华北农学报 ›› 2021, Vol. 36 ›› Issue (6): 63-70. doi: 10.7668/hbnxb.20192138

• 作物遗传育种·种质资源·生物技术 • 上一篇    下一篇

MsWRKY33蛋白的抗体制备及对非生物逆境的响应

张锦锦1, 王云平2, 王筱1, 张书兴1, 王学敏1   

  1. 1. 中国农业科学院 北京畜牧兽医研究所, 北京 100193;
    2. 北京汇诚绿洲园林工程有限公司, 北京 100125
  • 收稿日期:2021-09-16 出版日期:2021-12-28
  • 通讯作者: 王学敏(1976-),女,内蒙古锡林浩特人,研究员,博士,主要从事饲草种质资源与遗传育种研究。
  • 作者简介:张锦锦(1994-),女,新疆伊犁哈萨克人,硕士,主要从事紫花苜蓿分子遗传育种研究。
  • 基金资助:
    国家自然科学基金面上项目(31872410);财政部和农业农村部:国家现代农业产业技术体系(CARS-34);中国农业科学院科技创新工程(ASTIP-IAS10)

Antibody Preparation of MsWRKY33 Protein and Response to Abiotic Stress

ZHANG Jinjin1, WANG Yunping2, WANG Xiao1, ZHANG Shuxing1, WANG Xuemin1   

  1. 1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Beijing Huichenglüzhou Landscape Engineering Co., Ltd, Beijing 100125, China
  • Received:2021-09-16 Published:2021-12-28

摘要: 为在蛋白水平上探索紫花苜蓿MsWRKY33在抗逆性上的生物学功能,以紫花苜蓿品种中苜1号为材料,克隆了MsWRKY33抗原序列并对其性质和功能进行初步分析。对MsWRKY33蛋白的结构和特性进行生物信息学分析表明,MsWRKY33蛋白全长511 aa,主要由无规则卷曲和延伸链结构组成,不具有信号肽,是一个亲水性蛋白,免疫原性预测结果表明该蛋白免疫原性较强。结合序列的特异性选取适宜区段247~457 aa,利用该区段经蛋白纯化、免疫等步骤制备了MsWRKY33多克隆抗体。后续又采用Western Blotting的方法,对4种非生物胁迫(高盐、冷、PEG、ABA)下MsWRKY33蛋白的表达情况进行分析,发现4种胁迫均能诱导MsWRKY33蛋白表达增强,其中盐胁迫的诱导表达丰度最高,表明MsWRKY33在调控植物的高盐逆境响应中可能具有重要作用。试验结果为深入探索紫花苜蓿MsWRKY33基因功能及其对非生物逆境的抗性调控机制提供依据。

关键词: 紫花苜蓿, MsWRKY33, 多克隆抗体, 蛋白免疫印记, 盐胁迫

Abstract: To explore the biological function of alfalfa MsWRKY33 in stress tolerance at the protein level,by using Medicago sativa cv.Zhongmu No.1 as the material,MsWRKY33 antigen sequence was cloned and its properties and functions were analyzed.The structural characteristics analysis and immunogenicity prediction of MsWRKY33 protein were performed by using bioinformatics.The structure and characteristics of MsWRKY33 protein analysis results showed that the full length of MsWRKY33 protein was 511 aa,which was mainly composed of random coil and extended chain structure.MsWRKY33 protein had no signal peptide and was a hydrophilic protein.The immunogenicity prediction results indicated that the protein was highly immunogenic.According to the specificity of the binding sequence,the appropriate segment 247-457 aa was selected,and the MsWRKY33 polyclonal antibody was prepared by using this segment through protein purification,immunization and other steps.Subsequently,Western Blotting method was used to analyze the expression of MsWRKY33 protein under four abiotic stress treatments:salt,cold,PEG and ABA,all could enhance the MsWRKY33 protein expression.Among them,NaCl brought the highest expression level,indicated that MsWRKY33 might play an important role in salt stress regulation.The test results provided a reference for further exploration of the function of MsWRKY33 and the mechanism regulating the stress tolerance of alfalfa.

Key words: Medicago sativa L., MsWRKY33, Polyclonal antibody, Western Blotting, Salt stress

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引用本文

张锦锦, 王云平, 王筱, 张书兴, 王学敏. MsWRKY33蛋白的抗体制备及对非生物逆境的响应[J]. 华北农学报, 2021, 36(6): 63-70. doi: 10.7668/hbnxb.20192138.

ZHANG Jinjin, WANG Yunping, WANG Xiao, ZHANG Shuxing, WANG Xuemin. Antibody Preparation of MsWRKY33 Protein and Response to Abiotic Stress[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(6): 63-70. doi: 10.7668/hbnxb.20192138.

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