Abstract
Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominately in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.
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References
An G, Mitra A, Choi HK, Costa MA, An K, Thornburg RW, Ryan CA (1989) Functional analysis of the 3’ control region of the potato wound-inducible proteinase inhibitor II gene. Plant Cell 1:115–122
Armstrong CL, Green CE, Philips RL (1991) Development and availability of germplasm with high type II culture formation response. Maize Genet Coop News Lett 65:92–93
Asada K, Tanaka K, Kasai Z (1969) Formation of phytic acid in cereal grains. Ann NY Acad Sci 165:801–814
Austin S, Bingham ET, Koegel RG, Mathews DE, Shahan MN, Straub RJ (1994) An overview of a feasibility study for the production of industrial enzymes in transgenic alfalfa. In: Bajpai RK, Prokop A (eds) Recombinant DNA Technology II. New York Academy of Sciences, New York, pp 234–244
Brinch-Pedersen H, Olesen A, Rasmussen SK, Holm PB (2000) Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase. Mol Breed 6:195–206
Brinch-Pedersen H, Sorensen LD, Holm PB (2002) Engineering crop plants: getting a handle on phosphate. Trends Plant Sci 7:118–125
Chiera JM, Finer JJ, Grabau EA (2004) Ectopic expression of a soybean phytase in developing seeds of Glycine max to improve phosphorus availability. Plant Mol Biol 56:895–904
Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218
Coello P, Maughan JP, Mendoza A, Philip R, Bollinger DW, Veum TL, Vodkin LO, Polacco JC (2001) Generation of low phytic acid Arabidopsis seeds expressing an E. coli phytase during embryo development. Seed Sci Res 11:285–291
Cosgrove DJ (1966) The chemistry and biochemistry of inositol polyphosphates. Rev Pure Appl Chem 16:209–215
Denbow DM, Grabau EA, Lacy GH, Kornegay ET, Russell DR, Umbeck PF (1998) Soybeans transformed with a fungal phytase gene improve phosphorus availability for broilers. Poult Sci 77(6):878–881
Drakakaki G, Marcel S, Glahn RP, Lund EK, Pariagh S, Fisher R, Christou P, Stoger E (2005) Endosperm specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Mol Biol 59:869–880
Gibson DM, Ullah AH (1988) Purification and characterization of phytase from cotyledons of germinating soybean seeds. Arch Biochem Biophys 260:503–513
Greiner R, Konietzny U, Jany K (1993) Purification and characterisation of two phytases from Escherichia coli. Arch Biochem Biophys 303:107–113
Hegeman CE, Grabau EA (2001) A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings. Plant Physiol 126:1598–1608
Hong CY, Cheng KJ, Tseng TH, Wang CS, Liu LF, Yu SM (2004) Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds. Transgenic Res 13:29–39
Hood EE, Bailey MR, Beifuss K, Magallanes-Lundback M, Horn ME, Callaway E, Drees C, Delaney DE, Clough R, Howard JA (2003) Criteria for high-level expression of a fungal laccase gene in transgenic maize. Plant Biotechnol J 1:129–140
Joanin P, Gigot C, Philipps G (1992) Nucleotide sequence and expression of two cDNA coding for two histone H2B variants of maize. Plant Mol Biol 20:581–588
Jongbloed AW, Kemme PA, Mroz Z (1996) Phytase in swine rations: impact on nutrition and environment. In: BASF Technical Symposium. January 29, 1996, Des Moines, IA, BASF, Mount Olive, NJ, pp 44–69
Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti J (1998) Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl Environ Microbiol 64:2079–2085
Latta M, Eskin M (1980) A simple and rapid colorimetric method for phytate determination. J Agric Food Chem 28:1313–1315
Lei XG, Ku PK, Miller ER, Yokoyama MT, Ullrey DE (1994) Calcium level affects the efficacy of supplemental microbial phytase in corn-soybean meal diets of weanling pigs. J Anim Sci 72:139–143
Lei XG, Stahl CH (2001) Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl Microbiol Biotechnol 57:474–481
Li J, Hegeman CE, Hanlon RW, Lacy GH, Denbow MD, Grabau EA (1997) Secretion of active recombinant phytase from soybean cell-suspension cultures. Plant Physiol 114:1103–1111
Lucca P, Hurrell R, Potrykus I (2001) Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theor Appl Genet 102:392–397
Mitchell DB, Vogel K, Weimann B, Pasamontes L, van Loon APGM (1997) The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila. Microbiology 143:245–252
Mullaney EJ, Daly CB, Ullah AH (2000) Advances in phytase research. Adv Appl Microbiol 47:157–199
