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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (9): 2390-2399.doi: 10.3724/SP.J.1006.2022.11070

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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 Online:2022-09-12 Published:2022-07-15
  • Contact: FAN Qing-Qi,CHU Xiu-Sheng E-mail:lyb920327@sina.com;xschu2007@sina.cn
  • 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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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

Fig. 1

Titer analysis of ATG8 antiserum"

Fig. 2

Specific detection of ATG8 antibody The concentration of SDS gel is 12.5%."

Table 1

Identification of ATG8 protein by mass spectrometry"

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

Fig. 3

Western blot detection of ATG8 protein α-Tubulin is used as an internal control. The concentration of SDS gel is 15%; 3-MA: 3-MA+PEG-8000; the concentration is 10 mmol L-1; the treatment time is 48 h; the total protein load is 1 mg ; the error bar is the mean ± SD of three experimental data. Different letters represent significant differences."

Fig. 4

Detection of autophagy structures in wheat root cells The white arrow indicates autophagy structures, and the scale bar: 500 μm. The same parts of the roots of different treatments were selected, placed under a 10×10 microscope, and the number of autophagic structures in each field of view was statistically analyzed by ANOVA software. Different letters represent significant differences. The error is the mean ± SD of three experimental data."

Fig. 5

Statistical analysis of autophagy structures in wheat root cells The same parts of the roots of different treatments were selected, placed under a 10×10 microscope, and the number of autophagic structures in each field of view was statistically analyzed by ANOVA software. Different letters represent significant differences. The error is the mean ± SD of three experimental data."

Fig. 6

Detection of autophagy structures in wheat leaf cells The white arrow indicates autophagy structures, and the scale bar: 50 μm. The same parts of the leaves of different treatments were selected, and placed under a 10×20 microscope, and the number of autophagic structures in each field of view was statistically analyzed by ANOVA software. Different letters represent significant differences. The error is the mean ± SD of three experimental data."

Fig. 7

Statistical analysis of autophagy structures in wheat leaf cells The same parts of the leaves of different treatments were selected, and placed under a 10×20 microscope, and the number of autophagic structures in each field of view was statistically analyzed by ANOVA software. Different letters represent significant differences. The error is the mean ± SD of three experimental data."

Fig. 8

Detection of autophagy structures in wheat grains The short white arrow indicates autophagy structures in endosperm; the short yellow arrow indicates autophagy structures in pericarp; the long white arrows indicate the different structures of the grain, and the scale bar: 200 μm. Al: aleurone layer; En: endosperm; Pe: pericarp."

Fig. 9

Different types of wheat ATG8 protein expression, purification, and antibody recognition The concentration of SDS gel is 15%."

Fig. 10

Phylogenetic tree analysis of homology The red rectangular area is the new ATG8 protein, 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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