臺灣博碩士論文加值系統

綠竹筍乙烯受體BERSJ2之組胺酸激酶功能區塊類似於細菌之二元訊息傳導系統之組胺酸激酶，並包含所有決定激酶活性保守區域 (motifs)。本論文以 Pichia pastoris 表現BERSJ2中HK功能區塊 （BHK）之重組蛋白質以研究其功能，並進一步以N端定序確定重組蛋白質之正確性，且經實驗確定BHK具有自我磷酸化之功能。為了探討組胺酸激酶功能區塊中用以接受磷酸根所必須保守存在之組胺酸所扮演之角色，本論文亦利用點突變法將保守存在H box中之His置換為Gln，並表現此重組蛋白質- BHK(H23Q)。有趣的是，經進一步分析激酶活性時發現 BHK(H23Q) 並未因His被突變而抑制其自我磷酸化之功能。此外，磷酸化胺基酸之酸鹼穩定性之結果顯示，磷酸化之BHK與BHK(H23Q) 皆可穩定存在於3M NaOH 與 1M HCl 溶液中，由此可知，BHK內被磷酸化之胺基酸可能為serine、threonine或是tyrosine。因此仍需進一步證實綠竹筍乙烯受體BERSJ2之自我磷酸化反應確實有別於阿拉伯芥之乙烯受體ETR1。本論文亦利用BERSJ2內GAF功能區塊之重組蛋白質製備綠竹筍乙烯受體之抗體，並以此抗體分析綠竹筍內乙烯受體的表現情形。但由於綠竹筍內乙烯受體蛋白質的表現量過低，故無法直接以免疫染色鑑定綠竹筍內乙烯受體的含量。

The histidine kinase domain in ethylene receptor of bamboo, BERSJ2, contains all the conserved motifs present in a conventional HK of bacterial two-component system. To characterize the function of HK domain in BERSJ2 , the HK domain of bamboo (BHK) was expressed in Pichia pastoris as a c-myc-His-tagged fusion protein. The expressed recombinant BHK protein was identified by N-terminal sequencing and displayed the avtivity of autophosphorylation. To study the function of the conserved histidine that was thought as the phosphorylation site, a mutant BHK(H23Q) was generated. It is interesting that the mutation of histidine did not abolish the autophosphorylation activity . The result showed that BHK and BHK(H23Q) appeared to have higher phosphorylation activity in the presence of Mn2+ , and no activity in the presence of Ca2+. The phosphorylated residue(s) of BHK and BHK(H23Q) was stable in 3M NaOH and 1M HCl which indicated that the phosphorylated amino acids were most likely serine, threonine or tyrosine. Further experiments are needed to demonstrate that autophosp- horylation of BHK protein is indeed different from the one of the Arabidopsis ETR1 ethylene receptors. Polyclonal antibody against BERSJ2 was raised using the recombinant GAF domain as antigen. Due to the possible low yield of ethylene receptor protein, we can not identity the presence of ethylene receptor by Western blot in bamboo.

目錄…………………………………………………………………………………………………………I
縮寫表……………………………………………………………………………………………………IV
摘要………………………………………………………………………………………………………VI
Abstract…………………………………………………………………………………………………VII

第一章 緒論…………………………………………………………………………………………1

1.1.植物賀爾蒙與植物生長發育…………………………………………………………………1
1.1.1植物賀爾蒙-乙烯……………………………………………………………………………1
1.2 乙烯訊息傳遞路徑……………………………………………………………………………2
1.2.1 乙烯受體………………………………………………………………………………………3
1.2.2 乙烯訊息之傳遞……………………………………………………………………4
1.2.3 綠竹筍之乙烯受體……………………………………………………………………………8
1.3 激酶蛋白質………………………………………………………………………………………8
1.3.1 二元訊息傳導系統………………………………………………………………………9
1.3.1.1 組胺酸激酶……………………………………………………………………………10
1.4 研究材料、動機與目的………………………………………………………………………11
1.4.1竹………………………………………………………………………………………………12
1.4.1.1竹的特殊生理現象…………………………………………………………………12
1.4.1.2 綠竹筍…………………………………………………………………………………13
1.4.2 Pichia postoris………………………………………………………………………………13
1.4.23 研究組胺酸激酶之動機………………………………………………………………15
1.5.2以與酵母菌Pichia pastoris 表現綠竹筍組胺酸激酶之動機………………16

