Analysis On Rice Resistance Gene Analogue And Cloning Candidate Blast Resistance Gene

Rice blast disease,caused by fungal pathogen Pyricularia grisea Sacc.,is one of the most serious diseases of rice(Oryza sativa L.)in China and worldwide.This epidemic disease happen every year causing great loss in rice production.Occurrence and prevalence of rice blast also require the three key elements of epidemiology,the pathogen,host and environment.The breeding and utilization of disease resistance varieties not only could effectively cut off one of the three key elements of epidemiology,prevent occurrence of plant disease economically and efficiently,but also could avoid or reduce the pesticide residue and environmental pollution.Thus disease resistance has been one of the goals of plant breeding.The deep study on disease resistance breeding can provide a theoretical guide to plant resistance breeding.In this paper breeding genetic material of Sichuan province and nipponbare genomes are systematically studied using molecular biology and bioinformatics.A unique way of resistance cross breeding is also proposed in this study based on breeding practice.The main work is as follows.1.Through a way of fuzzy search,we identified 565 disease resistance protein-encoding genes in the rice genomic sequence of Nipponbare in the rice TIGR database.We indentified conserved domains and motifs,and transposable element in these genes protein sequences and DNA sequences. The results showed 14 of them annotations were error.The same time we drew distribution of R genes in the rice genome.Based on homology tree and distribution in chromosomes of these R genes,we think that both local and distant genetics duplication events shaped these R genes existing distribution and diversity.Transposable elements played an important role of R gene evolutionary.2.Comparison of clustering analysis was investigated utilizing spectrum of resistance to blast and polymorphism of resistance gene analog(RGA) in 25 varieties for blast resistance identification and 20 varieties (lines).The resistance spectrum clustering analysis showed that proposed the 45 varieties(lines) could be divided into3group A and group B with the genetic similarity coefficient of 0.450.Group A and group B could be divided into subclassâ…°,subclassâ…±,subclassâ…²,subclassâ…³respectively with 0.618 genetic similarity coefficient.The RGA-PCR clustering analysis showed that proposed the 45 varieties(lines) could be divided into groupâ… and groupâ…¡which clearly inclined the Indica-japonica Differentiation with 0.620 genetic similarity coefficient.Groupâ… could be divided into six subclasses and groupâ…¡could be divided into seven subclasses with 0.783 genetic similarity coefficient.The resistance spectrum clustering analysis showed that some varieties with similar resistance spectrum could be finely fallen into the same group,while the RGA-PCR clustering analysis showed some varieties with the similar genetic background could be fallen into the same group.For some varieties with low resistance frequency or high resistance frequency,there was a better corresponding relationship between the resistance spectrum clustering and the RGA-PCR clustering. In general comparison of clustering analysis showed that there was no parallelism relationship between group and group in two different types of the clustering.3.Rice maintainer Fuyi B with high resistance to blast was performed by RGA-PCR,utilizing the known degenerate primers N1N2 of plant disease resistance gene analogy(RGA) which amplified difference bands among indica and japonica rice varieties(lines).An open-reading NBS resistance gene analogue fragment has been obtained from the genome DNA of Fuyi B,and was named as Fuyi_NBS1,for the length of 540bp,encoding 176 amino acids.Bioinformational analysis showed that Fuyi NBSlin rice genome on chromosome 2 there were three copies,and two among those copies for disease resistance gene.It implied a structure area with conservative NB-ARC domain though Blastp comparison,with a high degree homology OsI₀05459 and CR273,and with the barley NBS-LRR class resistance gene homology CAD45026 rate of 58%.Homologous modeling analysis showed that Fuyi_NBS1 implied at least 7α-helixes and 3β-folds.4.Candidate gene sequences from GenBank database were utilized to design molecular primer.Sub-clone was performed with the primers between resistant cultivar and susceptible cultivar.In rice blast resistance Fuji B,two resistance gene analogues were obtained,and were recorded as "FY₂" and "FY₃",the size of the length of 2518bp and 4168bp. After bioinformatics analysis,FY₂ the length of the mRNA for 1092bp, encoding 634 amino acids.FY₃ the length of the mRNA for 226bp,encoding 742 amino acids.Through the analysis of their protein sequences,both of them belong to the CC-NBS types of resistance gene analogue.They and No.2 chromosome fragments shotgun OsI₀05457 in indica rice were a high degree of homology.Three-dimensional protein structure analysis,the end of the NBS domain in is obviously different between the NBS domain of FY₃ and the NBS domain of OsI₀05457 in C-terminal.This study was laid an important foundation for cloning resistance gene in Fuji B.5.According to the practice of rice blast resistance breeding,the strategy and method of blast resistance crossbreeding was suggested.That is summarized to cross between blast resistance and blast resistance, choice stress,wide choice cardinal number in low generations,choosing individual with excellent agronomic traits from individuals with blast resistance,consecutive evaluation,and choosing the best from individuals with blast resistance.