Cloning And Analysis Of NBS-type Resistance Gene Analogues In Ornamental Sunflower (Helianthus Annuus)

Sunflower, used for oil or food, is the best ornamental plants and maintaining ecological landscape. Its yield and quality will decrease, if sunflower susceptible to diseases. Resently, breeding for disease resistance and anti-pesticide rule is used for diseases control. But it also faces some problems, such as only single resistance of plants, and the potential risk of anti-pesticide rule to human health and living environment. With the continuous development of biotechnology, using transgenic technology for the improvement of plant varieties have been applied into the production of crops, and isolating resistance genes will be significant to breeding for disease resistance and depth understanding of resistance mechanism.This study included the following items: (1)Degenerate primers were designed basing on the NBS conserved motif(P-loop and GLPL). Resistance gene analogues (RGAs) of NBS were isolated from the genome of ornamental sunflower "Valentine" by polymerase chain reaction (PCR). The sequences were analyzed by bioinformatics method, and revealed the diversity and evolution relationship of NBS-LRR resistance genes from ornamental sunflower "Valentine". (2)Special primers were designed according to the RGAs isolated from "Valentine", and were used to analyze other sunflower varieties by PCR amplification. (3)Southern blotting was carried out with SFRGA-3 probe. The main results were as follows:1. 24 RGAs with uninterrupted open reading frames (ORFs) were obtained, named from SFRGA1 to SFRGA24.They were submitted to GenBank, and the accession number is EU352826~EU352849.The sequence analysis showed that putative amino sequences from 24 RGAs contained the same conserved motifs such as P-loop, Kinase-2, Kinase-3a and HD presented in known NBS-LRR resistance genes from other plants and belong to NBS-LRR resistance-like proteins. The results of identity searching indicated that nine RGAs share high similarity with the cloned resistance protein in Helianthus annuns. The others have similarity with cloned resistance gene and many other high score resistance-like protein in some other plants most of which were unknown fuctions. Sequence identity among the 24 RGA nucleotide sequences ranged from 41.1% to 99.4%, while identity of the deduced amino acid sequences from 24 RGAs ranged from 25.8% to 100%. The phylogenetic analysis for RGA nucleotide sequences and the deduced amino acids showed that RGAs of ornamental sunflower were divided into two classes,TIR type and non-TIR type.2. According to the RGAs isolated from "Valentine", three pairs of special primers were designed. All the ornamental sunflowers have the same results that amplified with primers RGA8-F1 and RGA8-R1 or RGA16-F1 and RGA16-R1. The special band can't be amplified by RGA3-F1 and RGA3-R1 in ornamental sunflower variety Sonja.The result indicated that Sonja maybe lost SFRGA3, or there is some mutation or recombination,3. Sunflower genome was digested with restriction endonuclease BamH I , EcoR I and Kpn I . Southern blotting was done using SFRGA3 as a probe. The result indicated that SFRGA3 -related sequences are presented as 1-3 members in Valentine genome.