Molecular biological study on bule light signaling system in stomatal guard cells.

研究了蚕豆保卫细胞原生质体中蛋白质磷酸化和去磷酸化在气孔蓝光反应中的分子机制。肌球蛋白轻链激酶(MLCK)抑制剂ML-7可抑制依赖蓝光的质子泵出，提示MLCk或MLCK样蛋白可能参与了这种气孔反应。结果表明，从鸡胆中提取的MLCK抗体可与分子质量分别为32kD和17kD的保卫细胞蛋白反应。从文库中分离到两个编码蛋白激酶的基因克隆VFPK1和VFPK2，并测定了它们的核苷酸序列。VFPK1和VFPK2推导的氨基酸序列分别含有271个和219个氨基酸残基，在重叠氨基酸水平上有88%的同源性。氨基酸序列分析表明，它们含有不变的蛋白激活区氨基酸残基，与已知的蛋白激酶略有相似之处(~lt；17%)。在大肠杆菌中表达了VFPK1与谷胱甘肽-S转移酶的融合蛋白，该蛋白具有蛋白激酶活性。这些结果表明，VFPK1基因编码新的蛋白激酶。然而，在气孔反应中的功能和作用还有待于阐明。此外，我们还发现保卫细胞对蓝光的反应中80kD蛋白发生了蛋白磷酸化。MLCK抑制剂抑制蓝光依赖蛋白的磷酸化，支持MLCK样蛋白可能参与气孔保卫细胞蓝光反应的观点。

Molecular mechanisms in blue light response of stomata were investigated regarding protein phosphorylation and dephosphorylation in guard cell protoplasts from Vicia faba. Blue light-dependent proton pumping has been shown to be inhibited by myosin light chain kinase (MLCK) inhibitor ML-7, suggesting that MLCk or MLCK-like protein may be involved in this stomatal response. We found that the antibody against MLCK from chicken gizzard reacted with guard cells proteins with molecular masses of 32 and 17kD.cDNA library of Vicia guard cells was immunoscreened using this polyclonal antibody. Two cDNA clones, VFPK1 and VFPK2, encoding putative protein kinases, were isolated from the library, and their nucleotide sequences were determined. The deduced amino acid sequences of VFPK1 and VFPK2 contain 271 and 219 amino acid residues, respectively, and are 88% identical at overlapping amino acid sequence level. The amino acid sequence revealed that they conserve invariant amino acid residues of kinase domain, and exhibited slight similarity to known protein kinases (<17%). Fusion protein of VFPK1 with glutathione-S-transferase was expressed in E.coli and the protein had protein kinase activity. These results suggest that VFPK1 cDNA encodes new protein kinase. However, the function and role in stomatal responses should be elucidated.Furthermore, we found protein phosphorylation of 80 kD protein in guard cells in response to blue light. The blue light-dependent protein phosphorylation was inhibited by MLCK inhibitor, supporting the notion that MLCK-like protein might be involved in blue light response of stomatal guard cells.