白粉菌诱导下小麦TaNHO1基因的克隆、表达分析及亚细胞定位

Cloning,Expression Analysis and Subcellular Localization of TaNHO1 Gene Induced by Powdery Mildew in Wheat (Triticum aestivum)

非寄主抗性基因NHO1(non-host resistance 1)编码的甘油激酶(glycerol kinase,GK),是甘油代谢中的限速酶之一,参与植物对多种病害的抵御过程。为进一步探究其响应小麦白粉菌侵染的表达模式,利用RT-PCR方法克隆获得了转录自抗病种质N9134的三个部分同源染色体的小麦NHO1基因(分别命名为TaNHO1-2A、TaNHO1-2B和TaNHO1-2D),分析三个TaNHO1同源基因的序列、启动子组成元件和表达模式,并明确了其亚细胞定位。序列分析结果表明,小麦TaNHO1-2A/2B/2D基因CDS区序列全长分别为1605、1599和1605 bp,分别编码534、532和534个氨基酸残基;3个同源基因均含有与AtNHO1(AT1G80460.1)基因以及OsNHO1(Os04G0647800)基因高度相似的FGGY_N端和FGGY_C端结构域。经对启动子区结构分析,该基因启动子区含有大量与植物激素及逆境响应相关的顺式作用元件,意味着TaNHO1可受到多种植物激素诱导并参与小麦抗病过程。qRT-PCR结果表明,在N9134抗病近等基因系响应白粉菌(Blumeria graminis f.sp.tritici,Bgt)侵染过程中,三个同源基因在不同时间点表达模式不同,其中TaNHO1-2A、TaNHO1-2B基因在侵染早期均上调表达,而TaNHO1-2D则表现出多次波动的表达模式;在N9134感病近等基因系遗传背景下,同源基因的表达水平在病菌侵染后48 h均表现出下调,说明该基因能够响应白粉菌侵染。经亚细胞定位分析,该基因主要作用于细胞质、细胞膜以及核膜。

Non-host resistance genes NHO1 encodes glycerol kinase(GK),which is one of the rate-limiting enzymes in glycerol metabolism.In order to explore its expression pattern in response to the infection of powdery mildew,the NHO1 gene transcribed from three partial homologous chromosomes was cloned from wheat germplasm N9134 by RT-PCR,and designated as TaNHO1-2A,2B and 2D.The promoter components,expression patterns and subcellular localization of TaNHO1 gene were further determined.The result of sequence analysis showed that the full length of CDS region of wheat TaNHO1-2A/2B/2D gene is 1605,1599 and 1605 bp,encoding 534,532 and 534 amino acids,respectively.Protein domain analysis showed that the three homologous genes contain FGGY_N-terminal domain and FGGY_C-terminal domain which are highly similar to AtNHO1(AT1G80460.1)gene and OsNHO1(Os04G0647800)gene.The structure analysis showed that the promoter region of TaNHO1 contains a large number of plant hormones and cis-acting elements in stress response,which meant that TaNHO1 could be induced by a variety of plant hormones and participates in the process of wheat disease resistance.The results of qRT-PCR showed that TaNHO1 is responsive to powdery mildew(Blumeria graminis f.sp.tritici,Bgt)infectionin the disease-resistant near isogenic line of N9134R/S,although the expression of the three homologous genes is different at different time points.TaNHO1-2A/2B gene showed up-regulated expression pattern at the early stage of inoculation,while TaNHO1-2D gene showed fluctuant expression pattern.Under the genetic background of N9134 susceptible near-isogenic line,the expression level of homologous gene was down-regulated at 48 h post inoculation,indicating that the gene responds to powdery mildew infection.The results of subcellular localization showed that the gene mainly acted in cytoplasm,cell membrane and nuclear membrane.The results of this study lay a foundation for further analysis of the specific function of TaNHO1 gene in wheat.