青稞HvnWRKY26基因的克隆及表达分析

Cloning and Expression Analysis of HvnWRKY26 Gene in Tibetan Hulless Barley

WRKY家族转录因子广泛参与植物的生物和非生物胁迫过程。为探索WRKY家族转录因子在青稞抗条纹病过程中的作用,本课题组前期利用条纹病菌分别侵染抗病青稞品种昆仑14号和感病品种Z1141,并进行转录组测序,发现一个在侵染时期差异表达的WRKY基因家族成员。序列分析发现,该基因的开放阅读框为765 bp,可编码255个氨基酸,具有典型的WRKY结构域,属于WRKY基因家族。Uniprot注释结果表明,该蛋白为HvnWRKY26。启动子区域预测表明,该区域含有脱落酸和茉莉酸响应元件以及与干旱、低温、盐胁迫和防御应激等逆境胁迫相关的顺式作用元件。序列比对分析显示,HvnWRKY26蛋白与其他物种中同源蛋白的氨基酸序列一致性均不高,但该蛋白与大麦HvWRKY26蛋白的WRKY结构域序列完全相同。系统进化分析表明,青稞HvnWRKY26与大麦WRKY26的亲缘关系最近;进一步对HvnWRKY26及与其亲缘关系较近的大麦、玉米、水稻WRKY蛋白进行进化分析,发现这些WRKY蛋白被分为A、B和C三大类,其中HvnWRKY26蛋白属于A类。RNA-seq和qRT-PCR分析结果表明,青稞在遭受条纹病菌侵染时,HvnWRKY26基因的相对表达量在抗病品种昆仑14号和感病品种Z1141中均显著上调表达,且抗病品种的表达量显著高于感病品种,推测HvnWRKY26基因在青稞抗条纹病过程中发挥重要作用。

WRKY transcription factors are widely involved in biotic and abiotic stresses in plant.In order to explore the role of WRKY transcription factor in Tibetan hulless barley resistance to leaf stripe,we obtained a differentially expressed gene from the transcriptional sequencing results of normal and susceptible leaves in a resistant Tibetan hulless barley variety Kunlun 14 and a susceptible variety Z1141.Sequence analysis showed that the gene contains a 765 bp open reading frame,encoding 255 amino acids,with a highly conserved WRKY domain,belonging to WRKY gene family.Uniprot annottation indicated that the protein was HvnWRKY26.Prediction analysis of the gene promoter region indicated that the region contained abscisic acid,jasmonic acid response elements,and multiple cis-acting elements associated with drought,low temperature,salt,and defense stress responses related to adversity stress.Sequence alignment analysis showed that amino acid sequence of HvnWRKY26 protein was not consistent with homologous proteins in other species,but the WRKY domain sequence of HvnWRKY26 protein was identical with that of HvWRKY26 protein in barley.The phylogenetic tree showed that HvnWRKY26 protein was most closely related to barley WRKY26.Further evolutionary analysis of HvnWRKY26 and WRKY proteins in barley,maize and rice showed that these WRKY proteins can be divided into A,B,and C groups,where HvnWRKY26 protein is located in group A.The results of RNA-seq and qRT-PCR showed that the expression level ofHvnWRKY26gene was significantly increased in the resistant variety Kunlun 14 and the susceptible variety Z1141,and the expression level of HvnWRKY26 gene in Kunlun 14 was significantly higher than that in Z1141.It is speculated thatHvnWRKY gene plays an important positive role in the resistance to stripe disease in Tibetan hulless barley.