抗病蛋白CkPGIP1关键氨基酸突变增强对大丽轮枝菌的抑制作用  

Enhanced inhibitory effect of mutations of key amino acids in the disease-resistance protein CkPGIP1 on Verticillium dahliae

作　　者：闫鑫 高琳颖 胡梦慧 邵苗苗 侯玉霞[1] YAN Xin;GAO Linying;HU Menghui;SHAO Miaomiao;HOU Yuxia(Department of Applied Chemistry,College of Science,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业大学理学院应用化学系,北京100193

出　　处：《农药学学报》2024年第5期922-931,共10页Chinese Journal of Pesticide Science

基　　金：国家自然科学基金项目(32272631)。

摘　　要：牛心朴子草多聚半乳糖醛酸酶抑制蛋白(CkPGIP1)能有效抑制大丽轮枝菌多聚半乳糖醛酸酶(VdPG1)的活性,为了进一步提高CkPGIP1的抗病性,本研究从CkPGIP1定点突变入手,研究其突变增强对大丽轮枝菌的抑制作用。通过重叠延伸聚合酶链式反应(PCR)成功构建了CkPGIP1蛋白突变体T152VCkPGIP1、D202SCkPGIP1和I250KCkPGIP1,构建原核表达载体对CkPGIP1及其突变体进行体外诱导表达和纯化,用3,5-二硝基水杨酸(DNS)法和琼脂糖径向扩散法测定突变蛋白对大丽轮枝菌VdPG1的抑制作用。DNS法测定结果表明:突变蛋白能有效抑制VdPG1活性,且呈剂量依赖性,其中,D202SCkPGIP1对VdPG1的抑制效果显著,T152VCkPGIP1抑制效果次之,I250KCkPGIP1抑制作用不明显。琼脂糖径向扩散结果与DNS法的一致,其中经D202SCkPGIP1处理的VdPG1扩散直径缩小最多,表明其抑制活性最为显著。进一步研究发现,突变蛋白可以抑制大丽轮枝菌在棉花叶片上的扩展,进一步证实CkPGIP1关键氨基酸突变增强了对大丽轮枝菌的抑制作用。The polygalacturonase inhibitory protein(CkPGIP1)of Cynanchum komarovii can effectively inhibit the activity of polygalacturonase(VdPG1)of Verticillium dahliae.To further enhance the disease resistance of CkPGIP1,this study started with site-directed mutagenesis of the disease-resistant protein CkPGIP1 to investigate its enhanced inhibitory effect on V.dahliae.Using overlapping extension polymerase chain reaction(PCR)technology,mutant variants T152VCkPGIP1,D202SCkPGIP1,and I250KCkPGIP1 of CkPGIP1 were successfully obtained.The corresponding prokaryotic expression vectors were constructed for the induction expression and purification of CkPGIP1 and its mutant proteins.The inhibitory effect of the mutant proteins on VdPG1 was determined by 3,5-dinitrosalicylic acid(DNS)method and agarose radial diffusion method.The results of DNS assay showed that the mutant proteins effectively inhibited the activity of VdPG1 in a dose-dependent manner.Among them,the inhibitory effect of D202SCkPGIP1 on VdPG1 was most significant,followed by T152VCkPGIP1,and the inhibitory effect of I250KCkPGIP1 was not obvious.The results of agarose radial diffusion were consistent with those of DNS assay.D202SCkGIP1-treated V.dahliae showed the most obvious decrease in the halo area of VdPG1,indicating that D202SCkPGIP1 had the most significant inhibitory activity.Further study revealed that the mutant proteins could inhibit the expansion of V.dahliae on cotton leaves,confirming that the key amino acid mutations in CkPGIP1 enhanced its inhibitory effect on V.dahliae.

关 键 词：牛心朴子草 大丽轮枝菌 多聚半乳糖醛酸酶抑制蛋白 多聚半乳糖醛酸酶 关键氨基酸突变 

分 类 号：S481[农业科学—农药学]