【作者】 周亮；

【导师】 杨远柱； 陈良碧；

【作者基本信息】 湖南师范大学 ， 植物学， 2012， 硕士

【摘要】 稻瘟病是由子囊菌Magnaporthe grisea(Hebert)Barr引起的,该病广泛分布于世界水稻种植区域,是对水稻生产最具毁灭性的病害之一。实践证明,利用分子标记与传统杂交育种相结合将单个或多个抗病基因转移到高产优质水稻中是防治稻瘟病经济、有效和安全的措施。水稻稻瘟病主效显性基因Pigm为广谱抗性基因,其抗谱明显强于广谱抗性基因Pi1、Pi2、Pi3、Pi9等,在抗病育种中具有更高的应用价值。本研究利用Pigm的供体亲本谷梅4号、95个水稻亲本及一些杂交材料,开发与Pigm共分离的分子标记,对加速抗源筛选,抗病基因的鉴定和分子标记辅助育种具有重大意义。另外,本研究试图分析Pigm与Pita在聚合后的纯合体材料中各自的表达量。主要研究结果如下：(1)根据Pigm定位标记在互联网上查找它们在水稻物理图谱上的位置,在它们所在的区域附近找到22个已定位于该区段的SSR标记。在22个SSR标记中,仅有RM276和RM3724两个标记在谷梅4号和多个亲本间具有多态性。RM3724较RM276与Pigm距离较近,因此利用RM3724在谷梅4号和95个亲本间检测多态性,结果表明,仅有R527、R624共2个亲本材料与谷梅四号具有多态性。且RM3724进行MAS选择的准确率仅为80%,表明RM3724为非共分离的分子标记。(2)根据谷梅4测序的结果以及籼稻9311的shot-gun序列、粳稻日本晴AP005659序列和Pigm等位的同源性很高的Pi2序列比对的结果,根据插入缺失区域序列总共设计了40对InDel引物,其中结合S29742、S1408、S2997、S2723这4对引物可以将谷梅4号和其它95个亲本完全分开。利用这些InDel标记对谷梅4号的杂交组合的F2群体进行分子辅助选择,并结合田间的抗性表现来判断MAS选择的准确性。分子检测和田间鉴定的结果均表明,标记S29742、S1408、 S2997、S2723分子检测的结果和田间抗性鉴定的结果是完全一致的,MAS选择的效率高达100%,表明这些标记与Pigm共分离,完全满足MAS的应用。(3)利用半定量RT-PCR进行了接种前和接种后4、8、12、24、48小时共六个时期Pigm、Pita基因各自的表达分析,结果表明, Pigm、 Pita基因的表达不受病原菌的诱导,而且接种后不同时间点的表达基本上是一致的,由此说明,Pigm、Pita属于组成型表达的基因。更多还原

【Abstract】 Rice blast, caused by fungal pathogen Magnaporthe grisea (Hebert) Barr, is one of the most devastating diseases in rice production all over the world. Developing resistant varieties to rice blast is one of the most economical, effective and safe methods to control rice blast disease with MAS and convention breeding methods.Pigm derived from Gu-mei4, is a dominant rice blast resistant gene, was characterized of broad-spectrum and durable resistance to rice blast. The spectrum of Pigm is broader than that of gene Pi-1, Pi-2, Pi9, Pi-3, has a higher value in rice breeding to rice blast. In this study, we use the donor Gu-mei4, some parents and some hybrid materials to develop molecular markers co-segregated with Pigm, which has important significance for selection cultivars with resistance gene, identification of resistance genes and MAS. In addition, this study also attempts to analyze the resistance the resistance in the nursery in material of polymerization of Pigm and Pita through the each expression of Pigm and Pita. The main results are as follows.(1)Based on others research, Pigm was finely mapped between markers C5483and C0428on chromosome6. According to markers C5483and C0428, we find22SSR markers located in the region, which are located at the two sides, searched from rice genome database on Internet. Among all22SSR markers, RM276and RM3724were polymorphic between Gu-mei4and parents. RM3724was closer linked to Pigm than RM276, so RM3724was chosen to detect polymorphism between Gu-mei4and parents. The results showed that only two parents R527, R624were polymorphism between Gu-mei4and the selection rate of RM3724is only80%, was non-co-segregated with Pigm.(2) The results of the sequencing compared with93-11, Nipponbare sequence by NCBI blast, together with the alignment squence of AP005659,93-11and Pi2which has high homologous with Pigm, primers was designed based on regions of insertion or deletion sequences. Together, We develop a total of40InDel markers, together with markers S29742, S1408, S2997and S2723, Gu-mei4was separated from the other95parents. These InDel markers were used in marker-assisted selection in the F2plants of the crosses Gu-mei4with other parent, combined with nursery identification to determine the accuracy rate of selection for Pigm, we find the rate of these InDel markers is100%. Therefore these markers were co-segregated with rice blast resistance gene Pigm, fully meet the needs of rice breeding.(3)To analyze each expression profile of the Pigm and Pita gene, the material were inoculated4,8,12,24and48h, was investigated by semi-quantitative RT-PCR analysis. The results revealed that no difference was observed for the gene Pigm and Pita among different time points. Thus, it was concluded that the each expression profile of Pigm and Pita is constitutive and is not induced by blast infection.更多还原