关键词: 大豆, phyA基因, 密码子优化, 遗传转化

Abstract:

Phytic acid is the main anti-nutritional components in the plant origin food. Reducing the phytic acid content of transgenic plants is an effective way to improve the nutrient utilization rate of soybean. In this research, the codons of Aspergillus ficuum phyA were optimized according to the codon usage bias in soybean. The phyA(b) gene with scheming DNA sequence was synthesized chemically, which was suitable for expression in soybean. The plant expression vector pCBPS-phyA(b) was constructed. In the vector, phyA(b) gene was driven by promoter of soybean lectin gene and signal peptide sequence, and transformed into Jilin 35 via Agrobacterium-mediated method. PCR detection indicated that the target gene was successfully integrated into soybean genome. The protein product of bar gene could be detected in all positive plants by LibertyLink strip analysis. Herbicide leaf painting showed that leaves of wild-type plants were lesioned, while there of transgenic plants remained green. In total, 13 phyA transgenic plants and 19 phyA(b) transgenic plants were verified by semi quantitative RT-PCR. The detection results of phytase activity, inorganic phosphorus content and phytic acid content in T3 transgenic soybean seeds, showed that the artificial phyA(b) gene was successfully expressed in soybean, and the phytase activity in phyA(b) gene transforming plants was significantly higher than that in phyA gene transforming plants. It is suggested that codon optimization can significantly improve the expression of foreign genes.

Key words: Soybean, phyA gene, Codon optimization, Genetic transformation