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作物学报 ›› 2016, Vol. 42 ›› Issue (12): 1798-1804.doi: 10.3724/SP.J.1006.2016.01798

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

植酸酶phyA基因的密码子优化及其在大豆中的表达

寇莹莹,宋英今,杨少辉,王洁华   

  1. 天津大学环境科学与工程学院, 天津 300072
  • 收稿日期:2016-02-28 修回日期:2016-06-20 出版日期:2016-12-12 网络出版日期:2016-06-27
  • 通讯作者: 杨少辉，E-mail: shaohuiyang77@tju.edu.cn
  • 作者简介:第一作者联系方式: E-mail: 15620071109@163.com
  • 基金资助:

    本研究由国家转基因生物新品种培育重大专项(2014ZX0800404B)资助。

Codon Optimization and Expression of phyA Gene in Soybean (Glycine max Merr.)

KOU Ying-Ying, SONG Ying-Jin, YANG Shao-Hui*, and WANG Jie-Hua   

  1. School of Environment Science and Engineering, Tianjin University, Tianjin 300072, China
  • Received:2016-02-28 Revised:2016-06-20 Published:2016-12-12 Published online:2016-06-27
  • Contact: YANG Shao-Hui，E-mail: shaohuiyang77@tju.edu.cn
  • Supported by:

    This work was supported by the National Transgenic Major Project of China (2014ZX0800404B).

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摘要：

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。

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

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