目录
摘要II
关键词II
AbstractIII
引言
引言1
1材料与方法2
1.1试验材料 2
1.1.1供试材料 2
1.1.2稻瘟病菌株 2
1.2试验方法 3
1.2.1种植方法3
1.2.2稻瘟病菌的培养与接种3
1.2.3表型鉴定3
1.2.4基因组DNA的提取与制备4
1.2.5标记的开发即及利用5
1.2.6引物的制备5
1.2.7PCR扩增及产物检测5
1.2.8转化载体的构建7
1.2.9载体、菌株、试剂盒、测序及引物合成公司9
2结果与分析9
2.1薄稻抗稻瘟病穗瘟主效基因pbbd1的精细定位9
2.2候选基因P1P6的CRISPR材料表型鉴定11
3讨论13
3.1薄稻抗稻瘟病穗瘟QTL主效位点pbbd1的精细定位13
3.2薄稻抗稻瘟病穗瘟QTL主效位点pbbd1候选基因的功能验证14
致谢14
参考文献14
附录A 马铃薯琼脂（PDA）培养基（1L）16
附录B 玉米粉稻秸秆琼脂培养基（1L）16
附录C TPS配方16
附录D 2×CTAB（200mL）16
附录E 大肠杆菌感受态DH5α转化方法16
太湖流域地方品种薄稻抗稻瘟病QTL位点pbbd1的候选基因的功能验证
摘 要
稻瘟病是水稻的一种真菌性病害，因其高致病性和广传播性，严重影响世界水稻产量及质量，被列为水稻的三大病害之一。培育具有稻瘟病广谱抗性的水稻品种是最经济有效的稻瘟病防治方法，也是从源头根除稻瘟病的方法，因此挖掘、评估具有稻瘟病抗性的种质资源和基因并应用于育种成为重要工作。实验室前期对太湖流域地方品种的稻瘟病抗性进行鉴定、评估时，发现薄稻穗瘟抗性表现优良。因此，本研究以具有广谱稻瘟病抗性的太湖流域地方品种薄稻作为研究对象，与感病品种苏御糯构建重组自交系（R *51今日免费论文网|www.jxszl.com +Q: ^351916072# 
ILs），构建次级群体BC5F5。设计多态性标记，并以BC5F5群体对薄稻抗性基因进行精细定位，最终将薄稻抗稻瘟病QTL主效位点Pbbd1定位在分子标记BS13、BS98之间的31.4kb区间内，并预测到存在于该区间内的4个候选基因P3、P4、P5、P6。同时，利用CRISPCas9技术构建候选基因的转基因植株，对候选基因进行功能验证，结果表明P3、P4突变体感病性更强，可能为目标基因。
FUNCTIONAL VERIFICATION OF CANDIDATE GENE FOR BLAST RESISTANCE TO QTL LOCUS PBBD1 OF LOCAL THIN RICE IN TAIHU BASIN
ABSTRACT
Rice blast is a fungal disease of rice, which seriously affects the world rice yield and quality due to its high pathogenicity and wide spread. Breeding rice varieties with broad spectrum resistance to rice blast is the most economical and effective method to control rice blast and to eradicate rice blast at the source.In the early laboratory, the resistance of rice blast of local varieties in taihu river basin was identified and evaluated.Therefore, in this study, the local thin rice with broadspectrum resistance to rice blast in the taihu basin was taken as the research object, and a recombinant inbred line (RILs) was constructed with the affected rice variety suminuo to construct a secondary population.Polymorphism markers were designed and BC5F5 population was used to accurately locate the thin rice resistance genes. Finally, pbbd1, the main QTL locus of thin rice resistance to blast disease, was located in the 31.4 KB interval between the molecular markers BS13 and BS98, and four candidate genes P3, P4, P5 and P6 were predicted. At the same time, CRISPCas9 technology was used to construct transgenic plants with candidate genes, and the function of the candidate genes was verified. The results showed that P3 and P4 mutants were more susceptible to diseases and might be the target genes.

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