Pen J, Verwoerd TC, van Paridin PA, Beukeder RF, van der Elzen PJM, Geerse K et al (1993) Phytase-containing transgenic seed as a novel feed additive for improved phosphorus utilization. Bio/Technol 11:811–814
Ponstein AS, Bade JB, Verwoerd TC, Molendijk L, Storms J, Beudeker RF et al (2002) Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product. Mol Breed 10:31–44
Ravindran V, Bryden WL, Kornegay ET (1995) Phytates: occurrence, bioavailability and implications in poultry nutrition. Poult Avain Bio Rev 6:125–143
Reddy NR, Sathe SK, Salunkhe DK (1982) Phytates in legumes and cereals. Adv Food Res 28:1–92
Richardson AE, Hadobas PA, Hayes JE (2001) Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant J 25:641–649
Schroder B, Breves G, Rodehutscord M (1996) Mechanisms of intestinal phosphorus absorption and availability of dietary phosphorus in pigs. Dtsch Tierarztl Wochenschr 103:209–214
Streatfield SJ, Lane JR, Brooks CA, Barker DK, Poage ML, Mayor JM, Lamphear BJ, Drees CF, Jilka JM, Hood EE, Howard JA (2003) Corn as a production system for human and animal vaccines. Vaccine 21:812–815
Tomes DT (1995) Direct DNA transfer into plant cell via microprojectile bombardment. In: Gamborg OL, Philipps GC (eds) Plant cell tissue and organ culture: fundamental methods. Springer-Verlag Publisher, Berlin, pp 197–213
Ullah AH (1988) Aspergillus ficuum phytase: partial primary structure, substrate selective, and kinetic characterization. Prep Biochem 18:459–461
Ullah AH, Sethumadhavan K, Mullaney EJ, Ziegelhoffer T, Austin-Phillips S (1999) Characterization of recombinant fungal phytase (phyA) expressed in tobacco leaves. Biochem Biophys Res Commun 264:201–206
Ullah AH, Sethumadhavan K, Mullaney EJ, Ziegelhoffer T, Austin-Phillips S (2002) Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase. Biochem Biophys Res Commun 290:1343–1348
Urbano G, Lopez-Jurado M, Aranda P, Vidal-Valverde C, Tenorio E, Porres J (2000) The role of phytic acid in legumes: antinutrient or beneficial function? J Physiol Biochem 56:283–294
Vaintraub IA, Lapteva NA (1988) Colorimetric determination of phytate in unpurified extracts of seeds and the products of their processing. Anal Biochem 175:227–230
Van Droogenbroeck B, Cao J, Stadlmann J, Altmann F, Colanesi S, Hillmer S, Robinson DG, Van Lerberge E, Terryn N, Van Montagu M, Liang M, Depicker A, De Jaeger G (2007) Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proc Natl Acad Sci USA 104:1430–1435
Verwoerd TC, van Paridon PA, van Ooyen AJ, van Lent JW, Hoekema A, Pen J (1995) Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol 109:1199–1205
Vohra A, Satyanarayana T (2003) Phytases: microbial sources, production, purification, and potential biotechnological applications. Crit Rev Biotechnol 23(1):29–60
Wodzinski RJ, Ullah AHJ (1996) Phytases. Adv Appl Microbiol 42:263–302
Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R, Oesterhelt G, Lehmann M, van Loon AP (1999a) Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance. Appl Environ Microbiol 65(2):359–366
Wyss M, Pasamontes L, Friedlein A, Remy R, Tessier M, Kronenberger A, Middendorf A, Lehmann M, Schnoebelen L, Rothlisberger U, Kusznir E, Wahl G, Muller F, Lahm HW, Vogel K, van Loon AP (1999b) Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl Environ Microbiol 65:367–373
Yao B, Yuan TZ, Wang YH. Cao SS, Wang YR, Shi XY, Fan YL (2001) Cloning of neutral phytase gene nphy from Bacillus subtilis and its expression in Escherichia coli. Sheng Wu Gong Cheng Xue Bao 17:11–15
Yao B, Zhang CY, Wang JH, Fan YL (1998) Recombinant Pichia pastoris overexpressing bioactive phytase. Sci China C 41:330–336
Zhang ZB, Kornegay ET, Radcliffe JS, Denbow DM, Veit HP, Larsen CT (2000) Comparison of genetically engineered Aspergillus and canola in weanling pig diets. J Anim Sci 78:2868–2878
Acknowledgments
We thank Mr. Li Denghai, Prof. Zhang Shihuang and Dr. Li Mingshun for their help. We acknowledge Key National Project of China Crop Genetic Resources and Gene Improvement for providing greenhouse and other facilities. This work was partially supported by the National Basic Research and development Program of China (973 Program) (No. 2005CB120905) and a research grant from Pioneer Hi-Bred International, a DuPont Company.
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Chen, R., Xue, G., Chen, P. et al. Transgenic maize plants expressing a fungal phytase gene. Transgenic Res 17, 633–643 (2008). https://doi.org/10.1007/s11248-007-9138-3
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DOI: https://doi.org/10.1007/s11248-007-9138-3