水稻抗褐飞虱基因Bph3调控蛋白B8AK41的外源表达、纯化及亚细胞定位  

Exogenous expression, purification and subcellular localization of the rice brown planthopper resistant gene Bph3 regulated protein B8AK41

作　　者：卿冬进 卢柏亦 彭宇婧 戴高兴[1] 陈韦韦[1] 李经成[1] 周维永[1] 黄所生[2] 潘英华[1] 黄娟[1] 邓国富[1] QING Dong-jin;LU Bai-yi;PENG Yu-jing;DAI Gao-xing;CHEN Wei-wei;LI Jing-cheng;ZHOU Wei-yong;HUANG Suo-sheng;PAN Ying-hua;HUANG Juan;DENG Guo-fu(Rice Research Institute,Guangxi Academy of Agricultural Sciences/Guangxi Key Laboratory of Rice Genetics and Breeding,Nanning 530007,China;Plant Protection Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,China)

机构地区：[1]广西农业科学院水稻研究所/广西水稻遗传育种重点实验室,南宁530007 [2]广西农业科学院植物保护研究所,南宁530007

出　　处：《西南农业学报》2025年第1期107-114,共8页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(32160645);广西自然科学基金重点项目(2023GXNSFDA026060)。

摘　　要：【目的】外源表达获得B8AK41蛋白,并检测B8AK41蛋白亚细胞定位,为研究该蛋白在水稻抗褐飞虱上的生物学功能提供基础。【方法】对B8AK41蛋白序列进行跨膜区预测,构建原核表达载体诱导外源蛋白表达,纯化目标蛋白并鉴定蛋白多肽序列;构建双元表达载体并转入水稻原生质体检测蛋白亚细胞定位。【结果】通过对B8AK41蛋白的跨膜结构域分析,发现该蛋白第47~99位氨基酸区域可能存在跨膜结构域,因此选取第100~374位氨基酸进行外源表达蛋白片段。通过SDS-PAGE电泳分析外源表达的B8AK41蛋白,结果显示B8AK41蛋白在28℃IPTG诱导下成功表达,采用亲和层析方法可纯化得到分子量在35~40 kD的His6-B8AK41-His6融合蛋白。通过质谱分析纯化得到的蛋白,其中有3条属于B8AK41蛋白的肽段,证明外源表达纯化的B8AK41蛋白完全正确。采用荧光共聚焦显微镜观察到B8AK41蛋白定位在细胞内质网上。【结论】水稻抗褐飞虱基因Bph3的正调控蛋白B8AK41的非跨膜区能够在大肠杆菌中表达,通过亲和层析方法纯化得到融合蛋白His6-B8AK41-His6,后续可将B8AK41蛋白用于抗体制备。B8AK41蛋白定位在水稻细胞内质网上,为研究蛋白功能提供基础。【Objective】B8AK41 protein was obtained by exogenous expression and the subcellular localization of B8AK41 protein was examined to provide the basis for studying the biological function of this protein on planthopper resistance in rice.【Method】Predicting the transmembrane region of the protein B8AK41 sequence and constructing the prokaryotic expression vector inducible expression of the exogenous protein, purifying the protein and verified peptide sequence.The binary vector and transferred into rice protoplasts were constructed to observe the protein subcellular localization.【Result】By analyzing the transmembrane domain of B8AK41 protein, it had been found that the protein might contain transmembrane domain from the 47th to 99th amino acid region, so the amino acids 100th to 374th were selected to express the protein fragment.SDS-PAGE electrophoresis analysis showed that B8AK41 protein could be expressed successfully at 28 ℃ with IPTG induction.The 35-40 kD fusion protein His_6-B8AK41-His_6 could be purified with affinity chromatography.The purified protein was validated using mass spectrometry, and three peptides were detected from B8AK41 protein, indicating that the exogenously expressed and purified B8AK41 protein was correct.The localization of B8AK41 protein on the endoplasmic reticulum was observed using fluorescence confocal microscopy.【Conclusion】The non-transmembrane region of the brown planthopper resistant gene Bph3 positive-regulated protein B8AK41 can be expressed in Escherichia coli.The expressed fusion protein His_6-B8AK41-His_6 is purified by affinity chromatography successfully.The B8AK41 protein can be used for antibody preparation in further study.The localization of B8AK41 protein on rice endoplasmic reticulum provides a basis for the study of protein function.

关 键 词：水稻 褐飞虱 Bph3基因 B8AK41蛋白 外源表达 亚细胞定位 

分 类 号：S511[农业科学—作物学]