节点文献

小麦TaARF20基因克隆及表达分析

Cloning and expression analysis of TaARF20 gene in wheat(Triticum aestivum L.)

  • 推荐 CAJ下载
  • PDF下载
  • 不支持迅雷等下载工具,请取消加速工具后下载。

【作者】 郭晓凤王超杰张莉莉景豆豆李政都晶晶马守才

【Author】 GUO Xiao-feng;WANG Chao-jie;ZHANG Li-li;JING Dou-dou;LI Zheng;DU Jing-jing;MA Shou-cai;College of Agronomy,Northwest A & F University/National Yangling Agricultural Biotechnology & Breeding Center/Yangling Branch of State Wheat Improvement Center/Wheat Breeding Engineering Research Center,Ministry of Education/Key laboratory of Crop Hetersis of Shaanxi Province;Institute of Crop Science,Chinese Academy of Agricultural Sciences/Centre for Crop Genetics and Breeding/Wheat Mutation Breeding Centre;College of Life Science,Henan University/State Key Laboratory of Crop Adversity Adaptation and Improvement;

【通讯作者】 马守才;

【机构】 西北农林科技大学农学院/国家杨陵农业生物技术育种中心/国家小麦改良中心杨陵分中心/小麦育种教育部工程研究中心/陕西省作物杂种优势研究与利用重点实验室中国农业科学院作物科学研究所/作物遗传育种中心/小麦诱变育种中心河南大学生命科学学院/作物逆境适应与改良国家重点实验室

【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。

【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.

【基金】 国家重点研发计划七大农作物育种专项(2018YFD0100904);陕西省重点研发计划项目(2020NY-048);陕西省重点实验室后补助项目(2018SZS-22)~~
  • 【文献出处】 南方农业学报 ,Journal of Southern Agriculture , 编辑部邮箱 ,2021年09期
  • 【分类号】S512.1
  • 【被引频次】1
  • 【下载频次】302
节点文献中: 

本文链接的文献网络图示:

本文的引文网络