丝瓜WRKY转录因子基因的分离与褐变分析

发布时间：2018-04-20 01:11

本文选题：丝瓜 + 转录组测序(RNA-seq)　；参考：《农业生物技术学报》2017年12期

【摘要】：WRKY转录因子是一类发现于高等植物中的转录因子超家族。许多研究表明,WRKY转录因子在植物生物和非生物胁迫中起作用,但是这些研究主要集中在拟南芥(Arabidopsis thaliana)等模式植物中,在甜瓜(Cucumis melo)及辣椒(Capsicum annuum)等蔬菜作物中也有报道,而丝瓜(Luffa cylindrical)WRKY转录因子在褐变中的研究还未见相关报道。前期利用转录组测序(RNA-seq)技术获得了鲜切丝瓜福丝3号果肉褐变前后转录组数据库,本研究从中筛选得到4个与植物WRKY转录因子蛋白一致性较高的Unigene序列。分析发现,4个丝瓜WRKY基因Unigene0018509、Unigene0021412、Unigene0025291和Unigene0034271均含1个完整的开放读码框(ORF),序列全长分别为2 189、1 990、2 365和2 274 bp,Gen Bank登录号为:KY621843~KY621846。Wolf Psort预测显示,4个Unigene编码的WRKY蛋白均定位于细胞核中。系统进化和基因保守结构域分析表明,4个WRKY转录因子均含有1个保守的WRKY结构域,且锌指结构为C2H2型,属于第(6)类WRKY蛋白家族。利用定量PCR技术分析了4个WRKY基因在丝瓜'福丝3号'不同组织(根、茎、叶、花和果肉)以及采后不同储藏时间下的表达情况,对4个WRKY基因在鲜切丝瓜果肉不同时间的表达模式与RNA-seq结果进行了分析。结果表明,4个丝瓜WRKY基因均具有组织表达特异性,且表达模式各不相同。4个WRKY基因在鲜切后q-PCR表达值与RNA-seq得到的表达量(fragments per kilobase million,FPKM)变化趋势基本一致,WRKY基因Unigene0018509、Unigene0021412和Unigene0025291在丝瓜鲜切后0~6 h内的表达量呈显著上调(P0.05),Unigene0034271在0~3 h内上调表达,6 h出现下调表达。分析发现,4个WRKY基因在丝瓜采后不同储藏褐变时间内的表达趋势总体上调,但4个WRKY基因的表达模式不尽相同。该研究为进一步阐明丝瓜褐变调节机理和品种的选育提供理论依据。
[Abstract]:WRKY transcription factors are a superfamily of transcription factors found in higher plants. Many studies have shown that WRKY transcription factors play a role in plant and abiotic stress, but these studies are mainly concentrated in model plants such as Arabidopsis thaliana, and are also reported in vegetable crops such as Cucumis melo) and Capsicum annuumum. However, the study of Luffa cylindrical)WRKY transcription factors in the process of browning has not been reported. Transcriptome database before and after browning of flesh of fresh-cut luffa gourd Fusi 3 was obtained by RNA-seq technique. Four Unigene sequences with high consistency with plant WRKY transcription factor protein were obtained in this study. It was found that four WRKY genes, Unigene0021412 Unigene0025291 and Unigene0034271, all contained a complete open reading frame, and the total length of the sequences was 218999 990,365 and 2 274 BP Bank respectively, which was predicted by the accession number of KY621843KY621846. Wolf Psort and the four WRKY proteins encoded by Unigene were all located in the nucleus. Phylogenetic analysis and gene conserved domain analysis showed that all the four WRKY transcription factors contained a conserved WRKY domain, and the zinc finger structure was C2H2 type, belonging to the sixth WRKY family. The expression of four WRKY genes in different tissues (root, stem, leaf, flower and pulp) and postharvest storage time of Luffa melon 'Fusi 3' were analyzed by quantitative PCR technique. The expression patterns and RNA-seq results of four WRKY genes in fresh cut loofah pulp were analyzed. The results showed that all of the four WRKY genes had tissue expression specificity. The expression patterns of the four WRKY genes were different after fresh cutting. The q-PCR expression values of the four WRKY genes were similar to those obtained from RNA-seq. The changes of WRKY gene, Unigene0018509, Unigene0021412 and Unigene0025291, were significantly up-regulated within 6 h after fresh cutting of loofah. The up-regulated expression was down-regulated at 6 h. It was found that the expression of four WRKY genes was up-regulated in different storage browning time after harvest, but the expression patterns of four WRKY genes were different. This study provides a theoretical basis for further elucidation of browning regulation mechanism and breeding of luffa.
【作者单位】：福建省农业科学院作物研究所/福建省农业科学院蔬菜研究中心/福建省蔬菜工程技术研究中心;
【基金】：福建省属公益类科研院所基本科研专项(No.2017R1026-3) 国家大宗蔬菜产业体系福州试验站(No.CARS-25-G-20) 福建省农业科学院创新团队PI项目"特色作物重要性状形成机理研究"(No.2016PI-40);福建省农业科学院青年人才创新基金(No.2015QC-6);福建省农业科学院蔬菜科技创新团队(STIT2017-1-2)
【分类号】：Q943.2;S642.4

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