Geetha S, Joshi J Beslin, Kumar K K, Arul L, Kokiladevi E, Balasubramanian P, Sudhakar D
Department of Plant Biotechnology, Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu India.
3 Biotech. 2019 Jun;9(6):208. doi: 10.1007/s13205-019-1731-7. Epub 2019 May 9.
A full-length cDNA of gene of , encoding phytase enzyme, was cloned and expressed in BL21 cells and assayed for its activity. The phyA cDNA consisted of 1404 bp, which encoded 467 amino acid residues. The phytase activity of purified phytase was 826.33 U/mL. The gene under the control of endosperm-specific promoters was transformed into an Indian maize inbred line, UMI29, using particle bombardment-mediated transformation method to generate transgenic maize plants over-expressing phytase in seeds. PCR and GUS analyses demonstrated the presence of transgenes in T transgenic plants and their stable inheritance in the T progenies. Three transgenic events expressing detectable level of phytase were characterized by western blot analysis. Phytase activity of 463.158 U/kg of seed was observed in one of the events, JB-UMI29-Z17/2. The phytase activity of transgenic maize seeds was 5.5- to 7-fold higher than the wild-type UMI29 seeds and, consequently, the seeds had 0.6- to 5-fold higher inorganic phosphorus content.
克隆了编码植酸酶的 基因的全长 cDNA,并在 BL21 细胞中进行表达及活性检测。phyA cDNA 由 1404 bp 组成,编码 467 个氨基酸残基。纯化后的植酸酶的植酸酶活性为 826.33 U/mL。利用粒子轰击介导的转化方法,将受胚乳特异性启动子控制的 基因导入印度玉米自交系 UMI29 中,以产生种子中过表达植酸酶的转基因玉米植株。PCR 和 GUS 分析证明 T 代转基因植株中存在转基因,且它们在 T 代子代中稳定遗传。通过蛋白质免疫印迹分析对三个表达可检测水平植酸酶的转基因事件进行了表征。在其中一个事件 JB-UMI29-Z17/2 中,观察到种子的植酸酶活性为 463.158 U/kg。转基因玉米种子的植酸酶活性比野生型 UMI29 种子高 5.5 至 7 倍,因此,种子的无机磷含量高 0.6 至 5 倍。