三个小麦防御素基因的克隆及序列分析

Cloning and Sequence Analysis of Three Defensin Genes in Wheat (Triticum aestivum L.)

本试验从济麦22幼叶中克隆得到三个小麦防御素基因Ta PDF(Triticum aestivum defensin),分别命名为Ta PDF32、Ta PDF33和Ta PDF34(Gen Bank登录号分别为BT009185、BT009167和BT009022)。序列分析发现,三个防御素基因各自含有一个长度为243、249 bp和225 bp的开放阅读框(open reading frame,ORF),依次编码长度为80、82个和74个氨基酸的蛋白。成熟蛋白的分子量均约为5.5 k D,等电点均大于7,含有8个保守的Cys残基。保守结构域分析发现,Ta PDF32含有一个γ-硫堇功能结构域,Ta PDF33和Ta PDF34均含有一个Knot1功能结构域,两者均是植物防御素的典型结构域,因此这三个Ta PDF属于典型的植物防御素蛋白。SWISS-MODEL在线软件分析表明,三个Ta PDF的三级结构均由三股反平行的β-折叠和一个α-螺旋组成。系统进化分析表明,Ta PDF32、Ta PDF33的氨基酸序列与玉米、粟米的防御素相似性较高,而Ta PDF34与大豆的防御素相似性较高,但均与已经报道的小麦防御素相似性较低,表明Ta PDF32、Ta PDF33和Ta PDF34是小麦三个新的防御素蛋白。荧光定量RT-PCR分析发现,Ta PDF32、Ta PDF33基因在小麦叶片中表达量最高,种子次之,根中最低;Ta PDF34基因在叶、种子和根中的表达量基本相同。

Three Triticum aestivum defensin（ Ta PDF） genes,Ta PDF32,Ta PDF33 and Ta PDF34（ Gen Bank accession No. BT009185,BT009167 and BT009022）,were cloned from the young leaves of Jimai22 cultivar. Sequence analysis showed that each had an open reading frame of 243,249 bp and 225 bp,which encoded the proteins with 80,82 and 74 amino acids respectively. The mature proteins,with the estimated molecular masses of all about 5. 5 k D and the isoelectric points all higher than 7,each contained eight conservative cysteine residues. Conserved domain analysis found that Ta PDF32 contained a strictly conserved gamma- thionin domain,while Ta PDF33 and Ta PDF34 each had a Knot1 function domain. Both of the domains are the typical characteristics of plant defensin. Thus,the three Ta PDFs all belong to typical plant de-fensin proteins. Study of the three dimensional structure using SWISS- MODEL showed that all of them contained a triple- stranded anti- parallel β- sheet and an α- helix stabilized by four disulfide bridges. Phylogenetic analysis showed that Ta PDF32 and Ta PDF33 were close to defensins of Zea mays and Setaria italica,while Ta PDF34 was close to Clycine max defensin. But they all owned low similarities with the other reported wheat defensins,which indicated that Ta PDF32,Ta PDF33 and Ta PDF34 were three new wheat defensins. Real- time PCR analysis revealed that Ta PDF32 and Ta PDF33 genes had the highest expression level in leaves,followed by seeds,and the lowest level in roots. Whereas Ta PDF34 expressed basically consistent in all tissues.