| Rice sheath blight is a worldwide rice disease,threatened the rice production seriously.It happens widespread in rice-plant countries,and usually causes a loss of ten percent to thirty percent in rice production,even fifty percent of production decreases when it is serious.Rice sheath blight is the most serious rice disease in our country now,and there is no rice line highly resistant were found.The pathogen is Rhizoctonia solani,it has a wide host range and distribution.It can not generate asexual spores in natural conditions and lives in soil by the form of sclerotia.According to hyphal fusion classification,R.solani was divided into 14 fusion groups,and the ascendant pathogenic flora for rice sheath blight is AG-1-IA subgroup.Along with the development of agrobacterium induced rice transgenic technology, T-DNA mutant library construction has been an important approach for gene function analysis and identification.Rice T-DNA mutants can be studied conveniently by the way of forward and reverse genetic means,so that can be materials in the study of resistance genes,and be regarded as new resistance resources.Suppression subtractive hybridization(SSH) was utilized for the comparison of differentially expressed genes in different physiological state,through selectively amplifying differentially expressed genes and inhibiting non-target genes.It can provide important information for life activities,so it is used widely in differential genes screening in species as effective tool in gene clone and expression,pathogenic genes especially.This study tried to find out resistance genes to sheath blight in rice T-DNA mutants,the main results were summarized as follows:(1) Sixty Rhizoctonia solani strains were isolated from rice sheath blight samples collected from distinctive ecological rice regions of Zhejiang Province,China.Hyphal fusion identification result revealed that all the 60 strains belonged to anastomosis group AG-1,for that they all fused with AG-1-â… A,AG-1-â… B and AG-1-â… C,which three groups are ISGs (intraspecific group) of AG-1 group.Pathogenicity identification analysis from leaves in vitro indicated that pathogenicity difference between the strains was significant.Further ITS analysis indicated that phylogenetic tree coincided with the result of hyphal fusion identification,and was irrelevant with pathogenicity difference.(2) 2576 rice mutant lines from Nipponbare T-DNA mutant library were identified to resistance to sheath blight(SB) by broadcasting inoculation.Most of them were found with high-susceptible to rice SB,only 36 lines reached moderate resistant or resistant level,which occupied only 1.4%,180 lines were moderately susceptible,occupied 7.0%.Further identification was done to these 216 lines with embedding inoculation,18 lines were found more resistant and differences among secondary screening lines significant.Final identification with micro-chamber inoculation to these 18 lines in greenhouse was carried out and the identification result indicated that there was no significant difference among 15 mutant lines,and 17 lines have significant differences with the wildtype.(3) The T-DNA insertion site of a Nipponbare T-DNA mutant was identified through Tail PCR,this mutant was more resistant to rice sheath blight than the other mutants.Suppression subtractive hybridization(SSH) was utilized for the isolation of differentially expressed genes in rice to sheath blight,and forward and reverse suppression subtractive cDNA library was constructed.Positive clones were sequenced and 309 nonredundant cDNA fragments were obtained after contig assembly.The homology searches of cDNA fragments were done in TIGR and Genbank using blastn and blastx,219 cDNA fragments got homologous sequences. The function sorting was done to these homologous genes on Gene Ontology. |
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