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

发布时间：2018-08-20 16:44

【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片段对高浓度SA响应能力进一步显著增强,对低浓度SA响应也有微弱提高;而长度为2 000 bp的核苷酸片段无论对高浓度SA还是对低浓度SA都具有显著响应能力,且达到最强。与2 000 bp片段相比,2 500 bp的核苷酸片段没有进一步增强启动子片段对SA的响应能力。超表达Md WRKY40蛋白对其自身的转录具有抑制作用。【结论】2 mmol·L-1 SA处理所影响的基因主要参与了苯丙烷类、类黄酮的生物合成,植物病原菌互作及植物激素信号转导途径。位于Md WRKY40开放阅读框上游的2 500 bp核苷酸序列,为对SA进行特异性响应的核苷酸启动子序列。在1 000—1 500 bp及1 500—2 000 bp具有显著提高启动子对SA敏感性的未知核苷酸序列,另外,Md WRKY40转录调控存在反馈抑制机制。
[Abstract]:[objective] to investigate the effect of salicylic acid (SA) on gene transcription in apple leaves, and to identify the SA signaling pathway and its regulatory gene. In order to provide theoretical basis for studying the molecular mechanism of disease resistance mediated by SA. [methods] the leaves of 'Gara' plantlets growing for 30 days were treated with 2 mmol L-1 salicylic acid (SA) for 12 h and CTRL (0.2% ethanol) as control. Illumina Hi Seq TM 2000 was used to sequence transcription sequence. The regulation genes of SA signaling pathway were screened by comprehensive bioinformatics analysis (differential gene screening, conditional specificity analysis, go classification and KEGG enrichment analysis). The promoter which was specifically induced by SA was cloned, and the promoter activity was identified by means of protoplast transformation of apple cells. [results] the original data of 750439 459 BP and 751,596,153 BP were obtained by CTRL and SA treatments, respectively, with 44.77% and 43.88% matching the genome of 'Golden Crown' apple, respectively. A total of 3,329 differentially expressed genes, including phenylpropane, were obtained. Genes related to the biosynthesis pathway of secondary metabolites such as flavonoids (such as CAD, Cad, P450, 尾 -1m3-glucanase associated with fungal resistance, etc.), and important functional genes (calmodulin) regulating the interaction of plant pathogens. Protein Ca M, resistance protein RPM1, heat shock protein HSP90 WRKY transcription factor, etc., and 33 conditional specific expression genes (NAC transcription factor NieMIN1, WRKY40, etc.). Among them, 1 085 genes were up-regulated and 2 244 genes were down-regulated. The differential genes are mainly involved in cellular processes, metabolic processes and gene binding, catalytic activity, etc. According to the results of transcriptome, the promoter sequence of SA responsive gene Md WRKY40 was cloned into the expression vector containing luciferase gene. When placed upstream of luciferase gene, the luciferase activity was 20.6 times higher than that of non-SA treated apple protoplast. The activity of luciferase was not affected by abscisic acid (ABA), jasmonic acid (JA) 1-aminocyclopropane carboxylic acid (ACC), which indicated that the promoter was the promoter of specific response to SA in apple. The response ability of different segments of promoter fragments to SA was different. From Md WRKY40 translation initiation site (ATG) to upstream, 500-1 000 BP could only respond to high concentration of SA, but not to low concentration of SA. The response of 1 500bp fragment to high concentration of SA was further enhanced, and the response to low concentration of SA was also slightly enhanced. The nucleotide fragment with a length of 2 000 BP was highly responsive to both high and low concentrations of SA. Compared with the 2 000 BP fragment, the 2 500 BP nucleotide fragment did not further enhance the response of the promoter fragment to SA. [conclusion] the genes affected by 2 mmol L-1 SA are involved in the biosynthesis of phenylpropanes, flavonoids, the interaction of plant pathogens and the signal transduction pathway of plant hormones. [conclusion] the overexpression of Md WRKY40 protein inhibits its own transcription. [conclusion] the genes affected by 2 mmol L-1 SA are mainly involved in the biosynthesis of phenylpropanes, flavonoids and plant pathogens. The 2 500bp nucleotide sequence located in the upper reaches of Md WRKY40 open reading box is a nucleotide promoter that responds specifically to SA. At 1 000-1 500 BP and 1 500-2 000 BP, the sequence of unknown nucleotides significantly increased the sensitivity of promoter to SA. In addition, there was a feedback inhibition mechanism in the transcriptional regulation of Md WRKY40.
【作者单位】：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;
【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)
【分类号】：Q943.2;S436.611

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