綠竹筍乙烯受體之組胺酸激酶功能區塊的表現與檢定

Expression and Characterization of the Histidine Kinase Domain of Bamboo Ethylene Receptor

呂靜芳(Ching-Fang Lu)

指導教授 ： 楊健志

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.10093

摘要

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。

關鍵字

乙烯受體 ； 組胺酸激

並列摘要

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.

並列關鍵字

ethylene ； histidine kinase

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