Screening of Brown Planthopper Resistance-related Genes from Rice (Oryza sativa L.) Mutants by Differential Diaplay

• Main Authors: Chi-Ying Chou, 周琪英
• Other Authors: Hsin-Mei Ku
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/99111996176073592772

碩士 === 國立中興大學 === 農藝學系 === 91 === Brown planthopper (Nilaparvata lugens Stal., BPH) usually cause serious damage in most rice production areas, breeding for resistant varieties is the best solution for the pest control. Two mutants, TM6 and TM268, derived from the BPH susceptible variety TNG67 by sodium azide (NaN3) mutagenesis showed BPH resistance and controlled by a single dominant gene. To clone the genes responsible for the BPH resistance in mutants, differential display (DDRT-PCR) strategy was applied to screen the differential expressed genes by comparing the BPH resistant mutants TM6 and TM268 with the susceptible variety, TNG67. A total of 95 differential displayed fragments were obtained by screening 78 sets of primers. These fragments were amplified and cloned into the dT vector, and characterized by sequencing analysis and BLAST characterization. They were further used as probes in the Southern analysis to investigate the genomic difference between the resistant and susceptible lines. Five clones, OSDD30, OSDD51, OSDD54, OSDD81, OSDD166, and OSDD220 related with the BPH resistance were found with polymorphisms by Southern analysis. The OSDD166 clone was identified as a phenylalanine ammonia-lyase (PAL) gene in rice. Clones OSDD51 and OSDD81 showed 42~46% identity to the CBS (cystathionine beta-synthase) domain protein. No similarity in either nucleotide or amino acid sequences of OSDD54 and OSDD220 were found in the database therefore, they maybe the new genes in rice related to the BPH resistance. During BPH attacked, the expression of the CBS clone, OSDD51, was slightly higher in the BPH resistant lines by northern hybridization. The difference in DNA methylation patterns in rice mutants were detected by four clones OSDD51, OSDD81, OSDD166 and OSDD220 in Southern analysis using restriction isoschizomers Hpa II and Msp I to digest the genomic DNAs. Fifteen of the differentially expressed clones were correlated with retrotransposon or its activity related proteins by nucleotide BLAST. It is hypothesized that the expression of gene in response to BPH challenge in the resistant mutants may associate with the activation of retrotransposon in the early mutagenesis stage and the changes of DNA methylation pattern. Further experiments will be conducted to clone the five BPH resistance-related genes as well as the retrotransposon related genes from mutants and to investigate their functions in order to unravel the mechanisms of mutagenesis and BPH resistance in rice mutants.