【作者】 刘燕；

【导师】 张改生； Hei Leung；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2010， 博士

【摘要】 长期以来,在中国的广东省,三黄占2号(SHZ-2)被育种家们公认为是具有广谱,持久抗水稻稻瘟病的优良品种。但是,由于育种家们对它的抗病分子遗传机制缺乏了解,所以很难将它的这种广谱,持久抗病性转移到其他品种中并长期利用它来抵抗因稻瘟病带来的产量损失。为了了解三黄占2号品种的这种抗病的分子遗传机制,本文我们用它与另一个具有中度感稻瘟病的品种特籼占13号(TXZ-13)杂交,并用F1与特籼占13号回交,通过单粒传和田间选择的方法,从BC3中筛选到一个不仅具有优良农艺性状,而且具有持久抗水稻稻瘟病的品种BC-10。BC-10在种植的8个季节中一直表现着很强的稻瘟病抗性。此外,为了发掘和研究与持久抗病相关的QTL,本文还通过单粒传的方法创建了一个具有244个株系的BC4F3群体。同时为了能做到对QTL的精细定位,又利用单粒传的方法创建了两个F2群体,其中一个是SHZ-2和TXZ-13杂交得到的含有343个单株的群体;另一个是用BC-10和TXZ-13杂交得到的含有600个单株的群体。本研究采用了SNP芯片技术,SSR分子标记技术和设计候选基因特异引物的方法来识别与持久抗稻瘟病相关的导入片段以及QTLs。同时还利用44K基因表达芯片技术研究了SHZ-2, BC-10和TXZ-13在稻瘟病感染24小时和48小时的基因表达情况。通过上述研究,获得下述重要结果:1、利用SNP芯片技术,SSR分子标记技术和候选基因特异引物的方法,在BC-10中找到了一些从SHZ-2中导入的片段,经过在BC4F3群体中分析,发现了3个分布在染色体2,6和9上的与持久抗病相关的新的QTLs, qBR2.1,qBR6.1和qBR9.1。它们对水稻叶瘟病抗性方面的贡献分别为16.2%,14.9%和22.3%。2、利用BC4F3中杂合的自交系创建了含有qBR2.1,qBR6.1和qBR9.1三个QTLs的近等基因系,对这些近等基因系进行了多季田间稻瘟病抗性鉴定,发现qBR9.1对稻瘟病具有完全抗性;qBR2.1或qBR6.1单独存在时并没有减少病害,而是两个QTLs结合在一起时,它们减少了水稻19.5%的病害,这表明具有部分抗性的QTL可以有效地减少稻瘟病疫情。3、利用两个F2群体,发现qBR9.1是一个新的具有广谱抗稻瘟病的主效基因,将其精细定位,被确定在染色体9上一个69.1 kb区间内,并证明BC-10携带的该基因确系来自其亲本SHZ-2。通过水稻基因组分析,发现在这一区间只有一个具有假定抗病功能的NBS-LRR基因。利用设计该基因特因引物,发现该基因与持久抗病性紧密连锁。鉴此,将该新的持久抗病基因命名为Pi46(t)。4、经过测序分析,发现Pi46(t)编码一个具有NBS-LRR结构域的蛋白质,该蛋白质含有743个氨基酸。而在TXZ-13中Pi46(t)的等位基因只编码一个具有537个氨基酸的蛋白质,并且缺失了LRR结构域。进一步的表达研究表明稻瘟病可以强烈诱导Pi46(t)基因的表达。同时,本研究还发展了三个新的与Pi46(t)紧密连锁的分子特异标记CRG4-1 CRG4-2和CRG4-3,该标记已被用于分子辅助育种计划中。5、为了充分发掘与稻瘟病抗性有关的基因并了解水稻稻瘟病抗病的分子机制,首次利用44K基因表达芯片技术对SHZ-2, BC-10和TXZ-13在感染稻瘟病菌24和48小时的转录本进行了分析,结果表明,在抗病品种SHZ-2和BC-10中,因感染稻瘟病菌而被大大激活的基因(DEGs)数量是感病品种中的两倍。对这些基因(DEGs)进行聚类分析发现,SHZ-2和BC-10的持久抗性可能与那些48小时在两个品种中同时被激活的基因(DEGs)有关。对DEGs的功能分析同时结合BC-10基因组中的导入片段的调查,发现和荷尔蒙JA信号调节途径在水稻稻瘟病抗病中起着重要的作用。同时,在抗病品种中,一些转录因子也因稻瘟病菌的入侵而被大大激活,例如WRKY, AP2和ZIM。还有一些与防卫反应相关的基因和一些与抗病相关基因的表达也大大增强。更多还原

