柑橘响应溃疡病菌转录因子CsBZIP40的克隆及功能分析  

Gene Cloning and Expression Analysis of Canker-Related Transcription Factor Cs BZIP40 in Citrus

作　　者：贾瑞瑞[1] 周鹏飞[1] 白晓晶[1] 陈善春[1] 许兰珍[1] 彭爱红[1] 雷天刚[1] 姚利晓[1] 陈敏[1] 何永睿[1] 李强[1] 

机构地区：[1]西南大学柑桔研究所/中国农业科学院柑桔研究所/国家柑桔工程技术研究中心,重庆400712

出　　处：《中国农业科学》2017年第13期2488-2497,共10页Scientia Agricultura Sinica

基　　金：国家现代农业柑桔产业技术体系(CARS-27);中央高校基本科研业务费(XDJK2015C089;SWU115025;XDJK2014A08)

摘　　要：【目的】分析柑橘BZIP转录因子家族并克隆柑橘溃疡病菌相关的转录因子Cs BZIP40,对其进行亚细胞定位并研究外源激素及机械损伤对该基因的诱导表达,确定其诱导表达模式,分析其与溃疡病菌侵染的关系。【方法】基于全基因组公共数据库,对柑橘BZIP转录因子进行综合专业注释以获得柑橘中BZIP家族的所有成员信息,并根据染色体定位对其进行命名;利用MEME分析其结构域;利用MEGA 6.0分析柑橘BZIP与拟南芥BZIP的系统发育关系,并根据系统发育关系对该基因家族进行分类,确定本研究关注的Cs BZIP40所属的类型;结合染色体定位和系统发育树确定该家族的基因复制情况;利用实时荧光定量PCR(q RT-PCR)方法验证感染溃疡病菌前后由柑橘转录组筛选出的Cs BZIP40表达模式;分析Cs BZIP40、启动子元件(plant CARE)和核定位信号(c NLSmapper),构建GFP融合载体,用洋葱表皮进行亚细胞定位分析,来对预测的核定位信息进行验证;利用q RT-PCR技术分析水杨酸(SA)、茉莉酸甲酯(Me JA)、乙烯利(ET)以及机械损伤(wounding)对该基因的诱导表达模式,揭示该转录因子与激素代谢途径的关系。【结果】从柑橘(甜橙)全基因组数据库中共注释到47个BZIP,这些BZIP基因位于9号染色体之外的所有染色体上,其中3号染色体基因密度最大为4.5×10-7个/Mb,2号染色体上的BZIP基因密度最小,仅占所有BZIP基因的2%;柑橘BZIP基因家族含有较少的基因复制事件,所以相对其他已测序物种其BZIP家族较小;Cs BZIP40基因全长5 756 bp,开放阅读框1 530 bp,编码509个氨基酸;经BZIP家族染色体定位、结构域和系统发育分析结果显示Cs BZIP40基因序列特异性较好,且Cs BZIP40与拟南芥中AT1g08320属于同源基因,属于柑橘10个亚家族中参与病菌防御的D亚类;上游启动子元件含多个与植物逆境或激素应答相关的顺式作用元件,例如Box-W1、HSE、ERE等;此基因含有核定【Objective】 The objective of this study is to annotate the BZIP family and clone the citrus canker related transcription factor Cs BZIP40. It is also aimed to confirm the subcellular localization and the expression profile reduced by exogenous hormone and mechanical damage and the relations between Cs BZIP40 and Xanthomonas citri subsp. citri（Xcc） infection. 【Method】Based on the public genome databases, the BZIP gene family was expertly and comprehensively annotated and named based on the chromosomal localization of all the members of BZIP; the motifs of the BZIPs were analyzed by MEME online tool. The phylogenetic tree of BZIPs in Citrus and Arabidopsis thaliana was constructed using software MEGA 6.0 based on which the category of BZIP family was obtained. Canker-related transitional factor Cs BZIP40 obtained from transcriptome data was also detected by q RT-PCR. Elements in the promoter and the nuclear localization signal were analyzed with database plant CARE and online tool c NLSmapper, respectively. And then the subcellular localization was confirmed by GFP fusion experiments in onion to confirm the prediction of nuclear localization analyzed with softwares. Expression profiles induced by salicylic acid（SA）, jasmonic acid methyl ester（Me JA）, ethylene（ET） and mechanical damage of Cs BZIP40 were checked with q RT-PCR. 【Result】A total of 47 BZIP genes were annotated from the whole genome of Citrus sinensis and all the BZIPs are located on every chromosome except the 9th one. The BZIP concentration on chromosome 3 are 4.5×10-7/Mb which is the highest while chromosome 2 is the lowest, contains only 2% of all BZIPs in citrus. There were fewer gene duplication events detected from BZIP family of citrus compared with other plants, such as Arabidopsis, grapevine and so on. That is why citrus has a smaller BZIP family size. The full-length of Cs BZIP40 is 5 756 bp with a 1 530 bp open reading frame which codes a protein containing 509 amino acids. It is closely related to AT1g08320 in A.

关 键 词：柑橘溃疡病 BZIP 转录因子 亚细胞定位 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]