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作物学报 ›› 2017, Vol. 43 ›› Issue (04): 620-628.doi: 10.3724/SP.J.1006.2017.00620

• 研究简报 • 上一篇    

白菜型冬油菜类甜蛋白的筛选、克隆及其在低温胁迫下的表达

马骊1**,袁金海1**,孙万仓1,*,刘自刚1,曾秀存2,武军艳1,方彦1,李学才1,陈奇1,许耀照2,蒲媛媛1,刘海卿1,杨刚1,刘林波1   

  1. 1 甘肃省作物遗传改良与种质创新重点实验室 / 甘肃省油菜工程技术研究中心 / 甘肃农业大学农学院, 甘肃兰州 730070；2 河西学院农业与生物技术学院, 甘肃张掖734000
  • 收稿日期:2016-01-19 修回日期:2016-11-03 出版日期:2017-04-12 网络出版日期:2016-12-14
  • 通讯作者: 孙万仓, E-mail:18293121851@163.com, Tel: 18293121851
  • 基金资助:

    本研究由国家现代农业产业技术体系建设专项(CARS-13), 国家自然科学基金项目(31460356, 31560397), 国家重点基础研究发展计划项目(973计划) (2015CB150206), 国家农业科技成果转化项目(2014G10000317), 甘肃省自然科学基金项目(145RJZG050), 科技部油菜杂种优势利用技术与强优势杂交种创制项目(2016YFD0101300)和丝绸之路经济带新疆核心区农田周年覆盖高效种植模式研究与示范项目(2016E02009)资助。

Selection and Cloning of Thaumatin-like Protein (TLP) Gene from Winter Brassica rapa and Its Expression under Low Temperature Stress

MA Li1,**,YUAN Jin-Hai1,**,SUN Wan-Cang1,LIU Zi-Gang1,ZENG Xiu-Cun2,WU Jun-Yan1,FANG Yan1,LI Xue-Cai1,CHEN Qi1,XU Yao-Zhao2,PU Yuan-Yuan1,LIU Hai-Qing1,YANG Gang1,LIU Lin-Bo1   

  1. 1 Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement / Rapeseed Engineering Research Center of Gansu Province / College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Agronomy and Biotechnology, Hexi University, Zhangye 734000, China
  • Received:2016-01-19 Revised:2016-11-03 Published:2017-04-12 Published online:2016-12-14
  • Contact: Sun Wanchang, E-mail:18293121851@163.com, Tel: 18293121851
  • Supported by:

    This study was supported by the China Agriculture Research System(CARS-13), the National Natural Science Foundation of China (31460356, 31560397), The National Basic Research Program of China (973 Program) (2015CB150206), the Natural Science Foundation of Gansu Province (145RJZG050), the part of National Key Research and Development Program(2016YFD0101300), and the research and demonstration project of high efficient planting pattern in the core area of the Silk Road Economic Zone in Xinjiang.

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摘要：

类甜蛋白是一种能在低温胁迫下诱导表达，增强植物抗逆性的关键蛋白质。本研究运用双向电泳结合质谱技术，筛选低温胁迫下陇油7号叶片差异蛋白点，从中分离出与抗寒密切相关的类甜蛋白。根据已发表植物类甜蛋白基因TLP的保守序列设计引物，采用RT-PCR扩增陇油7号的DNA，获得TLP基因开放阅读框，长度为732 bp，编码243个氨基酸的蛋白质。生物信息学分析显示，与甘蓝型油菜(Brassica napus)的蛋白质氨基酸序列同源性高达99.18%，该基因在进化上高度保守，其保守序列属于植物的GH69-TLP-SF超家族，TLP相对分子质量和理论等电点分别为26.02 kD和9.15。TLP含有一个信号肽，为亲水性蛋白，亚细胞定位预测其是在内质网中合成的蛋白。二级结构预测表明陇油7号的TLP是由不规则卷曲和延伸链组成的不稳定蛋白。实时荧光定量和半定量分析显示，在适当阈值的低温胁迫下TLP基因表达量上调，表明该基因在白菜型冬油菜陇油7号适应低温胁迫过程中发挥重要作用。

关键词: 白菜型冬油菜, 双向电泳, 低温, 类甜蛋白

Abstract:

Thaumatin-like protein (TLP) is an essential protein induced by low temperature stress and enhances plant resistance to adverse circumstances. In our study, we selected differential protein spots from Longyou 7 leaves under low temperature stress and separated TLP closely related to cold resistance by using two-dimensional electrophoresis and mass spectrometry. Using the primers of TLP cDNA sequences and reverse transcription PCR (RT-PCR), we cloned the complete open reading frame of TLP gene of winter Brassica rapa cultivar Longyou7. The sequence length of TLP from Brassica rapa was 732 bp, encoding a protein of 243 amino acid residues with a predicted molecular weight of 26.02 kD and a theoretical pI of 9.02. Bioinformatics analysis showed a similarity of 99.18% in TLP amino acid sequence between B. rapa and Brassica napus, the predicted TLP contained a conserved amino acid sequence belonging to the plant GH69-TLP-SF superfamily. The TLP had one signal peptide which belongs to hydrophilic protein. Moreover, the detection of subcellular localization suggested the synthetic process of this protein is in the endoplasmic reticulum. The prediction of the second structures indicated that TLP is a labile protein consisting of random coils and extended strands. The expression analysis of TLP gene by utilizing real time and Semi-quantitative RT-PCR suggested that the TLP expression could be up-regulated in response to lower temperature. In conclusion, TLP gene of the winter Brassica rapa cultivar Longyou 7 might play a significant role in response to cold stress.

Key words: Winter Brassica rapa, Two-dimensional gel electrophoresis, Lower temperature, TLP

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