Sinaga Desyanti Saulina, Ho Shin-Lon, Lu Chung-An, Yu Su-May, Huang Li-Fen
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan City, 320, Taiwan, ROC.
Department of Life Sciences, National Central University, Taoyuan City, 320, Taiwan, ROC.
Plant Methods. 2021 Sep 25;17(1):99. doi: 10.1186/s13007-021-00799-2.
Transgenic plant suspension cells show economic potential for the production of valuable bioproducts. The sugar starvation-inducible rice αAmy3 promoter, together with its signal peptide, is widely applied to produce recombinant proteins in rice suspension cells. The OsMYBS2 transcription factor was shown recently to reduce activation of the αAmy3 promoter by competing for the binding site of the TA box of the αAmy3 promoter with the potent OsMYBS1 activator. In this study, rice suspension cells were genetically engineered to silence OsMYBS2 to enhance the production of recombinant proteins.
The mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene was controlled by the αAmy3 promoter and expressed in OsMYBS2-silenced transgenic rice suspension cells. Transcript levels of the endogenous αAmy3 and the transgene mGM-CSF were increased in the OsMYBS2-silenced suspension cells. The highest yield of recombinant mGM-CSF protein attained in the OsMYBS2-silenced transgenic suspension cells was 69.8 µg/mL, which is 2.5-fold that of non-silenced control cells. The yield of recombinant mGM-CSF was further increased to 118.8 µg/mL in cultured cells derived from homozygous F seeds, which was 5.1 times higher than that of the control suspension cell line.
Our results demonstrate that knockdown of the transcription factor gene OsMYBS2 increased the activity of the αAmy3 promoter and improved the yield of recombinant proteins secreted in rice cell suspension cultures.
转基因植物悬浮细胞在生产有价值的生物制品方面显示出经济潜力。糖饥饿诱导型水稻αAmy3启动子及其信号肽被广泛应用于在水稻悬浮细胞中生产重组蛋白。最近研究表明，OsMYBS2转录因子通过与强效激活剂OsMYBS1竞争αAmy3启动子TA盒的结合位点来降低αAmy3启动子的活性。在本研究中，对水稻悬浮细胞进行基因工程改造以沉默OsMYBS2，从而提高重组蛋白的产量。
小鼠粒细胞巨噬细胞集落刺激因子（mGM-CSF）基因由αAmy3启动子控制，并在沉默OsMYBS2的转基因水稻悬浮细胞中表达。在沉默OsMYBS2的悬浮细胞中，内源性αAmy3和转基因mGM-CSF的转录水平均有所增加。在沉默OsMYBS2的转基因悬浮细胞中获得的重组mGM-CSF蛋白的最高产量为69.8μg/mL，是未沉默对照细胞的2.5倍。在来自纯合F种子的培养细胞中，重组mGM-CSF的产量进一步提高到118.8μg/mL，比对照悬浮细胞系高5.1倍。
我们的结果表明，转录因子基因OsMYBS2的敲低增加了αAmy3启动子的活性，并提高了水稻细胞悬浮培养物中分泌的重组蛋白的产量。
