外源抗稻瘟病基因在滇型杂交稻中的遗传与表达研究

The rice blast caused by Magnaporthe grisea, is a major destructive and uncontrollable fungal disease in rice. It is significant that new wide-spectrum resistant material introduced foreign resistant gene by biotechnology is a basis of rice breeding resistant to blast..Supported by The National Natural Science Foundation of China, plasmid pSR2-4 containing lysozyme gene from T4 phage and plasmid pBLGC containing chitinase gene from Phaseolus limensis and β-1,3- glucanase gene from Nicotiana tabacum were bombarded into the recovery-line "Nan29" of Dian-type hybrid rice (Oryza sativa L. ssp. japonica) from Yunnan province of South-west China. In the experiment of transferring lysozyme gene , 70 regenerants were obtained from 19 independent calli resistant against G418 (100 to 150 mg/L) on NB medium. 58 of 70 regenerants were identified to be transgenic plants by dot blotting using lysozyme gene as the probe. Southern blotting hybridization certified that the foreign gene construction was intergrated into rice genome. In the experiment of transferring chitinase gene and β-1,3- glucanase gene, 93 regenerants were obtained from the calli that were resistant to G418 (100 to 150 mg/L) on NB medium. Using β-1,3 glucanase gene as the probe, 17 of the regenerants were identified to be transgenic lines by dot blotting and the foreign genes construction were intergrated into the genomes of T1 lines by Southern blotting hybridization. Three foreign genes were inherited stably to T5 generation according to PCR results of the lines. 36 lines harboring lysozyme gene and 6 lines harboring chitinase gene and β-1,3- glucanase gene exhibited high resistance to rice blast in the diseased field..In order to estimate the resistant spectrum of transgenic rice against various rice blast races, 36 transgenic lines derived from ten T0 generation transgenic plants harboring lysozyme gene and 6 transgenic lines harboring gene and β-1,3- glucanase gene were challenged in the greenhouse by inoculating 63 isolates belonging to 48 races of Magnaporthe grisea from Yunnan Province. The results indicated that lysozyme gene and chitinase-β-1,3- glucanase genes were confirmed to possess resistant function to rice blast and the resistance of transgenic rice lines against rice blast were enhanced compared with the receptor cultivar Nan29 by inoculating and field evaluation.

应用PCR和Southern 杂交连续多代检测滇型杂交水稻恢复系转基因株系植物抗毒素、几丁质酶、葡聚糖酶等抗稻瘟病外源基因的存在与否，在云南不同生态条件下自然和人工接种鉴定转基因株系抗病性，研究这些基因表达的稳定性和抗性范围；选择抗病株系与滇榆一号不育系杂交，通过杂种一代育性、杂种优势、抗病性分析，明确外源基因在杂交稻育种中的价值。