Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola
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Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.
KeywordsAgrobacterium-mediated transformation Aspergillus phytase Brassica napus Codon modification Endoplasmic reticulum retention signal
modified phytase gene
pathogen-related protein S
scaffold attachment regions
This research was supported by the Science Commission of Shanghai, project number 993913002. We thank Lori Osburn for proof-reading the manuscript.
- Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405Google Scholar
- Peng RH, Huang XM, Li X, Shun AJ, Yao QH, Peng YL (2001) Construction of plant binary expression vector containing intron-kanamycin gene and transformation in Nictiana tabacum. Acta Phytophysiol Sin 27:55–60Google Scholar
- Raboy V (1997) Low phytic acid mutants and selection thereof. US patent 5,689,504 US PTOGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Harbor, NYGoogle Scholar
- Sun CC, Zhao H, Fang GH, Wang WR, Li YL, Qian XF (2000) Study on pure-breeding techniques of some rapeseed cultivars (Brassica napus L) with low erucic acid and low glucosinolates in Shanghai's ecological condition. Acta Agric Shanghai 16:45–49Google Scholar
- Yao QH, Huang XM, Peng RH (1999) Synthesis and sequence determination of ACC deaminase gene using successive expressive extension PCR method. Acta Agric Shanghai (Suppl) 15:11–16Google Scholar
- Zhang ZB, Kornegay ET, Radcliffe JS, Denbow DM, Veit HP, Larsen CT (2000a) Comparison of genetically engineered microbial and plant phytases for young broilers. Poultry Sci 79:709–717Google Scholar