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连肖华,陈 坚.SUSIRI基因的生物信息学分析及亚细胞定位[J].中国农学通报,2015,31(6):128-135.Lian Xiaohua,Chen Jian.Analysis of SUSIRI Gene from Rice by Bioinformatics and Subcellular Localization[J].Chinese Agricultural Science Bulletin,2015,31(6):128-135
SUSIRI基因的生物信息学分析及亚细胞定位
Analysis of SUSIRI Gene from Rice by Bioinformatics and Subcellular Localization
投稿时间:2014-11-17  修订日期:2015-02-10
DOI:10.11924/j.issn.1000-6850.casb14110097
中文关键词: 水稻  核蛋白  亚细胞定位  基因克隆  生物信息学
英文关键词: rice  nucleus protein  subcellular localization  gene clone  bioinformatics
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基金项目:福建省自然科学基金项目“SUSIRI过表达及RNAi对水稻生育性状的影响”(2011J01104)。
作者单位E-mail
连肖华 山西省长子县实验中学 lianxiaohua721@163.com 
陈 坚 福建省农业科学院生物技术研究所 chenjian3@hotmail.com 
中文摘要:
      转录因子是植物生长发育及其对外界环境的应答反应中起重要作用的蛋白调控因子,一般含有DNA结构域、转录调控域、寡聚化位点和核定位信号。WRKY蛋白是一类具有重要作用的转录调控因子。在水稻的基因组中预测到的WRKY基因多达103个,对这些基因的功能加以注释具有重要的意义。SUSIRI基因是前人从粳稻日本晴中克隆的一个WRKY家族的转录因子。其开放阅读框长度为1755 bp,可编码584个氨基酸,具有典型的双WRKY结构域。为了进一步研究SSUSIRI基因,阐明它在水稻中的生物调控功能。通过应用生物信息学的方法,对该基因的预测蛋白进行了结构与功能的分析。利用EBI的interpro数据库分析SUSIRI基因序列的基序(motif),并结合NCBI的CDD(conserved domains)数据库分析该蛋白的保守结构域,用DNAMAN软件和CBS的TMHMM软件分析蛋白氨基酸残基的亲(疏)水性特点和跨膜结构域,用CBS的Protcomp version9.0软件和Protfun软件分别分析了蛋白的亚细胞定位和功能预测;通过实验设计把SUSIRI基因与GFP基因相融合,构建了由actin启动子驱动的SUSIRI基因的荧光表达载体pNSUGFP。通过采用基因枪法把该载体转入洋葱表皮细胞进行亚细胞定位鉴定。结果表明,生物信息学预测值显示:SUSIRI基因具有转录、转录调控、信号转导类功能的可能性最高,预测值分别为0.973、1.598和0.602;同时,其参与翻译功能、中间代谢功能和脂肪酸代谢功能的可能性也较大,预测值分别为4.800、1.490和1.265;由于SUSIRI蛋白中含有的亲水性氨基酸残基较多,该蛋白不具有跨膜性,是一种非跨膜类蛋白。对其亚细胞定位预测表明:在其氨基酸序列中含有较强的核定位信号,所以定位于细胞核内的可能性很大。进一步用荧光显微镜对基因枪轰击的洋葱表皮细胞的观察发现:对照的GFP蛋白主要沿细胞壁表达,而SUSIRI-GFP融合蛋白在细胞核中有很强的表达。通过试验研究得出:SUSIRI基因可能是一个参与中间代谢调控功能的基因,主要在基因转录环节发挥作用,是一种核定位蛋白。
英文摘要:
      Transcription factors are a kind of proteins that play an important role in the regulation of plant growth and development, as well as in plant response to environmental variation. They usually contain a DNA-binding domain, a transcription regulation domain, an oligomerization site and a nuclear localization signal inside their DNA sequence. WRKY proteins had been identified as one of the important transcription factors in plants. There have been 103 WRKY genes which were predicted in rice genome by data mining and the annotation of the biological function of these genes had the important value for the functional genomics of rice. SUSIRI gene was one of the WRKY transcription factor genes cloned from the Oryza sativa sub. Japonica var. Nipponbare and its ORF was 1755 bp long, encoding a polypeptide of 584 amino acids with two typical WRKY domains. In this study, based on nucleotide sequence of the SUSIRI gene, its biological function in gene regulation in rice had been further elucidated. Firstly, the structure and functions of the predicted protein were analyzed by bioinformatics tools while the Motif sequence of SUSIRI gene was analyzed by using EBI interpro database, the conserved domains of the protein was analyzed by NCBI CDD database; the hydrophilicity (hydrophobicity) of amino acid residues and the transmembrane domains of SUSIRI protein were analyzed by DNAMAN and CBS TMHMM. By using CBS Protcomp version 9.0 and Protfun software, the subcellular localization and function of protein were predicted. Secondly, the fluorescence expression vector of pNSUGFP driven by actin promoter was constructed by fusing the SUSIRI gene and GFP gene and was used to transform into epidermis cell of onion by Gene gun for detecting gene subcellular location. The prediction from bioinformatics showed that the SUSIRI gene had the greatest probability in function of transcription, transcription regulation or signal transduction in the rice cell with the value in prediction of 0.973, 1.598 and 0.602. Similarly, it had the greatest probability in participation of translation, central intermediary metabolism, fatty acid metabolism with the predicted value of 4.800, 1.490 and 1.265. Because of high content of hydrophilic amino acid residues among peptide sequences, the SUSIRI protein had hardly inserted into the membrane in the cell when it was likely to be localized within the cell nucleus since the strong nucleus location signal was detected from the sequence of amino acids. By using the fluorescence microscopy to observe the onion epidermic cells, the GFP protein was shown to express along the cell wall in the control, but the fused protein of SUSIRI-GFP expressed strongly in the cell nucleus. The result indicated that the SUSIRI gene was likely to be the transcription factor regulating the intermediary metabolism in rice and playing the role during the gene transcription. SUSIRI protein could be localized in nucleolus which is an obvious feature of transcription factor of SUSIRI gene determined by the experiment.
作者简介:第一作者简介: 连肖华, 男, 1983年出生, 山西长治人, 中教二级, 硕士, 研究方向为作物分子遗传育种。通信地址: 046600 山西省长子县鹿谷东街 117号 长子县实验中学, E-mail: lianxiaohua721@163.com。 通讯作者: 陈坚, 男, 1965年出生, 福建永泰人, 研究员, 博士, 研究方向为作物生化与分子生物学, 主要从事植物基因资源评价以及植物生化与分子生物学等领域的研究。先后主持过福建省自然科学基金项目、 福建省重点科技项目及公益类项目 5项; 参加过国家级 “中国-瑞典” 国际合作项目 2项, 农业公益类项目 1项以及各类省级重大重点项目 5项。发表论文 29篇, 获省级科技进步三等奖 2项。通信地址: 350003 福建省福州市鼓楼区五四路 247号 福建省农科院生物技术研究所, E-mail: chenjian3@hotmail.com。
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