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鱼腥藻PCC7120细菌光敏色素缺失突变体的自催化重组研究

Study on the Auto-assembly of Truncated Phytochrome from Synechocystis sp.PCC7120

    摘要
  • 摘要: 采用聚合酶链式反应(PCR)从鱼腥藻PCC7120DNA中扩增出细菌光敏色素缺失突变体基因aphA(26-320)、aphA(27-320)、aphA(28-320)、aphA(29-320)和aphA(32-320),利用表达载体pET30a进行高效表达,获得的AphA缺失突变体脱辅基蛋白在一定的反应体系下与藻蓝胆素进行了体外重组的研究。研究表明:AphA(26-320)体外重组获得的色素蛋白具有与植物光敏色素相似的可逆光致变色效应,同时酸性尿素变性实验和Zn2+荧光电泳实验显示藻蓝胆素和以上蛋白质发生共价连接。AphA(26-320)与藻蓝胆素重组产物的Pr/Pfr吸收峰处于660/610nm。其他4个缺失突变体,AphA(27-320)、AphA(28-320)、AphA(29-320)、AphA(32-320)和藻蓝胆素的重组产物中则没有发现可逆光致变色信号,表明这些缺失突变体不能和藻蓝胆素发生自催化重组。维系细菌光敏色素AphA与色素自催化连接的裂合酶结构域位于AphA(26-320)包含的肽链之中。

     

    Abstract: Five truncated fragments of aphA gene were amplified from the genomic DNA of Synechocystis sp.PCC7120 by PCR,including aphA(26-320),aphA(27-320),aphA(28-320),aphA(29-320)and aphA(32-320).After expression via the vector pET30,reconstitutions of the expressed mutated apo-proteins with phycocyanobilin(PCB) were studied in vitro under certain conditions.The results showed:The chromoprotein that obtained from the reconstitution of AphA(26-320) had similar photochemical properties of plant phytochrome.The covalent binding of PCB with the chromoprotein was verified further by acid-urea denaturation and UV-induced fluorescence by Zn2+-SDS gel tests.The maximal Pr/Pfr absorption of AphA(26-320) was at 660/610 nm.The other four truncated fragments,AphA(27-320),AphA(28-320),AphA(29-320),AphA(32-320) could not auto-assembly with PCB in the similar conditions,as no reversible photochemistry was detected.The results implicates that the lyase domain of AphA existing in AphA(26-320).

     

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