Cloning And Expression Analysis Of TaNLR、TaNBS And TabZIP19 Genes In TcLr19

Leaf rust disease, caused by the fungal pathogen Puccinia triticina Eriks, is one of the most severe diseases of common wheat (Triticum aestivum L.) all over the world. Breeding and application of resistant cultivars are the most economical and environmentally safe approach to reduce the damage caused by wheat leaf rust disease. The resistance gene Lr19 transferred into wheat from Agropyron elongatum has expressed resistance to most pathotypes of P. triticina in most regions of the world. As a resistance gene to wheat leaf rust, Lr19 would have high potential in application at home and abroad. Herein, three ESTs sequences, which were picked in cDNA library from wheat TcLr19 leaves induced by avirulent isolate of P. triticina, were cloned using RACE (Rapid Amplification of cDNA Ends) technology. To further understand the molecules and their function in the interaction of stripe rust fungus and wheat, bioinformatic analysis and expression profile analysis were done. The main results are as follows:The gene specific primers were designed based on the sequence of Contig 914 and Contig 551, then two NBS-like complete sequences neamed of TaNLR and TaNBS were obtained through the RACE method.The full length of TaNLR gene was 3042 bp including a complete open reading frame encoding NBS-LRR protein of 912 amino acids, continuous poly A and a typical poly adenylation signal. The deduced amino acids of TaNLR protein consisted of a CC domain, a nucleotide binding site (NBS) domain near the N-terminal, and a leucine-rich repeats (LRR) domain at the C-terminal, which was the conserved domain of plant resistance genes. It suggested that the TaNLR gene should be a member of a class CC-NBS-LRR type gene. Phylogenetic tree indicated that TaNLR might share a common ancestor with Hordeum vulgare,whereas amang the cloned wheat leaf rust resisitance genes, it had a near relationship with Lr10,and far from Lr21. Real-time PCR showed that the transcription profile of TaNLR was down-regulated in the incompatible interaction rather than the compatible interaction. It was speculated that TaNLR was involved in the interaction of P. triticina and wheat by a negative regulator.The full length of TaNBS gene was 1805 bp including a complete open reading frame encoding NBS protein of 488 amino acids. The deduced amino acids of TaNBS protein consisted of a nucleotide binding site (NBS) domain near the N-terminal of 173-444 amino acids, which was the conserved domain of plant resistance genes. It suggested that the TaNBS gene should be a member of a class CC-NBS-LRR type gene. Phylogenetic tree indicated that TaNBS might share a common ancestor with Rice,whereas amang the cloned wheat leaf rust resisitance genes, it had a near relationship with Lr19-AG15,and far from Lr21. Real-time PCR showed that the transcription profile of TaNLR was down-regulated in the incompatible interaction rather than the compatible interaction. The gene specific primers were designed based on the sequence of Contig683, then one bZIP complete sequences neamed of TabZIP19 was obtained through RACE methods. The full length of TabZIP19 gene was 1783bp including a complete open reading frame encoding NBS protein of 323 amino acids. The deduced amino acids of TabZIP19 protein consisted of a BRLZ or bZIP domain which was the conserved domain of bZIP translantor genes. InterProscan suggested that the TabZIP19 gene should be a member of a class bZIP type gene. Phylogenetic tree indicated that TabZIP19 might share a common ancestor with Brachypodium distachyon distachyon(XP003575087), Rice(NP001047055), and has far relationship with cloned.wheat bZIP(GQ266689 and CAA40102). Real-time PCR showed that the transcription profile of TabZIP19 was down-regulated in the incompatible interaction and up-regulated in the compatible interaction. It was speculated that TabZIP19 was functioned as a negative regulator of energy or nutrient transport in the interaction of leaf rust fungus and wheat.