Enhanced Production of Granulocyte-Macrophage Colony-Stimulating Factor Production in Rice Suspension Cell by Knockdown of a Transcription Repressor, OsMYBS2

• Main Authors: Desyanti Saulina br. Sinaga, 辛娜嘉
• Other Authors: Chung-An Lu
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/nnbpvb

碩士 === 國立中央大學 === 生命科學系 === 105 === Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that functions in cell proliferation and differentiation, especially in the growth of white blood cells. In previous studies, the expression of recombinant protein of mGM-CSF (rmGM-CSF) was drove by the αAmy3 promoter in rice suspension cultured cells. The cells were scaled up successfully in a 2-L bioreactor and the highest yield of rmGM-CSF was 24.6 mg/L. Sugar regulates the rice αAmy3 promoter through cis-elements, the GC box, the G box, and the TATCCA element. Three MYB transcription factors OsMYBS1, OsMYBS2, and OsMYBS3, that belong to 1R-MYB, interact specifically with the TATCCA element of αAmy3 promoter and play roles in sugar-regulated αAmy3 expression. The OsMYBS2 is repressor, that is expressed in sugar fed cells, can bind to the TATCCA element, so only a low level of αAmy3 is expressed. To increase the production of rmGM-CSF, the RNAi strategy was used to repress expression of OsMYBS2. The production of the mGM-CSF by aAmy3 promoter-base rice recombinant protein expression system was examined in OsMYBS2 knockdown rice suspension cultured cells. Our results showed that the expression of mGM-CSF gene was higher 1.3 – 5.25 fold under sugar starvation and 1.13 – 4.4 fold in sugar available condition in the OsMYBS2 knockdown cells than wild type. The Western blotting analysis showed that rmGM-CSF protein in OsMYBS2 knockdown was much higher than wild type. In day 5, productioon of rmGM-CSF in OsMYBS2 knockdown cell lines were higher 1.7 – 2.5 fold than in wild-type cell line. The highest yield of recombinant rmGM-CSF was 29.90 µg/mL in 5 days. The high rmGM-CSF productions in OsMYBS2 knockdown cell lines were still able to detect in 11 days of sugar starvation, as compared to in wild type. These studies demonstrated that knockdown of a transcription repressor OsMYBS2 increase the αAmy3 drove mGM-CSF production in sugar starvation of suspension rice cells.