Production and characterization of transgenic maize plants designed to improve iron nutrition
Kendall R. Lamkey
The increasing need to find alternative solutions to the devastating effects of iron deficiency anemia has led to the generation of crops with improved nutritional quality traits. Iron is one of the required but limited mineral elements in most diets consumed by people in developing countries who have limited access to supplements or iron-rich diets. With much of the world's population dependent on maize as a major source of their daily calorie intake, research toward improving its nutritional value is deemed necessary. The major objective of this research was to produce and characterize transgenic maize lines with improved iron content. To do this, two studies were carried out. In the first study, maize seed endosperms expressing the soybean ferritin (SoyFer1, GenBank accession number M64337) and the E. coli phytase (appA, Genbank accession number L03375) transgenes, individually and in combination, were produced by stable genetic transformation using particle bombardment. The endosperm-specific super gamma zein promoter was used to drive transgene expression. Transgene presence was confirmed by polymerase chain reaction (PCR), while transgene expression was confirmed in seed samples by western blot analysis. The activity of the phytase enzyme was determined using enzyme bioassays specific for the phytase. The PCR analyses results confirmed the presence of the soybean ferritin and E. coli phytase transgene DNA, implying successful integration into the maize genome. Protein analysis results further confirmed the expression of the transgenes in the maize seed endosperms. The highest phytase enzyme activity obtained from maize seeds was 5.527 units of enzyme per gram of seed (U/g). This was significantly higher (P<0.01) than that of the non-transformed B73 (negative control) at 0.759 U/g of seed.
Because of the potential of transgenes to cause unintended effects on expression and transcription levels of endogenous genes, another study was designed to examine the effect of the soybean ferritin transgene on transcript and protein levels of endogenous maize genes. This was done by comparing changes in mRNA transcript levels in maize roots, leaves and seed endosperm of soybean ferritin PCR negative plants to those of PCR positive ones. High performance liquid chromatography (HPLC) was used for zein protein quantification while inductively coupled argon plasma (ICAP) was used to quantify iron and other divalent minerals in transgenic maize seeds. PCR results showed that the soybean ferritin transgene was successfully introduced into maize seed endosperms and protein analysis confirmed its effective expression in the intended tissue. Messenger RNA abundance of seven tested iron homeostasis genes differed significantly (P<0.001) between seed samples positive and negative for the soybean ferritin transgene. Zein protein levels showed qualitative and quantitative differences between soybean ferritin PCR positive and negative samples. While most peaks were eluted at the same time, one peak (no. 12) appeared in PCR negative samples while peak 13 appeared nearby in PCR positive samples. Some area peaks were significantly higher (P<0.005) in PCR negative samples than in their PCR positive counterparts. Also, there were relative differences in mean peak area of the zein proteins. PCR positive samples had significantly higher (P<0.05) concentrations of calcium, magnesium and iron compared to the PCR negative samples. Similarly, mean percent total nitrogen in PCR negative seed endosperm samples was significantly higher (P<0.05) than that of PCR positive samples. This study has identified some unintended consequences of transgene expression. This information is relevant in increasing our overall understanding of iron homeostasis in plants. The findings reported here offer a starting point for further studies to determine the potential of the transformed maize plants in enhancing iron bioavailability since the phytase expressing plants putatively contain lower content of iron chelating phytates and the ferritin expressing plants will potentially have enhanced amount of iron in their grains.