【摘要】 【目的】克隆Ta ARF20基因,并分析其表达特性,为解析小麦生长素响应因子(ARF)基因家族成员的调控机制提供理论依据。【方法】以普通小麦为材料,利用同源克隆技术获得TaARF20基因的cDNA全长序列,利用生物信息学软件分析其序列特征,并通过亚细胞定位试验明确Ta ARF20蛋白的作用部位。将TaARF20基因与表达载体PcoldTF连接,转化大肠杆菌中进行原核表达。采用实时荧光定量PCR检测TaARF20基因在普通小麦不同组织及普通小麦和多子房小麦不同发育时期幼穗中的表达模式。【结果】Ta ARF20基因包含1个内含子和2个外显子,编码区(CDS)长度为1116 bp,编码371个氨基酸残基,蛋白分子量约40.38 k D,理论等电点(pI)为4.96,脂溶性系数为19.80,疏水性系数为0.985,不稳定系数为50.51,属于不稳定蛋白,定位在细胞核中,含有ARF家族蛋白的保守结构域和B3 DNA结合域,主要由α-螺旋(18.06%)、无规则卷曲(59.57%)和延伸连(22.10%)组成。启动子区含有激素响应、光响应和低温响应等多个顺式作用元件。TaARF20蛋白与二粒小麦ARF20的氨基酸序列相似性最高,亲缘关系也最近。Ta ARF20融合蛋白在原核系统中成功表达。Ta ARF20基因在小麦的根、叶、幼穗和籽粒中均有表达,但在幼穗中的相对表达量最高。对于长度为3和4 cm的幼穗来说,普通小麦Ta ARF20基因的相对表达量低于多子房小麦,但对于长度为5、6、8和9 cm的幼穗,普通小麦TaARF20基因的相对表达量高于多子房小麦。【结论】TaARF20基因在小麦穗生长发育中发挥重要调控作用,推测其是调控穗型、穗大小或穗粒数的关键基因。更多还原

【Abstract】 【Objective】To provide theoretical basis for analyzing the regulation mechanism of Auxin response factor(ARF)genes family in wheat by cloing and analyzing expression characteristic of TaARF20 gene.【Method】The fulllength cDNA sequence of Ta ARF20 gene was obtained from common wheat by homologous cloning technique,and its sequence characteristics were analyzed by bioinformatics. The active site of TaARF20 protein was determined by subcellular localization experiment. The TaARF20 gene was ligated with the expression vector Pcold-TF and transformed into Escherichia coli for prokaryotic expression. The expression patterns of TaARF20 gene in different tissues of common wheat and immature spike of common wheat and multi-ovary wheat at different development stages were analyzed by real-time fluorescence quantitative PCR.【Result】Sequence analysis indicated that TaARF20 gene contained one intron and two exons,the coding region(CDS)was 1116 bp and encoded 371 amino acid residues. The molecular weight of Ta ARF20 protein was about 40.38 kD,theoretical isoelectric point(pI)was 4.96,fat solubility coefficient was 19.80,hydrophobicity coefficient was 0.985,and instability coefficient was 50.51 which indicated TaARF20 was an unstable protein. Ta ARF20 was located in the nucleus and contained ARF family domain and B3 DNA binding domain. Secondary structure analysis showed that the protein was mainly composed of alpha helix(18.06%),extended strand(59.57%),random coil(22.10%).The analysis of the promoter region revealed multiple cis-regulatory element responses,including hormonal responses,light and temperature responses. Alignment analysis of amino acid sequences and phylogenetic tree analysis showed that TaARF20 protein of common wheat had the highest amino acid sequence similarity and closest homology with Triticum dicoccoides ARF20. The result of SDS-PAGE showed that TaARF20 fusion protein was successfully expressed in prokaryotic cells in vitro. Real-time fluorescence quantitative PCR analysis showed that TaARF20 gene was expressed differently in root,leaf,immature spike and seed of wheat,and the expression in immature spike was significantly higher than that in other tissues. The relative expression level of TaARF20 gene in common wheat was lower than that in multi-ovary wheat for immature spike of 3 and 4 cm,but higher than that in multi-ovary wheat for immature spike of 5,6,8 and 9 cm in length.【Conclusion】Ta ARF20 gene plays an important regulatory role in immature spike of wheat,which is speculated that it may be a key gene regulating spike shape,spike size and grain number per spike.更多还原