Center for Integrated Animal Genomics

1. P198 P2P199 • Research Experience in Molecular Biotechnology & Genomics • Summer 2008 Center for Integrated Animal Genomics Mahwish A. Hafeez, Lorena B. Moeller, Kan Wang MOLECULAR ANALYSIS OF TRANSGENIC CORN EXPRESSING AN ENDO-1,4-BETA-GLUCANASE FROM RUMINOCOCCUS ALBUS INTRODUCTION The ethanol industry has been growing considerably as the demand for alternate fuel sources continues to rise. There is nonetheless considerable debate regarding the high production costs associated with bio-ethanol as a replacement for fossil fuels in vehicles and the energy and pollution balance associated with ethanol production and the substantial amount of land needed which is currently used for crops (Rothman et al., 1983) One attractive alternative to current methods for ethanol production is to use cellulosic biomass as starting material for enzymatic conversion of cellulose to fermentable sugars. The enzymes required for this process (endo-1,4-beta-glucanase, exo-cellobiohydrolase, beta-glucosidase) are currently produced using recombinant microorganisms which greatly add to the high production costs. Using genetic engineering, crops can be used to produce these polysaccharide-degrading enzymes (cellulases) in a more efficient and cost-effective manner (Biswas et al., 2006). Since maize is the most widely grown annual crop cultivated worldwide and 45% of its plant material is comprised of cellulose, it is an ideal candidate for cellulase production and subsequently for biomass fuel production (Biswas et al., 2006). This could also be used in biomass degradation for utilization as animal feed or a raw material in food industries (Kawazu et al., 1999). EgI is a thermostable endo-1,4--glucanase that cleaves soluble cellulose, releasing cellobiose (Ziegelhoffer et al., 2001; Roberts et al., 1988). The Wang lab has previously produced two transgenic maize lines, P198 and P2P199, through insertion of the mEgI gene which codes for an optimized endo-1,4--glucanase. The gene was taken from the rumen bacterium, Ruminococcus albus. The P198 and P2P199 constructs, respectively, are under regulation of the maize ubiquitin 1 promoter and maize gamma zein promoter which instigate constitutive expression (Christensen & Quail, 1996) and endosperm specific expression (Marks et al., 1985), respectively. Thirteen independent events of P198 and eighteen independent events of P2P199 have been generated and partially characterized molecularly through PCR, Southern blot and zymmogram analysis. RESULTS Figures 1 to 3. A microplate based protocol for glucanase activity determination was established using glucose standards to generate calibration curves. A baseline for endogenous glucanase activity was established using non-transgenic B73 lines as reference material, over a period of 3 consecutive days to test reproducibility. Enzyme activities were normalized on total aqueous extractable protein. 1 2 3 Figures 4 – 6. Endosperms of transgenic line P2P199 and leaves of transgenic line P198 show endoglucanase activity comparable or higher than that observed in non-transgenic B73 controls. 4 6 5 OBJECTIVES The purpose of this investigation is to analyze both lines for transgene expression. The objectives are to establish a method for quantification of glucanase activity in a microplate based format and then to investigate the extent to which enzyme activity is higher in the transgenic plants as compared to negative controls. Figures 7 – 8. Segregating endosperms of transgenic line P2P199-31-3-11 show endoglucanase activity and presence of the mEgI gene. PLANT MATERIALS AND METHODS Leaves of the line P198 and endosperms of the line P2P199 were used for protein extraction. Total soluble extractable protein was measured using the Bradford assay. Glucanase activity was measured in duplicate using a modified microplate-based method for reducing sugar determination using carboxymethylcellulose and dinitrosalycilic acid, based on Xiao, Storms and Tsang, 2005. Leaves of the line P198 and embryos of the line P2P199 were used for phenol-based DNA extraction. DNA was quantified using a nanodrop and 100-300ng of genomic DNA were used for PCR analysis. The oligonucleotide primers 5’-CTGACCTGGAGTGGTACTTC-3’ (forward) and 5’-ATTAAATGTATAATTGCGGG-3’ (reverse) were designed and used to amplify a fragment from the 3’ end terminus of the mEg1 gene into the 5’ end of the Nos terminator. The positive controls were plasmid pWL-2 and known positive P198 and P2P199 plant samples. The negative control was water without a template. PCR products were analyzed by electrophoresis in a 1% agarose gel with ethidium bromide and photographed under ultraviolet light. A band at 300bp revealed the presence of the mEg1 gene. Data analysis was carried out on in Excel. 7 8 CONCLUSIONS This investigation successfully concluded that in most transgenic lines in constructs P198 and P199, samples that had the mEgI gene as verified by PCR had higher endo-1,4-beta-glucanase activity than those that didn’t. This suggests the creation of valid genetically modified maize crops that can produce endo-1,4-betaglucanase for industrial and commercial purposes. Future work may include PCR analysis of more P198 and P199 samples as well as statistical analysis. SELECTED REFERENCES Biswas, G., Ransom, C. Sticklen, M. Expression of biologically active Acidothermus cellulolyticus endoglucanase in transgenic maize plants. Plant Science 171(5), 617-623. Rothman, H., Greenshields, R., & Rosillo Call, F. (1983). The alcohol economy : fuel ethanol and the Brazilian experience. London: F. Pinter. Xiao, Z., Storms, R., Tsang, A. (2005). Microplate-based carboxymethylcellulose assay for endoglucanase activity. Anal biochem(342), 176-178. Ziegelhoffer, T., Raasch, J., Austin-Phillips, S. (2001). Dramatic effects of truncation and sub-cellular targeting on the accumulation ofrecombinant microbial cellulase in tobacco. Molecular Breeding(8), 147-158. ACKNOWLEDGMENTS The author would like to thank the National Science Foundation’s REU Program and Iowa State University for the opportunity to do this research. This author is also very grateful to Dr. Kan Wang, Lorena Moeller, and the Plant Transformation Facility at Iowa State for their help and support Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371