一种体内检测ROP蛋白活性的方法及应用  

A method for ROP protein activity detection in vivo and its application

作　　者：张皓 刘雪莹 贺千可 王鹏 张绍铃[1] 吴巨友[1] ZHANG Hao;LIU Xueying;HE Qianke;WANG Peng;ZHANG Shaoling;WU Juyou(College of Horticulture,Nanjing Agricultural University/Pear Engineering Research Center of Jiangsu Province,Nanjing 210095,Jiangsu,China)

机构地区：[1]南京农业大学园艺学院·江苏省梨工程研究中心,南京210095

出　　处：《果树学报》2023年第10期2252-2262,共11页Journal of Fruit Science

基　　金：国家自然科学基金项目(32172543)。

摘　　要：【目的】建立一种体内检测ROP蛋白活性的方法,为进一步研究ROP蛋白的功能提供技术支持。【方法】该方法基于RIC作为ROP蛋白的效应子能特异地与活性形式的ROP结合这一特点,通过荧火素酶互补试验,借助烟草瞬时表达系统,利用植物活体成像仪和酶标仪进行定性或定量检测荧光强度,从而实现体内检测ROP蛋白的活性。【结果】以拟南芥AtRIC1为检测基因,以AtROP1为例来检测其蛋白活性,成功检测到了AtROP1与AtRIC1有荧光,表明它们有互作。同时对AtROP1蛋白进行了点突变试验,成功构建了持续激活态的ROP1(AtROP1-CA)和持续失活态的ROP1(AtROP1-DN)载体,通过定性定量检测,发现与AtROP1-DN相比,AtRIC1与AtROP1-CA有更强的荧光信号,表明AtRIC1可以作为检测蛋白来检测AtROP1蛋白的活性。此外,通过引入3个蛋白(以AtGAP1、AtGDI1和AtGEF1蛋白为例),利用该方法进一步检测,表明AtGAP1和AtGDI1作为AtROP1蛋白活性的抑制因子,明显降低了AtROP1蛋白的活性;而AtGEF1作为AtROP1蛋白活性的促进因子,明显提高了AtROP1蛋白的活性,说明该方法还可以通过At-RIC1检测其他蛋白对AtROP1蛋白活性的影响。同时,该方法还解决了之前单一的pull-down试验检测ROP蛋白活性变化的问题,增加了一种新的体内检测ROP蛋白活性的方法。【结论】与其他方法相比,该方法具有高灵敏度、可视化、可定量化和操作简单等特点,并且适用于检测其他蛋白对ROP蛋白活性的影响,所以该方法是一种可靠的体内检测ROP蛋白活性的新方法。【Objective】This work aimed to establish a method for detecting ROP protein activity in plants and to provide some technological support for further studies on the function of ROP proteins.【Methods】The working principle of the method was mainly based on the fact that RIC,as an effector of the ROP protein,specifically binds to the active form of ROP proteins.Through the luciferase complementation assay,with the help of the Agrobacterium-mediated tobacco(Nicotiana benthamiana)transient expression system,the plant live imager and microplate reader were used to qualitatively or quantitatively detect the fluorescence intensity,to achieve the detection of ROP protein activity in vivo.【Results】In this study,the AtRIC1 was used as the test gene and AtROP1 was used as an example to detect its protein activity.By this method,a strong fluorescent signal was successfully detected in the co-injection region of AtROP1 and AtRIC1.The test results indicated that AtROP1 had an interaction with At-RIC1 and this method could detect the activity of AtROP1 protein.In addition,the AtROP1 protein was subjected to a targeted mutation assay,in which glutamine(Gln or Q)at site 64 was mutated to leucine(Leu or L)to create a persistently activated ROP1(AtROP1-CA)vector,and aspartic acid(Asp or D)at site 121 was mutated to alanine(Ala or A)to create a persistently inactivated ROP1(AtROP1-DN)vector.The two vectors(AtROP1-CA-nLUC and AtROP1-DN-nLUC)were then successfully constructed and transformed into Agrobacterium GV3101.The changes in the activity of AtROP1 protein after the targeted mutation were tested again by this method.The qualitative and quantitative results showed that the luciferase fluorescence intensity and activity values were significantly enhanced in the AtROP1-CA treated group(6605.0±209.2)compared with the control AtROP1 proteins(4395.0±103.1),which indicated that the AtROP1-CA protein showed higher activity.In contrast,the luciferase fluorescence intensity and activity values of AtROP1-DN protein(1134.0±39.83

关 键 词：ROP蛋白 RIC蛋白 荧火素酶 

分 类 号：Q94[生物学—植物学]