應用降低表現OsMYBS2水稻轉錄調控因子的策略增加mGM-CSF外源蛋白產量之探討

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辛娜嘉(Desyanti Saulina br. Sinaga) 查詢紙本館藏

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論文名稱

應用降低表現OsMYBS2水稻轉錄調控因子的策略增加mGM-CSF外源蛋白產量之探討
(Enhanced Production of Granulocyte-Macrophage Colony-Stimulating Factor Production in Rice Suspension Cell by Knockdown of a Transcription Repressor, OsMYBS2)

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摘要(中)

中文摘要

粒細胞 - 巨噬細胞集落刺激因子（GM-CSF）是一種細胞因子，作用在白血球細胞生長增殖和分化中。在先前的研究中，小鼠GM-CSF (mGM-CSF) 重組蛋白（rmGM-CSF）已成功使用αAmy3 啟動子表達在水稻懸浮液中，細胞培養在2-L生物反應器中，mGM-CSF的最高產量可達24.6 mg / L。糖訊息可通過三個cis-elements，GC box，G box和TATCCA element調控αAmy3啟動子。屬於1R-MYB 家族中的三種MYB 轉錄因子OsMYBS1，OsMYBS2 和OsMYBS3 與αAmy3啟動子的TATCCA element 相互作用, 並在糖調控的αAmy3的表達。 OsMYBS2是repressor，它在含糖培養的水稻細胞中表達，並與TATCCA element結合，使得αAmy3表現降低。為了增加rmGM-CSF的表達，本論文使用RNAi 策略抑制OsMYBS2 的表達,藉由降低OsMYBS2在水稻懸浮培養細胞中表現量，來增加αAmy3 啟動子表達rmGM-CSF。我的研究結果顯示，OsMYBS2基因靜默細胞中，mGM-CSF基因的表達分別在含糖與缺糖情況下較野生型高出1.3〜5.25倍與1.13〜4.4 倍。檢查rmGM-CSF 蛋白質的表達顯示，OsMYBS2靜默細胞中的蛋白質遠高於野生型,在缺糖第5天的OsMYBS2 基因靜默細胞培養液中，rmGM-CSF 的產量比野生型高 1.7-2.5 倍，重組rmGM-CSF的產量最高為29.90μg/ mL.重組rmGM-CSF蛋白質在更長的缺糖處理時,直到第11 天,產量仍然多於野生型。這些研究顯示，降低轉錄因子OsMYBS2，可增加αAmy3所驅動的 mGM-CSF並產生更多的rmGM-CSF。

摘要(英)

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.

關鍵字(中)

★ 應用降低表現OsMYBS2水稻轉錄調控因子的策略增加mGM-CSF外源蛋白產量之探討
★ 辛娜嘉
★ 陸重安博士

關鍵字(英)

★ Enhanced Production of Granulocyte-Macrophage Colony-Stimulating Factor Production in Rice Suspension Cell by Knockdown of a Transcription Repressor, OsMYBS2
★ Desyanti Saulina br. Sinaga
★ Dr. Chung-An Lu

論文目次

Table of contents

Abstract.................................................................................................................i
中文摘要...............................................................................................................ii
Acknowledgement..............................................................................................iii
Table of cotents...................................................................................................iv
Figure list.............................................................................................................vi
Table list.............................................................................................................vii
Suplementarry list............................................................................................viii
1. Inrtoduction......................................................................................................1
1.1 Plant molecular farming..........................................................................................1
1.2 Rice protein expression system...............................................................................1
1.3 GM-CSF recombinant protein................................................................................4
1.4 Study goal.................................................................................................................6
2. Material and methods......................................................................................7
2.1 Plant materials and growth condition....................................................................7
2.2 Cross of transgenic lines mGM-CSF and OsMYBS2-RNAi.................................8
2.3 Primers.....................................................................................................................8
2.4 Genotyping analysis.................................................................................................8
2.5 Sugar starvation treatments...................................................................................8
2.6 RT-PCR analysis......................................................................................................9
2.7 Western blot analysis.............................................................................................10

3. Result..................................................................................................................................11
3.1 Generation of transgenic rice harbor the Ubi::mGM-CSF and Ubi::OsMYBS2RNAi chimeric genes...................................................................11
3.1.1 Description of the αAmy3p::mGM-CSF and the Ubip::OsMYBS2RNAi transgenic rice plants..................................................................................11
3.1.1.1 αAmy3p::mGM-CSF transgenic rice plants...................................11
3.1.1.2 Ubip::OsMYBS2RNAi transgenic rice plants................................11
3.1.2 Generation of OsMYBS2 knockdown and mGM-CSF expressing transgenic rice by dihybrid crossing..........................................................12
3.2 Production of recombinant mGM-CSF in OsMYBS2 knockdown expression of rice suspension cultured cells...............................................................................14
3.2.1 Developing of mGM-CSF and OsMYBS2RNAi dihybrided calli and suspension cells.....................................................................................................14
3.2.2 Expression patterns of mGM-CSF in OsMYBS2 knockdown expression rice suspension cultured cells...............................................................................14
3.2.3 Recombinant mGM-CSF protein production in OsMYBS2 knockdown expression cell lines...............................................................................................16
Discussion...........................................................................................................19
References..........................................................................................................23
Appendix............................................................................................................50
Appendix for chemical solution..................................................................................50

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指導教授

陸重安(Chung-An Lu)

審核日期

2017-6-28

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