【摘要】 以中国春小麦幼苗为材料,克隆构建了小麦质体乙酰辅酶A羧化酶(ACCase)的羧基转移酶(CT)重组质粒(RCP18-5),并实现了重组质粒在大肠杆菌中的可溶性高表达.对重组蛋白的性质研究表明,该蛋白具有较强的疏水性,稳定性不高.为改善这种状况,对CT功能域基因进行了截短和延长,同样于大肠杆菌中进行表达.结果表明,仅长度为2325 bp的完整CT功能域基因能以可溶形式表达;2109 bp基因可表达,但是以包涵体形式存在;而1869 bp及3501 bp基因则不能表达.通过圆二色光谱及差热扫描分析对纯化的CT功能域蛋白与除草剂的相互作用进行研究表明,在除草剂存在下,CT蛋白的光谱发生显著变化,说明除草剂与可溶性蛋白之间存在较强的相互作用.更多还原

【Abstract】 Acetyl-CoA carboxylase(ACC) is the key enzyme in fatty acid metabolism that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities,biotin carboxylase(BC) and carboxyltransferase(CT).CT domain has been recognized as the critical target site of herbicide.If the soluble CT protein could be obtained,it would be beneficial for us to investigate the recognition mechanism of ACCase,further to design and develop novel herbicides.In this work,the ACCase CT domain from wheat was cloned and over expressed in E.coli in a soluble form;however the expressed protein exhibited higher hydrophobility and lower stability.In order to improve the situation,different lengths of CT genes was cloned and expressed in E.coli.The results showed that only 2325 bp gene could be expressed in a soluble form,which selected as the material to carry on the subsequent investigation;the 2109 bp gene was expressed in inclusion body state,while the 1869 bp and the 3501 bp gene could not be expressed in E.coli.The interactions of the soluble recombinant CT domain with herbicide were investigated by circular dichroism spectra(CD) and differential scanning calorimeter(DSC).The CD results showed that the spectra of CT protein were changed obviously in the presence of herbicide,indicating there was a strong interaction between the CT domain and herbicide.The DSC assay indicated that the thermal stability of recombinant CT protein was increased in the presence of herbicide.These results will provide important references for rational designing novel herbicide.更多还原