臺灣博碩士論文加值系統

Harpin 蛋白是經植物病原細菌第三型分泌系統 (Type III secretion system, TTSS )所分泌，具熱穩定性且為translocon的組成蛋白之一，在植物上會引起過敏性反應 (hypersensitive response, HR)，亦有研究指出harpin蛋白可引發植物本身的系統性抗病 (systemic acquire resistance, SAR)以增加植物對於病害的抵抗能力。Pseudmonas syringae pv. averrhoi (Pav)所產生的harpin蛋白HopAK1及HrpZ1即為translocon蛋白群之一；本研究為探討harpin蛋白在病害防治上的應用性以及其於translocon中之功能，構築C端具有6X His短胜&;#32957;的HopAK1及HrpZ1蛋白表現載體，使融合蛋白能以親和性管柱 (Ni-NTA affinity column)進行純化以取得大量的純化蛋白，且純化後的融合蛋白可在非寄主植物菸草上誘發過敏性反應，但純化後的HopAK1-His6融合蛋白會在短時間內相互聚集後沉澱且無法再度回溶，經gel filtration實驗後發現HopAK1蛋白會聚集形成接近2000 kDa 的大型分子；以生物資訊軟體預測HopAK1的次級結構，發現胺基酸序列之特性與α-螺旋的組成比例與其他harpin明顯不同但HopAK1仍具有熱穩定性及引發過敏性反應之特性，因此可能為不同類型之harpin。將非水溶性的HopAK1-His6融合蛋白以含有8M Urea的緩衝液回溶進行純化後以階段透析置換成其緩衝液後，並無明顯沉澱現象，而在SDS-PAGE分析中有位移現象，顯示其電荷可能產生改變，但仍可引發過敏性反應；而HopAK1-His6與HrpZ1- His6融合蛋白引發過敏性反應的最低蛋白濃度為50 ng/μl；以剛果紅溶液分別與HopAK1-His6及HrpZ1- His6融合蛋白混合後以波長300nm至700nm偵測析光值，發現加入HrpZ1- His6融合蛋白後產生藍移現象，而加入HopAK1- His6融合蛋白則沒有改變。將濃度3μg/ml的HopAK1-His6與HrpZ1- His6融合蛋白質溶液以噴灑方式施用於番茄植株後，分別經1天或4天後進行挑戰接種番茄細菌性斑點病菌，實驗結果顯示4天後接種之植株發病情形與控制組相比明顯降低並且確實可以增加系統性抗病基因PR-1a的表現量。此外，利用純化HopAK1-His6融合蛋白製備其多株抗體，並以西方墨點法分析，顯示此HopAK1多株抗體可於稀釋倍率2500比1下偵測到濃度為1 pg/μl的HopAK1純化蛋白之訊號，並且也可偵測到Pav在Hrp minimal medium培養基誘導後產生的HopAK1蛋白的訊號；利用融合蛋白與不具有His tag之HopAK1及HrpZ1進行免疫共沉澱分析，結果顯示HopAK1與HrpZ1間確實存在交互作用。

