating that in some of the plants, á-zeins may be expressed, but not in great amounts. These results have been very difficult to repeat. In order to be certain these results are correct we intend to continue with the SDS-PAGE analysis and possibly try different á-zein specific antibodies in our Western blots. Until now, our experimental results in this area are not clear enough to allow any definite conclusions.
Other modifications to the á-zein gene are also being generated. Four á-zein genes with inserts containing multiple lysine codons have been subcloned. The subcloning procedures involved inserting the lysine insert into the á-zein gene and then cloning the gene in Escherichia coli. Once the gene was cloned in E. coli it was then cloned into A. tumafaciens for transformation into tobacco. The subcloning and transformation process was performed April 2000 – August 2000. Two of these new mutations have been transformed into tobacco plants and are currently growing in the Cluff Building greenhouses. Two others are still in the transformation process. Once these plants are mature, they too will be pollinated by ã-zein tobacco plants and the collected seed will be subjected to the same protein analysis procedures.
As of yet we cannot not make any definite conclusions as to whether or not our hypothesis is correct. Steps that must be taken to conclude this project include: verification of protein presence in the multiple lysine insert plants, and quantitative measurement of protein expressed (if any) in both types plants. The project has been very interesting and educational and I appreciate the opportunity to participate this research. I also appreciate ORCA for the research scholarship.
References
- Thompson, G.A., and Larkins, B.A. (1989). Structural elements regulating zein gene expression. Bioessays 10, 108-113.
- Lending, C.R., and Larkins, B.A. (1992). Effect of the floury-2 locus on protein body formation during maize endosperm development. Protoplasma 171, 123-133.
- Coleman, C.E., Herman, E.M., Takasaki, K., and Larkins, B.A. (1996). The maize ã-zein sequesters á-zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm. The Plant Cell 8, 2335-2345. 41