【Abstract】 SHZ-2, an indica cultivar with broad spectrum and durable resistance to multiple races of the blast pathogen, has been recognized as a cultivar for long time in Guangdong, China. However, it has been difficult to transfer its broad spectrum and durable resistance into other cultivars due to the poor understanding of the molecular genetic mechanism of its resistance. In order to understand its molecular genetic basis of the broad spectrum and durable resistance of SHZ-2, it was crossed to TXZ-13, a blast susceptible variety, to produce a BC3 line (BC-10) that exhibited strong to moderate blast resistance over eight cropping seasons in the field. To identify and dissect the QTL responsible for durable blast resistance, 244 BC4F3 lines were evaluated for blast resistance in the greenhouse and blast nursery. To fine mapping the QTLs, two populations of 343 F2 derived from SHZ-2×TXZ-13 and 600 F2 from SHZ-2×TXZ-13 were developed by single seed descent. A single feature polymorphism microarray, Golden Gate chips, SSR markers and gene-specific primers were used to identify the introgressions of SHZ-2 in BC-10, to decide the QTLs associated with the durable resistance to multiple races in Philippines and fine mapping the QTLs. The main results obtained are as followings:1. Chromosomal introgressions from SHZ-2 were identified by using a single feature polymorphism microarray, SSR markers and gene-specific primers. Segregation analysis of BC4F3 population indicated that three regions (QTLs) on chromosome 2, 6, and 9, designated as qBR2.1, qBR6.1 and qBR9.1 were associated with blast resistance and contributed to the reduction of diseased leaf area (DLA) by 16.2%, 14.9 and 22.3% respectively.2. We defined three QTLs using pairs of near-isogenic lines extracted from heterogeneous inbred families (HIF). Pairwise comparison of these lines enabled the dissection of the relative contributions of individual QTL. The qBR9.1 conferred strong resistance as expected because of the presence of NBS-LRR genes. Under field condition, qBR2.1 or qBR6.1 individually did not reduce disease but when combined together, they reduced disease by 19.5%, suggesting that small effect QTLs could be effective in reducing epidemics. The qBR6.1 and qBR9.1 region contains NBS-LRR sequences, whereas the qBR2.1 did not. The expression pattern of candidate genes within the QTL regions suggested functional roles of these genes in response to blast infection.3. A new major QTL of qBR9.1 conferring durable resistance to rice blast was finely mapped into a 69.1 kb region on the chromosome 9 that has been inherited from the resistant line SHZ-2 into backcross 3 lines BC-10. By annotation analysis, only one predicated disease resistance gene with NBS-LRR domain was found within this region. Using gene specific marker analysis, we found this gene co-segregated with blast resistance in F2 and F3 populations derived from SHZ-2. We tentatively designate it as Pi46(t).4. Sequence analysis revealed that the Pi46(t) encodes a nucleotide binding site and leucine-rich repeat (NBS–LRR) protein which is composed of 743 amino acid polypeptide, Pi46 was highly induced by blast infection in resistant lines SHZ-2 and BC-10.5. Three new gene specific markers, CRG4-1 CRG4-2 and CRG4-3, have also been developed, which are co-segregated with the Pi46(t) and have been used for MAS breeding and the introduction of the durable resistant gene Pi46(t) into IR64 and the development of near isogenic lines.6. To identify the genes involved in blast resistance to blast in rice and understand the molecular mechanism of plants resistance to blast, a comparative transcriptomic analysis were did among the backcross line BC-10 and its parental lines SHZ-2 and TXZ-13 using 44k microarray method.We found the number of genes responding to blast infection in resistance lines BC-10 and SHZ-2 was twice that in susceptible line TXZ-13 at 24 HAI and 48 HAI. Cluster analysis reveals that the blast resistance in SHZ-2 and BC-10 may relate to those DEGs which response to blast infection both in SHZ-2 and BC-10 at 48 HAI. The function analysis of the DEGs suggests that the JA signaling pathway plays important role in the rice resistance to blast. The expression of some of the transcription factors, such as WRKY, AP2 and ZIM were significantly up-regulated only in resistant line BC-10 and SHZ-2 by blast infection. And some of the defense related genes were activated only in resistant lines BC-10 and SHZ-2.更多还原