阿拉伯芥熱誘導啟動子於菸草毛狀根之表現

與動物細胞及微生物相較，植物細胞有類似動物細胞的蛋白質後轉譯修飾能力，且培養基成本低，無prion，oncogene及動物病毒或內毒素感染之風險，具有作為優良外源蛋白質生產系統之潛力 (Hellwing et al., 2004)。然而，使用持續性啟動子 (constitutive promoter) 大量表現外源蛋白質，由於某些產物對於宿主可能產生毒性，使外源蛋白無法順利表現。利用誘導性啟動子 (inducible promoter) 調控基因表現，以避免宿主生長受到抑制，將是一理想的途徑。本研究中，我們將阿拉伯芥熱休克蛋白HSP18.2之啟動子(-870∼+120, 990bp, +1 為轉錄起始點) 融合gusA作為報導基因之質體轉殖至菸草毛狀根組織中，嘗試建構外源蛋白質熱誘導表現系統。經抗生素耐性、PCR與GUS活性表現量等篩選程序，從436株毛狀根轉殖株獲得最高表現量細胞株GD-3。其最適誘導表現條件為42 °C、2小時熱處理。於回復27 °C培養後，即可測得所表現GUS之mRNA與蛋白質活性，並於24小時達最大比活性 267.6 nmol MU min-1 mg protein-1。此時，熱誘導的GUS表現量佔細胞內可溶性蛋白質之0.10 %。然而，連續加熱並不會使GUS表現量上升，且產生了GUS活性延遲表現之現象。此外，為確認下游轉譯出的N端序列是否會影響此段啟動子的表現，我們將此段啟動子的-870∼-678及+42∼+120剔除，此經剔除後的啟動子和原先啟動子皆具有相同的HSP18.2 啟動子序列以及5’未轉譯區。經過抗生素耐性、PCR與GUS活性表現量等篩選程序，從576株毛狀根轉殖株獲得最高表現量細胞株22-4，並比較GD-3及22-4熱誘導表現行為。結果顯示，在轉錄過程中，此段序列對於轉錄 GUS mRNA並無明顯差異，在轉譯過程中，則發現具有HSP18.2之N端序列的GUS融合蛋白質，其活性可延長至24小時。在本研究中，我們成功建立了毛狀根熱誘導表現的模式系統，此系統除了可以應用於重要外源蛋白質生產研究外，亦可經由熱誘導啟動子作為植物基因釣取的工具，及RNA干擾技術、或轉殖重要次級代謝酵素進入植物組織，以研究次級代謝路徑。

關鍵字

熱誘導啟動子；菸草；毛狀根；阿拉伯芥熱休克蛋白18.2

並列摘要

Plant cells, compared to microbes and mammalian cells, hold great promise as an excellent expression system of exogenous proteins. They possess the advantages of similar post-translational modification, safety, and low cost as compared to animal cells. However, over-expression of certain gene products may reduce cell vitality if the produced protein is harmful to the host. Therefore, it is ideal to control the expression of a gene via a highly specific mechanism such as an inducible promoter through a two-stage culture system to avoid the inhibition of cell growth. In this research, the expression of gusA gene encoding β-glucuronidase (GUS) fused to the Arabidopsis small heat shock protein 18.2 promoter (-870∼+120, 990 bp, HSP18.2 promoter) was investigated in liquid tobacco hairy root cultures. Cell line GD-3 was selected from 436 clones after antibiotic treatment, PCR confirmation, and GUS activity screening. Our results showed that the optimum heat inducible conditions were 42 °C for 2 hr, with a maximum (267.6 nmol MU/mg protein/min) at the 24th hr after recovering the culture at 27 °C. The GUS yield was up to 0.1 % of the total soluble protein in hairy roots. However, prolonging the heating time to 24 hr did not cause higher expression of GUS, and a phenomenon of delayed expression of GUS was observed. We also modified the promoter corresponding to internal deletions of -870 to +678 and +42 to +120 bp in the HSP18.2 promoter. The modified promoter contained the same HSP18.2 promoter sequence between -679 to +1 (transcription start) and the same 5’ UTR (+1∼+41) compared to the original promoter. After antibiotics screening, PCR confirmation and GUS productivity, 576 constructs were selected, and cell line 22-4 was finally selected for further investigation. We compared the heat shock response of the GD-3 and 22-4, and found that the transcription during heat-shock showed no significant difference between these two construct. However, these 77 nucleotides encoding extra amino acid sequences to the N-terminus of GUS were found to maintain the stability of GUS activity for a longer time (24 hr). In this study, a model system inducible for exogenous protein expression in hairy roots has been established. It is not only useful to extrogenous protein expression study, but also helpful for gene tagging and secondary metabolism research in plant biotechnology.

並列關鍵字

heat induced promoter,hairy root ； GUS,HSP18.2 ； tobacco ； Arabidopsis

參考文獻

Almoguera C, Rojas A, Jordano J (2002) Reversible heat-induced inactivation of chimeric β-glucuronidase in transgenic plants. Plant Physiol. 129: 333-341

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Binns AN (2002) T-DNA of Agrobacterium tumefaciens: 25 years and counting. Trends Plant Sci. 7: 231-233

Boetti H, Chevalier L, Denmat LA, Thomas D, Thomasset B (1999) Efficiency of physical (Light) or chemical (ABA, Tetracycline, CuSO4 or 2-CBSU)-stimulus-dependent gus gene expression in tobacco cell suspensions. Biotechnol. Bioeng. 64: 1-13

Chilton M-D, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempé J (1982) Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295: 432-434

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阿拉伯芥熱誘導啟動子於菸草毛狀根之表現

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