BcMid1蛋白原核表达及其与绿原酸的作用

Prokaryotic expression of BcMid1 and its interaction with chlorogenic acid

Mid1蛋白是真菌钙通道蛋白,在维持细胞钙稳态方面发挥着重要作用。文章为探讨绿原酸对灰霉(Botrytis cinerea)生长的抑制与钙之间的关系,利用平板实验观察绿原酸存在条件下外源钙离子对灰霉生长的影响,并构建BcMid1原核表达载体,在大肠杆菌Rosetta菌株中诱导BcMid1蛋白表达,利用绿原酸的荧光特性探究BcMid1蛋白与绿原酸的体外结合情况。结果显示,钙离子可以降低绿原酸对灰霉的生长抑制。成功构建的pET-28a∶∶BcMid1重组载体可以在Rosetta菌株中表达BcMid1蛋白,SDS-PAGE和荧光定量检测发现绿原酸和BcMid1蛋白可以结合,结合常数为3.93×10^(4)mol/L。结果表明,绿原酸对灰霉的抑制与钙摄入相关,并且BcMid1蛋白是绿原酸的潜在作用靶点。该文为进一步研究绿原酸对灰霉的抑制机理奠定了基础。

Mid1 protein is a fungal calcium channel protein that plays an important role in maintaining cellular calcium(Ca^(2+))homeostasis.To study the relationship between the inhibitory effect of chlorogenic acid on the growth of Botrytis cinerea and Ca^(2+),a plate inhibition experiment was applied to observing the effects of exogenous Ca^(2+)on B.cinerea growth with the presence of chlorogenic acid.A BcMid1 prokaryotic expression vector was constructed and the BcMid1 protein was expressed in E.coli Rosetta.The fluorescence characteristics of chlorogenic acid were used to investigate the in vitro binding of BcMid1 protein to chlorogenic acid.The results showed that Ca^(2+)could reduce the inhibition of chlorogenic acid on the growth of B.cinerea.The pET-28a∶∶BcMid1 recombinant vector was successfully constructed and BcMid1 protein was expressed in Rosetta strain.SDS-PAGE and fluorescence quantification showed that chlorogenic acid and BcMid1 protein could bind,and their binding affinity was 3.93×10^(4) mol/L.These results indicate that the inhibition of chlorogenic acid on B.cinerea is associated with Ca 2+influx of B.cinerea cells,and BcMid1 protein is a potential target of chlorogenic acid.It lays a foundation for further study of the antifungal mechanism of chlorogenic acid on B.cinerea.