玉米肌动蛋白解聚因子ZmADF4基因克隆、转录活性及表达分析

Cloning, transcription activity and expression analysis of actindepolymerizing factor 4, ZmADF4 in maize(Zea mays L.)

  • 摘要: 【目的】克隆玉米肌动蛋白解聚因子4(ZmADF4)基因,并对其转录活性和表达模式进行分析,为探究玉米ADF家族基因的功能及调控机制提供参考依据,也为玉米抵抗逆境胁迫研究提供潜在的基因资源。【方法】采用同源克隆技术克隆玉米叶片ZmADF4基因,运用生物信息学软件分析其编码蛋白的理化性质、保守结构域及二、三级结构;通过原生质体瞬时转化进行亚细胞定位;利用酵母自激活分析该基因转录活性,下载RNA-Seq数据分析其在不同组织中的表达特性,并利用实时荧光定量PCR检测其在不同逆境胁迫下的表达情况。【结果】从玉米叶片克隆获得的ZmADF4基因,编码区(CDS)长度为420 bp,编码139个氨基酸残基,蛋白分子量为15.855 kD,理论等电点(pI)为7.66,属于亲水性蛋白,具有典型的植物ADF蛋白家族的保守结构域ADF_gelsolin,主要定位于细胞质中。ZmADF4基因不具有转录自激活效应,且在根、茎、叶、花丝、花药、胚、胚乳和果皮等组织中呈差异性表达,在茎、果皮和叶片中表达量较高。ZmADF4基因对高温胁迫、低温胁迫、高盐胁迫、干旱胁迫及脱落酸处理均有响应,其中高温胁迫下,整体呈上调表达趋势,而低温胁迫和脱落酸处理下,整体呈下调表达趋势,干旱和高盐胁迫下则呈先上调再下调表达的趋势。【结论】ZmADF4基因属于ADF基因家族成员,不仅参与玉米茎、果皮和叶片的生长发育调控,还参与低温、高温、干旱、脱落酸、盐胁迫等逆境响应调控。

     

    Abstract: 【Objective】To provide reference for studying the function and regulation pattern of maize ADF family gene and provide potential gene resources for stress resistance in maize, the actin-depolymerizing factor 4(ZmADF4) gene was cloned and its transcription activity and expression pattern were analyzed.【Method】The ZmADF4 gene was cloned from maize leaves by homologous cloning technology. The physicochemical properties, conserved domains, secondary and tertiary structures of the encoded protein were predicted by bioinformatics analysis. Subcellular localization was carried out by transient transformation of protoplasts, and its transcriptional activity was analyzed by yeast self activation. The expressions of ZmADF4 gene in different tissues were analyzed by RNA-Seq data downloaded and its expressions under different stress treatments were detected by real-time fluorescence quantitative PCR.【Result】The results showed that the ZmADF4 gene was successfully cloned from maize leaf. The full length coding region(CDS) of ZmADF4 was 420 bp, which encoded a 139 amino acids residues. The molecular weight of the protein was 15.855 kD and its theoretical isoelectric point(pI) was 7.66. The protein encoded by ZmADF4 gene belonged to hydrophilic protein, which had conserved domain ADF_gelsolin of typical ADF protein family and mainly located in cytoplasm. ZmADF4 gene had no self activating activity by transcriptional activity analysis. Further studies showed that the gene was differentially expressed in maize roots, stems, leaves, silks, anthers, embryo, endosperm and pericarps, with expressed highly in stems, pericarps and leaves. Moreover, the expressions of ZmADF4 gene changed under high temperature, low temperature, high salt stress, drought stress and abscisic acid(ABA) stress. The expression of ZmADF4 gene was up-regulated under high temperature, while down-regulated under low temperature and ABA stress. Under drought and high salt stress, the expression was first upregulated and then down-regulated.【Conclusion】ZmADF4 gene, belonging to ADF gene family, not only participates in the growth and development regulation of maize stalks, peel and leaves, but also in the adverse response regulation of low temperature, high temperature, drought and ABA and salt stresses.

     

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