第二章 材料與方法……………………………………………………………………………17

2.1 儀器設備…………………………………………………………………………………………17
2.2 材料…………………………………………………………………………………………………18
2.2.1 載體……………………………………………………………………………………………18
2.2.2 菌株……………………………………………………………………………………………18
2.2.2.1 大腸桿菌(E.coli)……………………………………………………………………18
2.2.2.2 酵母菌(Pachia pastoris)……………………………………………………………19
2.2.4 實驗動物 (Experiment animal) ………………………………………………………19
2.3 實驗藥品…………………………………………………………………………………………19
2.3.1 培養基………………………………………………………………………………………19
2.3.1.1 E. coli用培養基…………………………………………………………………………19
2.3.1.2 Pachia pastoris用培養基…………………………………………………………………………
2.3.1.3 動物用培養基……………………………………………………………………………21
2.3.2 藥品試劑………………………………………………………………………………………21
2.4 實驗方法…………………………………………………………………………………………24
2.4.1 聚合酶鏈鎖反應…………………………………………………………………………24
2.4.2 洋菜膠體電泳………………………………………………………………………………24
2.4.3 DNA 之分離與純化……………………………………………………………………24
2.4.4 T-A clonging…………………………………………………………………………………25
2.4.5 質體轉形……………………………………………………………………………………26
2.4.6 轉形菌株的篩選…………………………………………………………………………26
2.4.7 質體的抽取與鑑定………………………………………………………………………27
2.4.8組胺酸激酶表現載體之構築…………………………………………………………28
2.4.8.1尋找乙烯受體中之組胺酸激酶功能區塊……………………………………28
2.4.8.2 引子對的設計…………………………………………………………………………28
2.4.8.3 pGHK之構築…………………………………………………………………………29
2.4.8.4 pPHK 之構築……………………………………………………………………………29
2.4.9以定位點突變法構築組胺酸激酶突變株之表現質體………………………………29
2.4.9.1 引子對的設計……………………………………………………………………………29
2.4.9.2 pPH23Q 之構築………………………………………………………………………30
2.4.10 於P. pastoris中表現重組蛋白質………………………………………………………30
2.4.10.1 酵母菌轉形……………………………………………………………………………30
2.4.11 酵母菌轉形株之鑑定……………………………………………………………………31
2.4.12表現、純化及鑑定重組蛋白質…………………………………………………………32
2.4.12.1 重組蛋白質之表現……………………………………………………………………32
2.4.12.2 重組蛋白質之純化……………………………………………………………………32
2.4.13 蛋白質電泳分離及染色…………………………………………………………………33
2.4.13.1蛋白質電泳………………………………………………………………………………33
2.4.13.2 蛋白質膠片之染色及保存…………………………………………………………34
2.4.13.3 蛋白質免疫染色………………………………………………………………………34
2.4.14 蛋白質 N 端定序…………………………………………………………………………35
2.4.14.1 以胰蛋白酶切除重組蛋白質之N端修飾………………………………………35
2.4.14.2 蛋白質 N 端定序樣品之製備……………………………………………………35
2.4.15 組胺酸激酶之活性鑑定…………………………………………………………………36
2.4.15.1 自我磷酸化鑑定………………………………………………………………………36
2.4.15.2 磷酸化組胺酸之酸鹼穩定度分析………………………………………………36
2.4.16 乙烯受體之專一性抗體製備……………………………………………………………36
2.4.16.1 抗原製備………………………………………………………………………………36
2.4.16.2 抗原乳化………………………………………………………………………………37
2.4.16.3 小鼠免疫………………………………………………………………………………37
2.4.16.4 單株抗體之製備………………………………………………………………………38

第三章 結果與討論……………………………………………………………………………40

3.1 尋找綠竹筍乙烯受體之組胺酸激酶功能區塊 (BHK) ………………………………40
3.2 BHK 之功能預測………………………………………………………………………………40
3.3 以 pichia pastoris 表現 BHK 之重組蛋白質…………………………………………40
3.3.1 組胺酸激酶表現載體之構築……………………………………………………………40
3.3.2 酵母菌之轉形及鑑定……………………………………………………………………41
3.3.2.1 轉形測試………………………………………………………………………………42
3.3.2.2 酵母菌之轉形…………………………………………………………………………42
3.3.2.3 轉形酵母菌株之鑑定………………………………………………………………42
3.3.3 BHK 重組蛋白質之表現與鑑定………………………………………………………43
3.3.3.1 蛋白質之小量表現……………………………………………………………………44
3.3.3.2 最適表現之鑑定………………………………………………………………………44
3.3.3.3 BHK 之純化…………………………………………………………………………45
3.3.3.4 蛋白質定序……………………………………………………………………………46
3.3.3.4.1 胰蛋白酶部分切割BHK………………………………………………………46
3.3.4 BHK(H23Q) 重組蛋白質之表現與鑑定……………………………………………47
3.4 重組蛋白質分析………………………………………………………………………………48
3.4.1 重組蛋白質的構成分析…………………………………………………………………48
3.4.2 重組蛋白質之活性分析…………………………………………………………………48
3.4.2.1 BHK之活性分析………………………………………………………………………48
3.4.2.2 BHK(H23Q)之活性分析………………………………………………………………49
3.5 專一性抗體之製備及檢測……………………………………………………………………50
3.5.1 抗原製備………………………………………………………………………………………50
3.5.2 抗體製備………………………………………………………………………………………51
3.5.2.1 多株抗體之取得及單株抗體之製備………………………………………………51
3.5.2.2 綠竹筍內乙烯受體之分析……………………………………………………………52

第四章………………………………………………………………………………………………53

4.1 總結………………………………………………………………………………………………53
4.2 未來展望……………………………………………………………………………………………55
4.2.1自我磷酸化之受質胺基酸的鑑定………………………………………………………55
4.2.3單株抗體的製備………………………………………………………………………………56

參考文獻…………………………………………………………………………………………57

圖與表…………………………………………………………………………………………………62

附錄

附錄一、ZeocinTM……………………………………………………………………………………92
附錄二、Pichia pastoris 所使用之培養基介紹與使用目的………………………………93
附錄三、Condon usage of Pichia pastoris………………………………………………………94
附錄四、Integration of expression vector into Pichia genome at AOX1…………………95
附錄五、硫酸銨分劃表………………………………………………………………………………96

碩士論文口試問答摘要………………………………………………………………………………97

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