【作者】 谭炎宁；

【导师】 易自力； 蒋建雄；

【作者基本信息】 湖南农业大学 ， 植物学， 2006， 硕士

【摘要】 稻瘟病是水稻稻作区一种严重的真菌性病害。培育抗病品种是解决这一病害最有效的途径，通过生物技术手段与常规选育相结合创制抗病新种质预示了良好的应用前景。本研究以携带外源溶菌酶基因的ZH9(R)(高抗稻瘟病)为抗源供体，以优良的超级杂交稻恢复系明恢63(MH63)为受体，通过对MH63回交转育转移溶菌酶基因，获得抗性改良的新恢复系。作者在获得回交2-4代材料的基础上，通过抗生素辅助筛选体系进一步鉴定出转基因纯合系；对转育后代目的基因的追踪及抗病性鉴定、抗性纯合系的人工接种鉴定，分析外源基因的遗传规律、转育材料的抗性表现及稻瘟病抗性的遗传稳定性；对转育后代主要农艺性状的考察，分析转育后代农艺性状的遗传稳定性，并通过定向选择筛选出综合性状优良的抗病株系。 1．明恢63转育后代中抗性纯合系的筛选 在本课题组先前建立的转基因水稻快速检测技术的基础上，通过G418对抗生素标记基因的辅助筛选(种子筛选和叶片筛选)，从转育后代中快速鉴定出外源溶菌酶基因纯合的转育株系。 研究发现，在一定浓度的G418胁迫条件下，转育株系各器官、组织对抗生素具有不同的抗感反应。在种子筛选中，根据G418对水稻种子发芽特性(发芽率、生根率、根伸长受阻等抑制效应)筛选出高抗抗生素类株系。结果发现，TD-3、TD-10、TD-18三个株系发芽率、生根率、长根类种子比率都达90％左右，死亡率在10％以下，说明群体受抗生素毒害小，推测此株系为npt-Ⅱ基因纯合系，继续选其叶片做抗生素筛选试验及分子检测。 随后对这些材料做抗生素胁迫下的离体叶片鉴定，经x~2检测阳性株与阴性株分离比为1：0；PCR检测结果进一步证实同一株系内各单株不发生分离，因此这三个株系最终被确认为npt-Ⅱ基因、溶菌酶基因纯合系。 通过对回交转育后代株系的离体叶片筛选，共获得19个抗性纯系，其中BC4F3代3个，BC3F4代10个、BC2F5代6个。 2．明恢63转育后代抗稻瘟病遗传稳定性研究 通过各转育世代部分株系的外源基因检测，转育后代稻瘟病抗性的大田鉴定及抗性纯合系的人工接种鉴定，分析溶菌酶基因的遗传规律、转育后代的抗性表现及抗性遗传稳定性，并评价溶菌酶基因的抗性效果。得出如下结论： 1) 对由同一系谱来的分属于BC4F1、BC4F2、BC4F3代3个株系做叶片抗生素检测，经遗传分析，得出外源基因为单基因显性遗传，符合孟德尔遗传规律。对纯合系TD-10与不育系测交后代进行溶菌酶基因分子检测，结果都扩增出了特异性条带，由此说更多还原

【Abstract】 The rice blast is one of the most serious fungus disease in the world. It proved that the selecting of resistant varieties by biological approaches in combination with traditional approaches can take good effect to provide the crop the high resistance to fungs.The transferring researches by backcrossing had been done between the non-recurrent parent ZH9 (R) harbored lysozyme gene and recurrent parent Minghui63.On the basis of the acquired materials(2-4 backcossing generations),futher research was done including identifying the homozygous lines of lysozyme gene,selecting good characteristic lines by field neck resistance test, inoculating isolates and investigating the agronomic characters. So that the hereditary stability of forigen lysozyme and the resistance of transferring offspring could be well studied.1. Screening of the resistant homozygous lines from the offspring of Minghui63 backcrossed with transgenic riceThe resistant homozygous lines were identified out of the offsping with the antibiotic G418,by means of the screening method established by our research group previously, which can quickly screen the transgenic rice.Studies found that the different tissues of transferred lines showed different reactions to. The principle of identification of seeds is :the normal seeds can’t germinate ,grow roots and its roots cann’t extend when put in the G418 solution ,while the transgeice ones harboring npt- Ⅱ genes can normally grow.LinesTD-3 、 TD-10、 TD-18 ,resistant to G418 ,were screened using the seeds-test method ,which has 90% germination rate, root-growing rate and 10% fatal rate.Then ,the detached leaves from the above lines were put into G418 solution according to the leaf-test method.By x~2 test,the segregation of the resistant to the sensitive was 1:0, which is in accord with the PCR results. From this, it can be concluded the leaf-test method can be used to classify the transgenic homozygous lines. Thus,TD-3、TD-10、TD-18 were considered as the homozygous containing lysozyme gene, and npt- Ⅱ geneBy means of leaf-test method, 19 resistant homozygous lines were screened:3 for BC4F3 ,10 for BC3F4 and 6 for BC2F5.更多还原