Abstract
BackgroundTransgenic 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.ResultsThe 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 F5 seeds, which was 5.1 times higher than that of the control suspension cell line.ConclusionsOur 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.
Highlights
Transgenic plant suspension cells show economic potential for the production of valuable bioprod‐ ucts
Was used as the female plant, which was crossed with the Maize ubiquitin promoter (Ubip)::OsMYBS2RNAi (Fig. 1A) homozygous transgenic plant line which was used as the male plant
Given that both parental transgenic rice lines were generated under the genetic background of the rice cultivar Tainung 67 (TNG67) by Agrobacterium-mediated transformation, the progenies derived from the dihybrid crossing event were assumed to have only difference of the transgene copy numbers
Summary
Transgenic plant suspension cells show economic potential for the production of valuable bioprod‐ ucts. The sugar starvation-inducible rice αAmy promoter, together with its signal peptide, is widely applied to pro‐ duce recombinant proteins in rice suspension cells. The best-known system of transgenic rice cell suspension culture is based on the rice ALPHA-AMYLASE 3 gene (αAmy, termed RAmy3D) promoter, which is induced strongly by sugar starvation [7]. Several recombinant proteins have been produced using the αAmy promoter and signal peptide in cultured cells of transgenic rice cell suspensions [9,10,11,12,13,14,15,16]. The αAmy promoter has been used widely for sugar-regulated recombinant protein production [9], rice cells have been genetically engineered to improve the αAmy promoter based-recombinant production system. Knockdown of endogenous αAmy expression increased recombinant human GM-CSF production 1.9-fold in transgenic rice cells [17]; silencing of the expression of the CYSTEINE PROTEASE gene in transgenic rice cells resulted in an increase in the yield of recombinant human GM-CSF [18]
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