Journal of Nuclear Agricultural Sciences ›› 2020, Vol. 34 ›› Issue (10): 2168-2177.DOI: 10.11869/j.issn.100-8551.2020.10.2168

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

Cloning and Expression Analysis of SbJAZ1 in Sorghum

JIANG Junmei1,**, CHEN Meiqing1,**, NING Na1, FANG Yuanpeng1, GUO Jiyuan3, LUO Liting1, REN Mingjian1,2, XIE Xin1,2,*   

  1. 1College of Agricultural, Guizhou University, Guiyang, Guizhou 550025;
    2Guizhou Sub-Center of National Wheat Improvement Center, Guiyang, Guizhou 550025;
    3Department of Resources and Environmental Science and Engineering, Moutai Institute, Zunyi, Guizhou 564507
  • Received:2019-12-06 Online:2020-10-10 Published:2020-08-11

高粱SbJAZ1基因克隆与表达分析

蒋君梅1,**, 陈美晴1,**, 宁娜1, 方远鹏1, 郭继元3, 罗丽婷1, 任明见1,2, 谢鑫1,2,*   

  1. 1贵州大学农学院,贵州 贵阳 550025;
    2国家小麦改良中心贵州分中心,贵州 贵阳 550025;
    3茅台学院资源环境系,贵州 遵义 564507
  • 通讯作者: * 谢鑫,男,副教授,主要从事植物抗病基因功能研究。E-mail:xiexin2097757@163.com
  • 作者简介:蒋君梅,女,主要从事高粱抗病基因功能研究。E-mail:jjmguangan@163.com;陈美晴,女,主要从事高梁抗病基因功能研究。E-mail:c18212878410@163.com。同为第一作者。
  • 基金资助:
    国家自然科学基金资助项目(31801691),贵州省科技计划项目(黔科合支撑[2019]2408号),贵州省高层次留学人才创新创业择优资助项目([2018]02号),贵州大学引进人才科研基金(贵大人基合字[2017]54号)

Abstract: JAZ1 is one of the genes encoding JAZs protein of TIFY family. In order to clone SbJA2 gene from Sorghum and to study its expression characteristics under stress treatment, in this study, the total RNA was extracted from sorghum BTx623, and cDNA of SbJAZ1 was amplified by reverse transcription. Subsequently, bioinformatics analysis was conducted to characterize SbJAZ1 and the prokaryotic system was employed to express SbJAZ1 protein. The results showed that the full length of SbJAZ1 gene was 606 bp, encoding 201 amino acids, the isoelectric point of predicted protein was 7.70, and the molecular weight was 21.15 kDa. SbJAZ1 is a hydrophilic protein with high homology to maize JAZ1. In secondary structure, alpha helix accounted for 25.37%, elongation chain 9.95%, β angle 4.98%, and irregular curl 59.70%. qRT-PCR analysis demonstrated that SbJAZ1 could express in sorghum root, stalk, shoot and leaves, but mainly expressed in stalk. The expression level of SbJAZ1 was induced at 1 hour after plant hormone jasmonic acid (JA) treatment. Besides, PEG-6000 and plant hormone indole acetic acid (IAA) also enhance the expression of SbJAZ1. In order to obtain the soluble expression protein of SbJAZ1, the prokaryotic expression vector of SbJAZ1 gene was constructed and the expressed strains, induction temperatures and concentrations of IPTG (isopropyl-β-D-thiopyranogalactoside) were optimized. The results demonstrated that SbJAZ1 was highly expressed in Rosetta (DE3) strain at 30℃ with 0.8 mmol·L-1IPTG. This study provides a basis understanding for the functional study of SbJAZ1.

Key words: sorghum, JAZ1, gene cloning, expression analysis, prokaryotic expression

摘要: JAZ1基因是编码TIFY家族JAZs蛋白的基因之一。为克隆高粱SbJAZ1基因及研究其响应胁迫表达特性,本试验以高粱品种BTx623为研究材料,提取其总RNA,反转录获得cDNA,以cDNA为模板扩增SbJAZ1基因,并对其进行生物信息学、基因表达和原核表达分析。结果显示,SbJAZ1基因全长606 bp,编码201个氨基酸,蛋白质等电点为7.70,分子量大小为21.15 kDa;SbJAZ1与玉米相应同源基因亲缘关系最近,为亲水性蛋白;二级结构中α螺旋占25.37%、延伸链占9.95%、β转角占4.98%,无规则卷曲占59.70%。qRT-PCR分析表明,SbJAZ1在高粱根、茎、芽和叶组织中均有表达,且具有组织表达特异性,在茎中表达量最高;茉莉酸(JA)处理1 h后,SbJAZ1在高粱茎中表达量最高;吲哚乙酸(IAA)和PEG-6000可诱导SbJAZ1的表达。原核表达结果显示,SbJAZ1在大肠杆菌Rosetta(DE3)菌株诱导表达的最佳条件为:温度30℃,异丙基硫代半乳糖苷(IPTG)浓度0.8 mmol·L-1。本研究结果为SbJAZ1基因的生物学功能研究提供了基础。

关键词: 高粱, JAZ1, 基因克隆, 表达分析, 原核表达