| 引用本文: | 赵菲佚, 焦成瑾, 陈 荃, 马伟超, 安建平, 呼丽萍.拟南芥PKS5激酶点突变体外表达与磷酸化测试[J].广西植物,2015,35(3):408-413.[点击复制] |
| ZHAO Fei-Yi, JIAO Cheng-Jin, CHEN Quan,
MA Wei-Chao, AN Jian-Ping, HU Li-Ping.In vitro expression and determination of phosphorylation activity of point mutants of the PKS5 kinase in Arabidopsis[J].Guihaia,2015,35(3):408-413.[点击复制] |
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| 拟南芥PKS5激酶点突变体外表达与磷酸化测试 |
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赵菲佚1,2, 焦成瑾1, 陈 荃1, 马伟超1, 安建平1, 呼丽萍1,2
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1. 天水师范学院 生命科学与化学学院, 甘肃 天水 741000;2. 甘肃大樱桃工程技术中心, 甘肃 天水 741000
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| 摘要: |
| PKS5(protein kinase SOS2-like 5)虽为拟南芥(Arabidopsis thaliana)中介导植物响应外界高pH的蛋白激酶,但其关键功能结构域尚未被确定。该研究用PCR对PKS5不同位置点突变形式进行克隆,并在原核系统中进行表达,得到PKS5不同的点突变蛋白; 使用激酶通用底物MBP(myelin basic protein)及PKS5体内特异底物AHA2(A. thaliana isoform of the PM H+-ATPase,拟南芥质膜质子泵等位形式之一)对PKS5点突变蛋白磷酸化活性进行了测试。结果表明:点突变PKS5-2失去了激酶活性,PKS5-4、PKS5-5、PKS5-9自磷酸化与MBP磷酸化活性与PKS5相比无差异; 而与PKS5相比,点突变PKS5-6和PKS5-7自磷酸化及对AHA2的磷酸化活性升高,且PKS5-7活性高于PKS5-6。说明PKS5特定位置点突变改变PKS5的自磷酸化及底物磷酸化活性水平,不同位置的点突变对其磷酸化活性的影响存在差异。研究结果可为确定PKS5功能结构域及体内作用机理提供依据。 |
| 关键词: PKS5 点突变 蛋白表达 磷酸化活性 |
| DOI：10.11931/guihaia.gxzw201306008 |
| 分类号:Q948.112 |
| 基金项目: |
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| In vitro expression and determination of phosphorylation activity of point mutants of the PKS5 kinase in Arabidopsis |
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ZHAO Fei-Yi1,2, JIAO Cheng-Jin1, CHEN Quan1,
MA Wei-Chao1, AN Jian-Ping1, HU Li-Ping1, 2
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1. School of Life Sciences and Chemistry, Tianshui Normal Universiry, Tianshui 741000, China;2. Gansu Engineering and Research Center for Sweet Cherry, Tianshui 741000, China
1. School of Life Sciences and Chemistry, Tianshui Normal Universiry, Tianshui 741000, China;
2. Gansu Engineering and Research Center for Sweet Cherry, Tianshui 741000, China
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| Abstract: |
| In Arabidopsis,PKS5(protein kinase SOS2-like 5), a serine-threonine kinase, involves in the response to the external high pH stress based on the study of its loss-of-function mutant. Whereas, the fine functions of the domains resided in PKS5 are not currently well determined. We report here the dissection of domains of PKS5 in the activity of phosphorylation against MBP(myelin basic protein)and AHA2(one of the Arabidopsis thaliana isoform of PM H+-ATPases), which is the specific substrate of PKS5 in vivo, using the assay of phosphorlation in vitro via expressing the distinct PKS5 mutant versions in bacteria using the PKS5 cloning from plants employing PCR approach. The results showed that the point mutated PKS5-2 lost its activity, PKS5-4, PKS5-5 and PKS5-9 displayed no difference in autophosphorylation and the MBP phosphorylation. Moreover,autophosphorylation and the AHA2 phosphorylation of the point mutated PKS5-6 and PKS5-7 increased compared with PKS5 and the PKS5-7 activity was higher than PKS5-6. Taken together, the specific point mutations in PKS5 altered its activity of autophosphorylation and the substrate phosphoylation of BMP and AHA2. The effects due to the alteration of mutations resided in PKS5 on the activity of phosphorylation were comparable within the various PKS5 mutated protein versions. The results of this study would provide the basis for pinpointing the functional domains of PKS5 and the mechanism of functions of PKS5 in planta. |
| Key words: PKS5 point mutant protein expression phosphorylation activity |
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