Molecular Cloning And Characterization Of NBS Orthologues Of Rust Resistance Gene From Sunflower

Sunflower(Helianthus annuus) is an oil plant of great importance, but it is often infected by various pathogens in production. Sunflower rust, one of the most serious sunflower pathogens, restricts the growth and production of sunflower severely. In order to find some resistance genes of sunflower to this disease, homology-based candidate gene cloning method was adopted to isolate Heliamhus annuus resistance gene analogs (HaRGAs). Several HaRGAs were obtained and analyzed in this study. And the main results of this study are as follows:1. Degenerated primers were designed based on two conserved regions of NBS-LRR genes. P-loop and GLPL, to clone the HaRGAs from the cDNA of infected resistant cultivar CM29leaves. And10HaRGAs with continuous open reading frame were segregated. They were classified into two types, the TIR-NBS-LRR type and non-TIR-NBS-LRR type, by phylogenetic analysis. Multiple sequences alignment of the amino acid sequences of them showed these HaRGAs had typical domains of NBS-LRR resistance genes, such as P-loop, kinase-2a. kinase-3a and GPLP. The homology analysis results demonstrated that these HaRGAs had18.1%～-51.1%in homology with known NBS-LRR resistance genes. All the results above indicated that the cloned HaRGAs had a close relationship with resistance genes. These10HaRGAs had been submitted to the GenBank. and the accession numbers are from KJ620870to KJ620879.2. The expression files of the HaRGAs in sunflower after inoculated with sunflower rust pathogen and treated with different hormones (salicylic acid, ethylene and H;O;) were analyzed by real-time fluorescent quantitative PCR. The results showed HaRGA1and HaRGA4were up-regulated in sunflower leaves inoculated with rust pathogen, and they did not change significantly in the uninoculated leaves. After treatment with hormones. HaRGA1and HaRGA4both showed obvious changes. The expression files of HaRGA1increased rapidly and maintained for a long period after treated with H2O2Meanwhile HaRGA4was more sensitive to salicylic acid and ethylene. especially it expressed abundantly after the salicylic acid treatment. These results indicated these two HaRGAs had responses to different hormones, thus they might be involved in different resistance pathways of sunflower. These results will provide a theoretical basis for study on sunflower rust resistance genes in the future.