小麦转录因子基因TaERF7的克隆及其表达分析
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西北农林科技大学农学院

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国家科技支撑计划(2015BAD27B01)、国家自然科学基金(31371697)、陕西省科技统筹创新工程计划(2014KTZB02-01-02)、国家小麦产业技术体系项目(K3070217023)


Cloning and Expression Analysis of Transcription Factor Gene TaERF7 in Wheat(Triticum aestivum)
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    摘要:

    温光敏核不育小麦(Triticum aestivum)BNS(Bainong sterility)是一种良好的小麦杂种优势利用材料,利用其低温不育、高温可育的特性可以实现 “两系法”杂交小麦育种。本研究为找到BNS育性转换的关键基因,从已有BNS不育和可育花药的基因芯片数据中,鉴定到表达存在差异的TaERF7基因,并通过设计引物克隆了TaERF7的cDNA和启动子序列。 生物信息学分析显示,(1)TaERF7基因的CDS区有660 bp,编码219个氨基酸;(2)TaERF7蛋白含有AP2结构域和两个EAR motif,属于第二类乙烯响应因子(Ethylene response factors, ERFs);(3) TaERF7的氨基酸序列与拟南芥(Arabidopsis thaliana)AtERF4同源,可能是一种转录抑制因子;(4)TaERF7启动子区含有多个光响应和低温响应的顺式作用元件。原核表达结果显示TaERF7蛋白具有良好的可溶性,最佳的IPTG诱导浓度是0.5 mmol/L,纯化蛋白时最适的咪唑洗脱浓度为50 mmol/L。qRT-PCR结果表明,TaERF7在BNS的不同组织与器官中均有表达;在药隔期幼穗和减数分裂期花药中,表现为在不育系中上调表达,而在可育系中下调表达;不同播期的BNS花药中表达量存在显著性差异。分析TaERF7响应不同温度和光照的变化,结果表明长日照(14 h)下,TaERF7在BNS中的表达量下调约0.47倍,而在短日照(10 h)处理下,TaERF7在BNS中的表达量上调约1.14倍;4℃的低温处理使TaERF7表达量在2 h内上调了约25.7倍,并且在48 h内一直保持较高的水平,而在37℃下虽然可以使TaERF7表达量在1 h内上调约0.71倍,但2 h后便急剧下调,到12 h时其表达量与对照相比已下调了约0.85倍。通过以上研究结果,我们认为TaERF7在药隔期和减数分裂期的上调表达可能是引起BNS雄性不育的重要因素。

    Abstract:

    Bainong?male sterility(BNS)is an efficient thermo-photo-sensitive genic male sterile line of wheat(Triticum aestivum). BNS is sterile in low temperature and fertile in high temperature. With these features, BNS was widely used in hybrid wheat breeding. In this study, based on the gene chip data of the anthers of BNS sterile line and fertile line, we found that the transcription factor TaERF7 was differentially expressed between the two lines, and cloned the cDNA and promoter sequences of TaERF7. Bioinformatics analysis revealed that the coding sequences(CDS)of TaERF7 is 660 bp and encoding of 219 amino acids. TaERF7 protein contains AP2 domain and two EAR motifs. It belongs to class II Ethylene response factors (ERFs). The amino acid sequence of TaERF7 is homologous to Arabidopsis(Arabidopsis thaliana)AtERF4, which is a transcriptional repressor. The promoter region contained multiple cis-acting elements with light and low temperature response. Prokaryotic expression results showed that TaERF7 protein had good solubility in water. The best concentration of IPTG induction was 0.5 mmol/L, and the best Imidazole Elution concentration was 50 mmol/L for protein purification. The qRT-PCR results showed that TaERF7 expressed in different tissues of BNS. In young spikes at anther separation stage and anthers at meiosis phases, TaERF7 was up-regulated in sterile lines and down-regulated in fertile lines. TaERF7 was differentially expressed in anthers of BNS across with different sowing dates. Along with this, we analyzed TaERF7 response to several temperatures and daylight hours. The results showed that the expression of TaERF7 was down-regulated 0.47 fold under long daylight(14 h), while it was up-regulated 1.14 fold under short day(10 h). The low temperature treatment at 4°C increased the expression of TaERF7 about 25.7 fold in 2 h, and maintained a high level upto 48 h. The high temperature treatment at 37°C increased the expression of TaERF7 about 0.71 fold in 1 h, but it was sharply down-regulated to 0.85 fold after 2 h. According to the above results, we believe that the up-regulated expression of TaERF7 in anther seperation and meiosis stage may cause the male sterility in BNS.

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  • 收稿日期:2019-12-22
  • 最后修改日期:2020-02-02
  • 录用日期:2020-02-26
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