小麦自噬相关因子ATG5的原核表达和抗血清制备

doi:10.13560/j.cnki.biotech.bull.1985.2017-0020

摘要/Abstract

摘要： 细胞自噬在植物生长发育和环境响应中扮演了重要角色，ATG5是参与自噬小体组装的核心因子之一。在前期工作中我们克隆了两个小麦ATG5基因TaATG5a和TaATG5b，并对其开展了初步的功能分析。鉴于后续基因功能研究中的免疫学方法涉及对特异性抗体的需求，通过载体构建、原核表达和亲和层析过程获得了TaATG5a的重组蛋白，经兔免疫过程制备了TaATG5a的抗血清，采用ELISA和Western杂交等方法鉴定了抗体的效价和特异性。结果表明，克隆于pET30a载体上的TaATG5a在大肠杆菌中能够被IPTG高效诱导表达，重组蛋白的表观分子量与理论值基本一致，表达量在诱导0-8 h范围内逐渐增加。纯化的重组蛋白纯度较高，满足抗体制备要求。制备的抗血清能特异性识别原核表达和小麦内源的TaATG5a，效价达到1∶25 600。此外，制备的抗血清通过Western杂交还能够识别共价连接的小麦ATG12-ATG5复合物，该复合物是小麦叶片中ATG5的主要存在形式。

关键词: 小麦, 自噬相关因子ATG5, 原核表达, 抗血清

Abstract: Autophagy is closely implicated in plant growth and development，as well as responses to environmental stimuli. ATG5 is one of the core factors involved in autophagosome assembly. Two wheat ATG5-encoding genes，TaATG5a and TaATG5b were cloned previously and are under deep functional study in our lab. In order to meet the need of specific antibodies in immunological methods used in further functional gene and protein study，a recombinant protein TaATG5a was acquired by vector construction，prokaryotic expression，and purification via the Ni-column affinity chromatography method，then the antiserum was prepared in rabbit immunization. The titer and specificity of the prepared antiserum were tested by the ELISA and Western blot assays. The results showed that the expression of TaATG5a cloned in vector pET30a was efficiently induced in Escherichia coli by IPTG，and gradually increased with the extension of IPTG induction time over 0 - 8 h. The apparent molecular weight of recombinant protein TaATG5a was close to its theoretical value. The purified recombinant protein was in high-purity that satisfied the requirements for antibody preparation. The prepared antiserum had a high titer of 1∶25 600 and specifically recognized TaATG5a in E. coli or wheat total protein. In addition，the ATG12-ATG5 in conjugated form，i.e.，the main form of wheat ATG5 in leaves，was also recognized by the prepared antiserum through Western blot assay.

Key words: wheat, ATG5, prokaryotic expression, antiserum

张加姿,李开心,于宝佳,岳洁瑜,王华忠. 小麦自噬相关因子ATG5的原核表达和抗血清制备[J]. 生物技术通报, 2017, 33(7): 69-74.

ZHANG Jia-zi ,LI Kai-xin ,YU Bao-jia ,YUE Jie-yu, WANG Hua-zhong. Prokaryotic Expression of Wheat Autophagy-related Factor ATG5 and Preparation of Its Antiserum in Rabbits[J]. Biotechnology Bulletin, 2017, 33(7): 69-74.

参考文献

[1] Liu Y, Bassham DC. Autophagy：pathways for self-eating in plant cells[J] . Annu Rev Plant Biol, 2012, 63：215-237.
[2] Li F, Vierstra RD. Autophagy：a multifaceted intracellular system for bulk and selective recycling[J] . Trends Plant Sci, 2012, 17：526-537.
[3] Hanada T, Noda NN, Satomi Y, et al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy[J] . J Biol Chem, 2007, 282：37298-37302.
[4] Romanov J, Walczak M, Ibiricu I, et al. Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation[J] . EMBO J, 2012, 31：4304-4317.
[5] Nakatogawa H, Ichimura Y, Ohsumi Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion[J] . Cell, 2007, 130：165-178.
[6] Xie Z, Nair U, Klionsky DJ. Atg8 controls phagophore expansion during autophagosome formation[J] . Mol Biol Cell, 2008, 19：3290-3298.
[7] Le Bars R, Marion J, Le Borgne R, et al. ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants[J] . Nat Commun, 2014, 20, 5：4121.
[8] Nishida Y, Arakawa S, Fujitani K, et al. Discovery of Atg5/Atg7-independent alternative macroautophagy[J] . Nature, 2009, 461（7264）：654-658.
[9] Murrow L, Malhotra R, Debnath J. ATG12-ATG3 Interacts with Alix to promote basal autophagic flux and late endosome function[J] . Nat Cell Biol, 2015, 17（3）：300-310.
[10] Jounai N, Takeshita F, Kobiyama K, et al. The Atg5-Atg12 conjugate associates with innate antiviral immune responses[J] . Proc Natl Acad Sci USA, 2007, 28, 104（35）：14050-14055.
[11] Yousefi S, Perozzo R, Schmid I, et al. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis[J] . Nat Cell Biol, 2006, 8（10）：1124-1132.
[12] Maskey D, Yousefi S, Schmid I, et al. ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy[J] . Nat Commun, 2013, 4：2130.
[13] Chung T, Suttangkakul A, Vierstra RD. The ATG autophagic conjugation system in maize：ATG transcripts and abundance of the ATG8-lipid adduct are regulated by development and nutrient availability[J] . Plant Physiol, 2009, 149：220-234.
[14] 王燕, 刘玉乐. 植物细胞自噬研究进展[J] . 中国细胞生物学学报, 2010, 32（5）：677-689.
[15] Xia K, Liu T, Ouyang J, et al. Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice（Oryza sativa L.）[J] . DNA Res, 2011, 18：363-377.
[16] Han S, Yu B, Wang Y, et al. Role of plant autophagy in stress response[J] . Protein Cell, 2011, 2（10）：784-791.
[17] Zhou J, Yu JQ, Chen Z. The perplexing role of autophagy in plant innate immune responses[J] . Mol Plant Pathol, 2014, 15（6）：637-645.
[18] Yang X, Bassham DC. New insight into the mechanism and function of autophagy in plant cells[J] . Int Rev Cell Mol Biol, 2015, 320：1-40.
[19] 孙鸿, 张微, 卫晓静, 等. 小麦重要自噬相关基因ATG18的鉴定和表达分析[J] . 中国农业科学, 2014, 47（9）：1657-1669.
[20] 张微, 孙鸿, 邢莉萍, 等. 小麦自噬相关基因ATG10的克隆及白粉菌侵染诱导的ATG10的表达[J] . 作物学报, 2014, 40 （8）：1392-1402.
［21］Pei D, Zhang W, Sun H, et al. Identification of autophagy-related genes ATG4 and ATG8 from wheat（Triticum aestivum L.）and profiling of their expression patterns responding to biotic and abiotic stresses［J］. Plant Cell Rep, 2014, 33：1697-1710.
［22］Yue JY, Sun H, Zhang W, et al. Wheat homologs of yeast ATG6 function in autophagy and are implicated in powdery mildew immunity［J］. BMC Plant Biol, 2015, 15：95.
［23］Phillips AR, Suttangkakul A, Vierstra RD. The ATG12-conjugating enzyme ATG10 is essential for autophagic vesicle formation in Arabidopsis thaliana［J］. Genetics, 2008, 178（3）：1339-1353.
［24］Codogno P, Meijer AJ. Atg5：more than an autophagy factor［J］. Nat Cell Biol, 2006, 8（10）：1045-1047.