华北农学报 ›› 2018, Vol. 33 ›› Issue (6): 17-23. doi: 10.7668/hbnxb.2018.06.003

所属专题: 玉米

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玉米AGPL2蛋白多克隆抗体制备及时空表达分析

吕亚楠1, 余国武1, 黄玉碧1   

  1. 1. 四川农业大学 农学院, 四川 成都 611130;
    2. 四川农业大学 农学院 作物科学实验教学中心, 四川 成都 611130
  • 收稿日期:2018-07-11 出版日期:2018-12-28
  • 通讯作者: 余国武(1980-),男,湖北荆门人,副教授,博士后,主要从事玉米淀粉合成调控研究;黄玉碧(1963-),男,四川南充人,教授,博士,主要从事玉米淀粉合成调控研究。
  • 作者简介:吕亚楠(1991-),女,河南南阳人,硕士,主要从事生物化学与分子生物学研究。
  • 基金资助:
    国家自然科学基金项目(31501322);留学回国人员科技活动择优资助项目(00124300);四川省博士后特别资助项目(03130104)

Preparation of Polyclonal Antibody of Maize AGPL2 Protein and Analysis of Its Spatiotemporal Expression Analysis

LÜ Yanan1, YU Guowu1, HUANG Yubi1   

  1. 1. College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China;
    2. Center for Crop Science Experimental Teaching, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
  • Received:2018-07-11 Published:2018-12-28

摘要: 为探讨玉米AGPL2在籽粒发育时期淀粉积累过程中的功能机制,通过AGPL2基因克隆和构建带有GST标签的原核表达载体PGEX-6T-1-AGPL2,并利用大肠杆菌诱导表达体系,用0.5 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)在28℃条件下诱导表达6 h后,GST-AGPL2融合蛋白能够得到高水平表达,然后利用GST(Glutathione S-transferase)标签蛋白纯化介质进行亲和层析纯化,能够得到质量较高的GST-AGPL2融合蛋白;利用纯化所得的GST-AGPL2重组蛋白免疫新西兰大白兔制备了AGPL2多克隆抗体,分离并纯化抗血清,然后用rTEV Protease去除抗原GST-AGPL2融合蛋白的GST标签,并用纯化后的AGPL2多克隆抗体进行Western Blot检测,结果发现,AGPL2多克隆抗体具有较高特异性和灵敏性,可用于检测纳克级抗原蛋白;并利用Western Blot方法研究了AGPL2蛋白在玉米不同组织和不同授粉时期胚乳中的分布和表达模式,结果显示,AGL2蛋白在玉米不同组织中的表达具有组织特异性,且在胚乳中含量最高。AGPL2蛋白在玉米胚乳不同授粉时期表达量先增加后降低,且在授粉中期达到最大值。其结果与玉米籽粒发育过程中淀粉的积累规律一致,说明AGPL2主要存在于玉米胚乳中,且可能参与淀粉的合成,也说明AGPL2多克隆抗体具有很好的特异性,能够识别玉米体内的AGPL2抗原。

关键词: AGPL2蛋白, 基因克隆, 原核表达, Western Blotting

Abstract: In order to investigate the functional mechanism of maize AGPL2 in starch accumulation during grain development,this study cloned AGPL2 gene and constructed the GST-tagged prokaryotic expression vector PGEX-6T-1-AGPL2. Using to the inducing expression system of Escherichia coli,GST-AGPL2 fusion protein was able to be obtained expression at high levels by inducing with 0.5 mmol/L was opropyl-β-D-thiogalactoside (IPTG) at 28℃ for 6 h. Then GST (Glutathione S-transferase) tag protein purification medium was used to obtain higher quality GST-AGPL2 fusion protein by affinity chromatography. The AGPL2 polyclonal antibody was prepared by immunizing New Zealand white rabbit with purified GST-AGPL2 recombinant protein,and the antiserum was isolated and purified. Then the GST tag of the antigen GST-AGPL2 fusion protein was removed by rTEV Protease,and Western Blot assayed with the purified AGPL2 polyclonal antibody showed that AGPL2 polyclonal antibody had high specificity and sensitivity and could be used to detect nanogram antigen protein. The distribution and expression patterns of AGPL2 protein in different tissues and pollination stages of maize was studied by Western Blot. The results showed that the expression of AGL2 protein in different tissues of maize was tissue-specific and the highest in endosperm. The expression level of AGPL2 protein increased first and then decreased during the different pollination period of maize endosperm,and reached the maximum in the middle pollination. The results were consistent with the accumulation of starch during the development of maize kernels,and indicated that AGPL2 was mainly present in maize endosperm and might be involved in the synthesis of starch. It also indicated that AGPL2 polyclonal antibody was highly specific and could recognize AGPL2 antigen in corn.

Key words: AGPL2 protein, Gene cloning, Prokaryotic expression, Western Blotting

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引用本文

吕亚楠, 余国武, 黄玉碧. 玉米AGPL2蛋白多克隆抗体制备及时空表达分析[J]. 华北农学报, 2018, 33(6): 17-23. doi: 10.7668/hbnxb.2018.06.003.

LÜ Yanan, YU Guowu, HUANG Yubi. Preparation of Polyclonal Antibody of Maize AGPL2 Protein and Analysis of Its Spatiotemporal Expression Analysis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(6): 17-23. doi: 10.7668/hbnxb.2018.06.003.

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