小麦TaGSK5基因的克隆与表达分析

Cloning and expression analysis of TaGSK5 gene in wheat

【目的】糖原合成激酶(glycogen synthase kinase,GSK)在响应植物非生物胁迫和参与植物生长发育调控方面起重要作用.克隆小麦GSK基因家族成员,分析其序列结构和表达模式,为进一步解析小麦GSK基因功能提供理论依据.【方法】通过同源克隆从小麦品种陇中1号中获得TaGSK5的4个同源基因cDNA序列,利用生物信息学结合qRT-PCR方法对TaGSK5进行基因序列和蛋白结构预测,以及基因表达模式分析.【结果】小麦TaGSK54个部分同源基因分别位于小麦1A、4A、5B和5D染色体上,分别命名为TaGSK5-1A,TaGSK5-4A,TaGSK5-5B和TaGSK5-5D.本研究克隆该基因的cDNA全长为1281 bp,包含12个外显子和11个内含子,编码426个氨基酸,具有激酶结构域,其蛋白结构与粗山羊草亲缘关系较近.TaGSK5编码的蛋白为可溶性蛋白,等电点分别为8.53、8.57、8.79和8.54,分子量分别为48226.51、47602.69、48237.52和48314.58.二级结构元件以α-螺旋和不规则卷曲为主.TaGSK5基因在小麦根、茎、叶和穗中均有表达,但在穗中表达量较高;该基因在干旱胁迫处理后0~6 h表达下调,在盐胁迫下表达量没有显著变化.【结论】小麦TaGSK5包含4个同源基因,位于不同的染色体上,其编码氨基酸序列高度相似,该基因可能负调控小麦耐旱性,与盐胁迫无关.

【Objective】Glycogen synthase kinase(GSK)plays an important role in response to abiotic stress and in the regulation of plant growth and development.The GSK family genes in wheat were cloned,followed by analyzing their sequence structure and expression pattern,for the purpose of providing theoretical basis for further analysis of GSK gene function in wheat.【Method】Four homologous cDNA of TaGSK5 were obtained from wheat cultivar‘Longzhong 1’by the method of homologous cloning.Gene sequence,protein structures and expression pattern of TaGSK5 were analyzed and predicted by the way of bioinformatics combined with qRT-PCR.【Result】Four partially homologous genes of TaGSK5 were located on the chromosomes 1 A,4 A,5 B and 5 D in wheat,which were named as TaGSK5-1 A,TaGSK5-4 A,TaGSK5-5 B and TaGSK5-5 D,respectively.The full-length cDNA of these genes was 1281 bp,including 12 exons and 11 introns,which encoded 426 amino acids with a kinase domain.Its protein structure was close to that of Aegilops tauschii.The protein encoded by TaGSK5 was soluble,with isoelectric points of 8.53,8.57,8.79 and 8.54 and molecular weights of 48226.51,47602.69,48237.52 and 48314.58,respectively.The secondary structure elements of these proteins were mainlyα-helix and irregular coil.TaGSK5 gene was expressed in root,stem,leaf and spike of wheat,and its expression level was relatively high in spike.The expression level of TaGSK5 gene was down-regulated at 0~6 h after drought stress,while there was no significant change under salt stress.【Conclusion】There were four homologous genes in TaGSK5 family,which are located on different chromosomes and have highly similar amino acid sequences.TaGSK5 negatively regulates drought tolerance of wheat,and it had seemly nothing to do with salt stress.