基于转录组分析苹果水杨酸特异响应基因MdWRKY40的启动子鉴定  

Identification of MdWRKY40 Promoter Specific Response to Salicylic Acid by Transcriptome Sequencing

作　　者：邱化荣 周茜茜 何晓文 张宗营[1] 张世忠[1] 陈学森[1] 吴树敬[1] QIU HuaRong;ZHOU QianQian;HE XiaoWen;ZHANG ZongYing;ZHANG ShiZhong;CHEN XueSen;WU ShuJing(College of Horticultural Science and Engineering, Shandong Agricultural University/State Key Laboratory of Crop Biology, Tai＇ an 271018, Shandong)

机构地区：[1]山东农业大学园艺科学与工程学院/作物生物学国家重点实验室

出　　处：《中国农业科学》2017年第20期3970-3990,共21页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31272132);山东省泰山学者工程启动基金(tshw20120712)

摘　　要：【目的】探明水杨酸(SA)对苹果叶片基因转录调控的影响,鉴定SA信号途径及其调控基因,为研究SA介导的抗病分子机制提供理论依据。【方法】生长30 d的‘嘎啦’组培苗叶片用2 mmol·L-1水杨酸(SA)处理12 h,以CTRL(0.2%乙醇)处理作为对照,利用Illumina Hi Seq TM 2000进行转录组测序,通过综合的生物信息学分析(差异基因筛选、条件特异性分析、GO分类及KEGG富集分析等)筛选SA信号途径的调控基因。克隆受SA特异性诱导表达基因的启动子,利用苹果细胞原生质体转化技术,进行启动子活性鉴定,确定对SA进行特异性响应的核苷酸序列。【结果】CTRL和SA处理分别获得750 439 459 bp和751 596 153 bp的原始数据,分别有44.77%和43.88%与‘金冠’苹果基因组完全匹配。获得3 329个显著性差异基因,包括苯丙烷类、类黄酮等次生代谢物生物合成途径的相关基因(如木质素合成关键酶CAD、细胞色素P450、真菌抗性相关的β-1,3-葡聚糖酶等),调控植物病原菌互作途径重要功能基因(钙调蛋白Ca M、抗病蛋白RPM1、热激蛋白HSP90、WRKY转录因子等)以及33个条件特异性诱导表达基因(NAC转录因子、NIMIN1、WRKY40、ERF转录因子等)。其中1 085个基因上调,2 244个基因下调。差异基因主要涉及细胞过程、代谢过程和基因绑定、催化活性等;根据转录组学的结果,将SA响应基因Md WRKY40的启动子序列克隆到含有荧光素酶基因的表达载体中,置于荧光素酶基因的上游,转化苹果原生质体细胞。SA处理的原生质体细胞,荧光素酶的活性为未经SA处理的20.6倍,而脱落酸(ABA)、茉莉酸(JA)、1-氨基环丙烷羧酸(ACC)对荧光素酶的活性没有影响,说明该启动子为苹果中对SA进行特异性响应的启动子序列。不同区段的启动子片段对SA响应能力不同,从Md WRKY40翻译起始位点ATG向上游500—1 000 bp只能响应高浓度SA,而对低浓度SA不具有响应能力,1 500 bp片�[Objective] In order to explain the theoretical basis for disease resistance molecular mechanism mediated by SA, the influence of transcriptional regulation of apple leaves response to salicylic acid was studied, and the SA signaling pathway and its regulated genes were identified.[Method] The leaves of tissue culture apple ‘gala＇ seedling growing under 24℃ for 30 d were treated with 2 mmol·L-1 SA（0.2% ethanol treatment as control） for 12 h. Then the transcriptome libraries was constructed by using Illumina Hi Seq TM 2000 sequencing technique, and the regulated genes of SA signaling pathway were screened by integrated bioinformatics analysis which included differential genes screening, condition specificity analysis, GO classification and KEGG enrichment analysis. The promotor of the gene which was specific response to SA was cloned and promotor activity was identified by using protoplast transformation technique. The specific response of different nucleotide fragment of promotor was verified.[Result]The original data of 750 439 459 and 751 596 153 bp sequences was obtained from CTRL and SA treatment samples, Which were 44.77% and 43.88% perfect match with the ‘golden delicious＇ apple genome sequence, respectively. The transcriptome data of SA treated samples suggested that 3 329 genes were significantly expressed, including the genes related to biosynthesis of secondary metabolites pathway（the key enzyme of lignin synthetic pathway CAD, cytochrome P450, β-1,3-glucanase related to fungal resistance）, the important functional genes involved in plant-pathogen pathway（calmodulin Ca M, disease resistance protein RPM1, heat shock protein HSP90, WRKY transcription factors） and 33 condition specificity genes（NAC transcription factors, NIM1, WRKY40, Ethylene responsive factors and so on）. Among them, 1 085 genes were up-regulated and 2 244 genes were down-regulated. Differentially expressed genes were participated in cellular process, metabolic process, binding, catalytic activity and so on. A

关 键 词：SA 苹果 转录组 信号转导 植物病原菌互作 

分 类 号：Q943.2[生物学—植物学] S436.611[农业科学—农业昆虫与害虫防治]