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 k D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。
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 c DNA sequences and reverse transcription PCR（RT-PCR）, we cloned the complete open reading frame of TLP gene of winter Brassica rapa cultivar Longyou 7. The sequence length of TLP from B. rapa was 732 bp, encoding a protein of 243 amino acid residues with a predicted molecular weight of 26.02 k D and a theoretical p I 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 B. rapa cultivar Longyou 7 might play a significant role in response to cold stress.
出处
《作物学报》
CAS
CSCD
北大核心
2017年第4期620-628,共9页
Acta Agronomica Sinica
基金
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)
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~~
关键词
白菜型冬油菜
双向电泳
低温
类甜蛋白
Winter Brassica rapa
Two-dimensional gel electrophoresis
Lower temperature
TLP
作者简介
第一作者联系方式：马骊，E-mail：348369277@qq．com，Tel：18189560623
第一作者联系方式：袁金海，E-mail：954152476@qq．com
通讯作者：孙万仓，E-mail：18293121851@163．com，Tel：18293121851
