| Rice is one of the most important food crops in the world, but the rice yield is limited by many biotic and abiotic stress factors. Sheath blight, one of the three diseases of rice, leads to severe yield loss each year and it has become the most serious one in some regions in China. It is crucial for us to analysis the genetic mechanism of the disease and clone resistance related genes so as to improve the resistance level of rice in the future.In this study, we have constructed two genetic population. Phenotypic identification of sheath blight resisitance and genotyping of the population were then carried out for QTLs mapping. By screening genes whose expressing level changed significantly in the microarray (fold change≥2.0), two candidate genes which located in the interval of the main effect QTLs were cloned. Over-expression vectors of the genes were constructed for rice transformation and molecular function were studied. The main results of the study were:1. An F2 population consisted of 286 individuals and a recombinant inbred lines (RILs) population consisted of 121 lines were constructed and they were both derived from the across of HH1B and RSB03, the latter of which was a deep-water rice cultivar from Guangxi, China. Inoculation and resistance evaluation for the population and parents were carried out and the indices for resistance identification of sheath blight included disease grade (DG), lesion length (LL), lesion height (LH), relative lesion length (RLL) and relative lesion height (RLH) etc.2. Phenotypic identifications for the parents and RILs population have been carried out in four environments, which were Hainan and Shanghai in 2008 and 2009, respectively. The results have indicated that the resistance level of RSB03 was higher than that of HH1B. The average values of DG, LL and RLL for HH1B in four environments were 5.2,23.7cm and 28.1%, respectively, whereas RSB03 were 3.3,23.7cm and 12.7%, respectively. A total of 16,12,4 and 4 additive QTLs for disease resistance which distributed on 10 different chromosomes were identified in four environments and several QTLs were co-located with each other. Besides that, a total of 15,25,23 and 22 epistatic QTLs were identified in four environments. There are QTLs which detected in E4, combinated QTLs in E2 and E4, combinated QTLs in four environments and they were located in the marker interval of RM5704-RM202 in chromosome 11.3. We have evaluated the disease resistance for the F2 population at Hainan in 2007 and the disease grade (DG) of HH1B and RSB03 were 5.4 and 4.3, respectivly. Normal distributions were observed for the traits of the population and twelve main effect QTLs have been detected. Among them, two QTLs for DG which located on chromosome 11 and 12 were identified and they could explain 18.07% and 12.27% of the phenotypic variation, respectively. The QTL located on chromosome 11 also influenced the traits of RLL and RLH and the resistant alleles were both come from the parent of RSB03.4. The parent of RSB03 was selected for inoculation and RNA of the sheaths 36 hours post inoculation and blank sheaths was extracted for microarray analysis. A total of 383 genes whose expression level was significantly changed (fold change>2.0) were identified and they could be classified into 21 categories referring to physiological process, metabolism, cell part and biological regulation, etc.5. Combinating the results of microarray and QTL mapping of the F2 population, nine of the 383 significantly changed genes were identified which located in the marker interval of RM5389-RM202 on chromosome 11. Quantitative RT-PCR was then conducted to analysis the expression level of these genes. The results showed that the expression patterns of five genes were concordant with those in the microarray. And among them,2 genes were up-regulated and 3 were down-regulated by the attack of pathogen of sheath blight.6. Two candidate genes, Cg3 and Cg8, which located in the interval of RM5389-RM202 were cloned from the cDNA of RSB03. Over-expression vectors and genetic transformation were then conducted and transgenic plants were obtained for molecular function analysis.7. The full length of the open reading frame (ORF) of Cg3 was 930bp. Compared with Nipponbare, only 2 bp were different, but it did not lead to difference on amino acid level. As a transmembrane-like protein, the protein that Cg3 encoded contains three mitochondrial transporter protein domains. The expression level of Cg3 in the transgenic plants was increased significantly. Compared with Nipponbare, the plants of T1 generation had a better performance on the traits of disease resistance. The average value of DG was 5.1-5.5 and LL 14.0-21.3cm, whereas the DG and LL of Nipponbare were 6.5 and 29.9cm, respectively. Significant difference was identified between the transgenic plants and CK. Disease resistance identification for the plants of T2 generation was carried out at Shanghai in 2010. The average value of DG was 4.0, which was lower than that of CK- 4.3 on average, but the difference was not significant. Because of the abnormal climatic conditions at Shanghai in 2010, the exact resistance level of the transgenic plants needs to be confirmed in the future.8. The full length of the ORF for Cg8 was 825bp. A total of 32 amino acids were different between the proteins encoded by the gene in RSB03 and Nipponbare. The protein contained signal peptide, transmembrane domain and leucine repeat domain, etc., and it was also classified as a number of membrane-like proteins. The quantitative RT-PCR results showed that the expression level of Cg8 in the progenies of transgenic plants was significantly higher than that of Nipponbare. Except that the plant height of the T1 generation was higher than that of the wild type (WT), no significant differences of the disease resistance related traits were identified. Further study for the T2 plants also indicated that the transgenic plants and WT were not significantly different and the disease resistance of the transgenic plants has not been improved. |
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