植酸是植物源食品中的主要抗营养成分，降低植酸含量可有效提高大豆的营养利用率。本文根据大豆密码子使用偏好性，对无花果曲霉植酸酶 phyA 基因进行密码子优化，人工合成了适合在大豆中表达的 phyA(b)基因。以pCAMBIA3301为骨架，构建由大豆凝集素基因启动子和信号肽序列调控的植物表达载体pCBPS-phyA(b)。用农杆菌介导法遗传转化吉林35大豆子叶节。PCR检测表明目的基因已初步整合至大豆基因组中； bar试纸条表明所有阳性植株中均能检测到 bar 基因的蛋白产物；除草剂叶片涂抹显示野生型的叶片出现黄化或枯萎现象，而转基因植株叶片表现正常，具除草剂抗性；以半定量RT-PCR共筛选到13株转phyA和19株转phyA(b)阳性转基因大豆植株。通过对转基因大豆T3种子中植酸酶活性、无机磷和植酸磷含量等检测，证明人工基因phyA(b)比phyA在大豆种子中所表达的植酸酶具有更高的活性，说明密码子优化有利于提高外源基因的表达。%Phytic acid is the main anti-nutritional components in the plant origin food. Reducing the phytic acid content of trans-genic plants is an effective way to improve the nutrient utilization rate of soybean. In this research, the codons ofAspergillus ficuumphyA were optimized according to the codon usage bias in soybean. ThephyA(b)gene with scheming DNA sequence was synthesized chemically, which was suitable for expression in soybean. The plant expression vector pCBPS-phyA(b)was con-structed. In the vector,phyA(b) gene was driven by promoter of soybean lectin gene and signal peptide sequence, and transformed into Jilin 35 viaAgrobacterium-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 19phyA(b) transgenic plants were verified by semi quantitative RT-PCR. The detection results of phy-tase 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 inphyA(b)gene transforming plants was signifi-cantly higher than that inphyAgene transforming plants. It is suggested that codon optimization can significantly improve the expression of foreign genes.
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