摘要
目的:蒙古黄芪病程相关蛋白(Astragalus membranaceus pathogenesis-related protein-10,AmPR-10)具有核酸酶活性,对黄芪生长发育的抗病过程有重要作用。为进一步提高野生型AmPR-10的活性,设计了相应突变体,并分析其表达量和活性较野生型的变化。方法:通过理性分析发现野生型AmPR-10的Ser(84)、Gly(140)、Glu(141)突变后会影响目标蛋白的空间结构、降低底物单核苷酸的对接自由能,据此设计合成了两个突变体M1:S84D+G140V、M2:S84D+G140V+E141M,并对野生型AmPR-10与M1、M2分别进行外源低温诱导表达,同时测定了他们的核酸酶活性和抗菌抑菌作用。结果:突变体M1、M2经诱导后表达量和核酸酶活性皆高于野生型,其中M1、M2核酸酶活性分别为野生型的1.46倍及1.45倍;突变体M1可对金黄色葡萄球菌产生抑菌作用,抑菌环大小约为5 mm。结论:AmPR-10突变体的活性较野生型有所提高,这一结果有利于深入探讨AmPR-10的相关活性机制,为抗病黄芪的育种奠定理论依据。
Objective:Astragalus membranaceus pathogenesis related protein-10(AmPR-10)has nuclease activity and plays an important role in the growth of Astragalus membranaceus. In order to further improve the activity of the wild type of AmPR-10,the mutants were designed and the changes of expression and activity compared with wild-type were analyzed.Methods:Through rational analysis,it was found that Ser(84),Gly(140)and Glu(141)mutations of wild-type AmPR-10would affect the spatial structure of target protein and reduce the docking free energy of single nucleotide with substrate.Based on this,two mutants M1:S84D+G140Vand M2:S84D+G140V+E141M were designed and synthesized,and the wildtype AmPR-10,M1 and M2 were exogenous expressed under low temperature,and their nuclease activity and antibacterial activity were determined. Results:The expression level and nuclease activity of M1 and M2 mutants were higher than the wild type,and the nuclease activities of M1 and M2 mutants were 1.46 and 1.45 times higher than wild type respectively. The mutant M1 could inhibit staphylococcus aureus,and the size of inhibition ring was about 5 mm. Conclusion:It was proved that the activities of the designed mutantswere higher than that of the wild type,which is conducive to further explore the related activity mechanism of AmPR-10 and established a theoretical basis for the breeding of disease resistant Astragalus membranaceus.
作者
王珏
吴娜
张育敏
翟丽娟
胡丽丽
Wang Jue;Wu Na;Zhang Yumin;Zhai Lijuan;Hu Lili(Department of Basic Medicine,Shanxi University of Chinese medicine,Jinzhong Shanxi 030619)
出处
《山西中医药大学学报》
2022年第2期111-115,共5页
Journal of Shanxi University of Chinese Medicine
基金
山西省自然科学基金面上项目(201901D111339)
山西省高等学校科技创新项目(2020L0407,2020L0420)
山西中医药大学科技创新能力培育计划(2020PY-JC-06,2021PY-JC-06)。
作者简介
王珏,硕士,讲师,Email:wj.happysmile@163.com;通讯作者:胡丽丽,博士,副教授,Email:251480072@qq.com。