The harpin proteins of phytobacterial pathogenes are secreted via Type III secretion system (TTSS),which are heat stable and are components of translocon. The previous studies indicated that harpin proteins trigger the hypersensitive response (HR) and systemic acquire resistance (SAR). The harpin proteins, HopAK1 and HrpZ1 of Pseudmonas syringae pv. averrhoi HL1 are components of translocon, To investigate the application of harpin protein on bacterial disease control and the interaction with other translocon proteins, we constructed the HopAK1 and HrpZ1 fused with 6X His short peptide in C terminus which could be purified by using Ni-NTA affinity column. Results indicated the purified HopAK1-His6 and HrpZ1-His6 fusion protein could elicit the HR, but the purified HopAK1-His6 fusion protein were aggregated and precipitated. The results of gel filtration experment showed that HopAK1 was congregated into a complex of 2000 kDa in size. According to the prediction of bioinformatics software, the ratio of α-helix in amino acid sequence of HopAK1 is different from other harpins; nevertheless, it still has harpin activity. The HopAK1 in non-soluble fraction was restored by using buffer containing 8M Urea, then replaced the original buffer by dialysis. Then the precipitation of HopAK1 did not occurr, but molecular weight shown in SDS-PAGE analysis was shiffed to be larger molecular weight, this result revealed that the renatured HopAK1 protein might change the charge, and still could elicit the HR. The threshold concentration of HR elicitation was about 50 ng/μl. In the congo red binding assay, HrpZ1-His6 fusion protein showed the blue shift in peak of absorbance, while the HopAK1-His6 fusion protein had no change in absorbance. In the experiment for the controlling bacterial spot diease of tomato Xanthomonas vesicatoria 1 or 4 days after spraying the purified HopAK1-His6 and HrpZ1-His6, the result indicated that the disease severity of bacterial spot decreased 4 days and the expression of systemic acquired resistance related gene PR-1a was induced after harpin treatment than that without treatment. The western blotting probed with HopAK1 antibody showed that the anti-HopAK1 polyclonal antibodies could detect the purified HopAK1 under concentration 1pg/μl and also detect the secretion of HopAK1 of Pav HL1 grown on Hrp minimal medium, and the co-immunpreciptation assay indicated that HopAK1could interact with HrpZ1.

中文摘要…………………………………………………………………………i
Abstract………………………………………………………………………………………ii
縮寫字對照表…………………………………………………………iii
壹、 緒言……………………………………………………………1
貳、 材料與方法……………………………………………………6
一、 供試菌株、質體及菌體生長環境……………………………6
二、 重組DNA技術……………………………………………………6
三、 聚合酵素連鎖反應 (PCR) …………………………………6
四、 勝任細胞 (competent cell) 的製備………………………7
五、 核酸定序及以蛋白質預測軟體分析結構與功能……………7
六、 利用T7 RNA polymerase dependent system於E. coli表達HopAK1-His6與HrpZ-His6融合蛋白……………………………………7
七、 HopAK1-His6與HrpZ-His6融合蛋白之純化…………………8
八、 製備HopAK1多株抗體 (polyclonal antibody) …………10
九、 西方墨點法 (Western blotting)…………………………11
十、 誘導與偵測HopAK1蛋白在楊桃細菌性斑點病菌內的表現…11
十一、 菸草過敏性反應測定…………………………………………12
十二、 植物RNA萃取及RT-PCR分析…………………………………12
十三、 番茄接種試驗…………………………………………………13
十四、 蛋白質免疫共沉澱 (Co-immueprecipitation) …………14
十五、 剛果紅結合測試(Congo red binding assay) ……………15
參、 結果…………………………………………………………16
一、HopAK1-His6與HrpZ1-His6融合蛋白之純化與分析
(一) 利用T7 RNA polymerase dependent system於E. coli表達HopAK1-His6與HrpZ1- His6融合蛋白…………………………………16
(二) HopAK1-His6與HrpZ1- His6融合蛋白可藉由Ni-NTA層析管柱進行純…化……………………………………………………16
(三) 以gel filtration進一步純化HopAK1- His6融合蛋白……16
(四) 純化之HopAK1-His6與HrpZ1-His6融合蛋白能誘發菸草葉片之過敏性反應……………………………………………………………17
(五) 以生物資訊軟體分析HopAK1與HrpZ1蛋白的次級結構組成…………………………………………………………………………18
(六) HopAK1-His6融合蛋白結構回復性測試及較適緩衝液……18
(七) 剛果紅結合測試………………………………………………18
二、誘導表現HopAK1-His6融合蛋白質以製備HopAK1多株抗體及力價測試…………………………………………………………………19
三、利用anti-HopAK1多株抗體偵測HopAK1在Pav HL1內表現情形…………………………………………………………………………19
四、HopAK1-His6與HrpZ1- His6融合蛋白誘導植物系統性抗病及挑戰接種測試……………………………………………………………19
五、Tranlocon組成蛋白交互作用分析…………………………20
肆、 討論……………………………………………………………22
伍、 參考文獻………………………………………………………26
陸、 圖表……………………………………………………………33

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