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作物学报 ›› 2022, Vol. 48 ›› Issue (9): 2390-2399.doi: 10.3724/SP.J.1006.2022.11070

• 研究简报 • 上一篇    下一篇

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

李永波1(), 崔德周1, 黄琛1, 隋新霞1, 樊庆琦1,*(), 楚秀生1,2,*   

  1. 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
  • 收稿日期:2021-08-06 接受日期:2021-11-30 出版日期:2022-09-12 网络出版日期:2022-07-15
  • 通讯作者: 樊庆琦,楚秀生
  • 作者简介:E-mail: lyb920327@sina.com
  • 基金资助:
    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)

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

LI Yong-Bo1(), CUI De-Zhou1, HUANG Chen1, SUI Xin-Xia1, FAN Qing-Qi1,*(), CHU Xiu-Sheng1,2,*   

  1. 1. 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
    2. School of Life Science, Shandong Normal University, Jinan 250000, Shandong, China
  • Received:2021-08-06 Accepted:2021-11-30 Published:2022-09-12 Published online:2022-07-15
  • Contact: FAN Qing-Qi,CHU Xiu-Sheng
  • Supported by:
    Agricultural Variety Improvement Project of Shandong Province(2019LZGC01702);National Natural Science Foundation of China (Youth Project)(32001542);Shandong Natural Science Foundation (Youth Project)(ZR2020QC114);“Key Crop Breeding Technology”(CXGC2021A09)

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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。

关键词: 小麦, 自噬, 多克隆抗体, ATG8蛋白

Abstract:

Autophagy is an evolutionarily highly conserved lysosomal/vacuolar degradation pathway, which is of great significance for the body to adapt to various biotic or abiotic stresses and maintain its normal growth and development. Autophagy-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 antibody 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.

Key words: wheat, autophagy, polyclonal antibody, autophagy-related protein 8

图1

ATG8抗血清效价分析"

图2

ATG8抗体特异性检测 SDS胶浓度为12.5%。"

表1

质谱鉴定ATG8蛋白"

起始
Start		 序列
Sequence		 结束
End		 蛋白鉴定
Protein ID		 名称
Name
4 SSFKLEHPLER 14 U3Q019 Autophagy-related protein 8
4 TCFKTEHPLER 14 Q7XY24 Autophagy-related protein 8
24 EKYSDRIPVIVEK 36 U3Q019 Autophagy-related protein 8
24 EKYADRIPVIVEK 36 Q7XY24 Autophagy-related protein 8
37 ADKSDVPEIDKKK 49 Q7XY24 Autophagy-related protein 8
37 AGKSDIPDIDK 47 U3Q019 Autophagy-related protein 8
49 KYLVPADLTVGQFVYVVR 66 U3Q019 Autophagy-related protein 8
69 IKLSAEKAIFIFVK 82 U3Q019 Autophagy-related protein 8
83 NTLPPTAALMSAIYEENKDEDGFLYMTYSGENTFG 117 U3Q019 Autophagy-related protein 8

图3

Western blot检测ATG8蛋白 α-Tubulin用作内参, SDS胶浓度为15%; 3-MA: 3-MA+PEG-8000; 浓度为10 mmol L-1; 处理时间为48 h; 总蛋白上样量均为1 mg; 误差是3次实验数据平均值±SD。不同的字母代表显著性差异。."

图4

小麦根部细胞自噬结构检测 白色箭头指示的是自噬结构, 标尺为500 μm。选取不同处理根部的相同部位, 置于10×10倍显微镜下, 并通过ANOVA软件对每个视野中的自噬结构数量进行统计学分析。不同的字母代表显著性差异。误差是3次实验数据平均值±SD。"

图5

小麦根部细胞自噬结构统计分析 选取不同处理根部的相同部位, 置于10×10倍显微镜下, 并通过ANOVA软件对每个视野中的自噬结构数量进行统计学分析。不同的字母代表显著性差异。误差是3次实验数据平均值±SD。"

图6

小麦叶部细胞自噬结构检测 白色箭头指示自噬结构, 标尺: 50 μm。选取不同处理叶部的相同部位, 置于10×20倍显微镜下, 并通过ANOVA软件对每个视野中的自噬结构数量进行统计学分析, 不同的字母代表显著性差异。误差是3次实验数据平均值±SD。"

图7

小麦叶部细胞自噬结构统计分析 选取不同处理叶部的相同部位, 置于10×20倍显微镜下, 并通过ANOVA软件对每个视野中的自噬结构数量进行统计学分析, 不同的字母代表显著性差异。误差是3次实验数据平均值±SD。"

图8

小麦籽粒自噬结构检测 白色短箭头指示胚乳中的自噬结构, 黄色短箭头指示果皮中的自噬结构, 长箭头指示籽粒的不同结构; 标尺为200 μm。Al (Aleurone layer): 糊粉层; En (Endosperm): 胚乳; Pe (Pericarp): 果皮。"

图9

不同类型的小麦ATG8蛋白表达、纯化及抗体识别 SDS胶浓度为15%。"

图10

同源系统进化树分析 红色矩形区域为ATG8新型蛋白, ATG8j; ATG8a (AGW81789.1), ATG8b (AGW81790.1), ATG8c (AGW81791.1), ATG8d (AGW81792.1), ATG8e (AGW81793.1), ATG8f (AGW81794.1), ATG8g (AGW81795.1), ATG8h (AGW81796.1), ATG8j (A0A3B6PL31)。"

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