高度特异性小麦ATG8抗体的研制及其在细胞自噬检测中的应用  

Preparation of highly specific wheat ATG8 antibody and its application in the detection of autophagy

作　　者：李永波[1] 崔德周[1] 黄琛 隋新霞[1] 樊庆琦[1] 楚秀生[1,2] LI Yong-Bo;CUI De-Zhou;HUANG Chen;SUI Xin-Xia;FAN Qing-Qi;CHU Xiu-Sheng(Crop Research Institute,Shandong Academy of Agricultural Sciences/Key Laboratory of Wheat Biology and Genetic improvement in North Huang-Huai River Valley,Ministry of Agriculture and Rural Affairs/National Engineering Laboratory for Wheat and Maize,Jinan 250100,Shandong,China;School of Life Science,Shandong Normal University,Jinan 250000,Shandong,China)

机构地区：[1]山东省农业科学院作物研究所/黄淮北部小麦生物学与遗传育种重点实验室/小麦玉米国家工程实验室,山东济南250100 [2]山东师范大学生命科学学院,山东济南250014

出　　处：《作物学报》2022年第9期2390-2399,共10页Acta Agronomica Sinica

基　　金：山东省农业良种工程项目(2019LZGC01702);国家自然科学基金青年项目(32001542);山东省自然科学基金青年项目(ZR2020QC114);山东省农科院创新工程:“主要农作物育种关键技术”项目(CXGC2021A09)资助。

摘　　要：细胞自噬是一种进化上高度保守的溶酶体/液泡降解途径,对机体适应各种生物或非生物胁迫,以及维持自身正常生长发育具有重要的意义。自噬相关蛋白8(autophagy-related protein 8,ATG8)是检测自噬的金标准。目前由于缺乏特异性好的小麦ATG8抗体,导致小麦细胞自噬研究进展缓慢。本研究通过人工合成小麦ATG8蛋白序列上的一段18个氨基酸的多肽作为抗原去免疫兔子,成功获得了高度特异性的小麦ATG8多克隆抗体。该抗体不仅能够识别小麦根、叶及种子中的ATG8蛋白条带,而且可以在根、叶中检测到细胞自噬结构,并首次在小麦籽粒中实现了细胞自噬结构的检测。本研究研制的小麦ATG8抗体,除了能够检测到小麦中常见的ATG8a-h等8种典型类型外,还能检测到一种非典型的新的ATG8蛋白,为深入开展植物细胞自噬调控机制研究以及优异新基因挖掘提供了最佳方法学基础。Autophagy is an evolutionarily highly conserved lysosomal/vacuolar degradation pathway,which is of great signifi-cance for the body to adapt to various biotic or abiotic stresses and maintain its normal growth and development.Auto-phagy-related protein 8(ATG8)is the gold standard for detecting autophagy.At present,the lack of specific wheat ATG8 antibody has led to slow progress in the research of wheat autophagy.In this study,an 18-amino acid polypeptide of wheat ATG8 protein sequence was artificially synthesized as an antigen to immunize rabbits,and a highly specific wheat ATG8 polyclonal antibody was successfully obtained.The antibody can not only recognize the ATG8 protein bands in roots,leaves,and seeds of wheat,but also can detect autophagy structures in roots and leaves,and autophagy structures in wheat grains were detected for the first time.Besides eight typical types of ATG8a-h antibody found in wheat,a typical new ATG8 protein was detected by wheat ATG8 anti-body developed in this study,These results provide the best methodological basis for further study on the regulation mechanism of plant autophagy and excavation of excellent genes.

关 键 词：小麦 自噬 多克隆抗体 ATG8蛋白 

分 类 号：S512.1[农业科学—作物学]