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

• Main Authors: Ching-Fang Lu, 呂靜芳
• Other Authors: 楊健志
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/03376730886152031340

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 93 === 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.