水稻抗褐飞虱基因QBph4.2的克隆及其育种利用研究

The brown planthopper (Nilaparvata lugens Stål; BPH) has been one of the most devastating pests of rice. Host-plant resistance is the most effective and environment friendly strategy in the management of BPH. However, there were few BPH resistance genes used in rice breeding, and the molecular mechanisms of rice resistance to the BPH have not yet been clearly elucidated. In our previous work, a major QTL for BPH resistance was identified from a introgression line derived from Oryza australiensis, which could explain 46% of phenotypic variation. Subsequently QBph4.2 was delimited to a 46-Kb region. 1-2 candidates of QBph4.2 will be selected according to further fine mapping, parental comparative sequencing and gene annotations by bioinformatics. Then genetic complementary test will be conducted, and transgenic plants will be investigated for co-segregation analysis. Ultimately we definitely claim that QBph4.2 will have been successfully cloned. Further, it will be performed to examine the temporal and spatial expression patterns (typically in phloem of sheath) for the two alleles of QBph4.2 before and after BPH infestation, which will explain if QBph4.2 mediated interaction of host and insect and elucidate the molecular mechanisms of rice resistance to the BPH. Furthermore, gene effects of QBph4.2 under the backgrounds of 9311 and Zhenshan 97 near isogenic lines (NILs) will be evaluated. The new development of this work will provide a new valuable resistance gene and functional markers in rice breeding, which will facilitate the control of the BPH.

褐飞虱是水稻最主要的害虫之一，利用水稻内源抗性基因培育高抗品种是防控褐飞虱最为经济有效的方法。目前在育种上可用的抗性基因很少且对水稻抗性的分子机制也知之甚少。我们在澳洲野生稻的抗虫导入系中定位了一个新的抗性QTL，QBph4.2，解释46%的表型变异，并进一步将其精细定位至46 kb区域。在此基础上，拟先通过进一步精细定位和亲本比较测序等方法确认1–2个候选基因，然后通过功能互补试验，确认QBph4.2的克隆。然后，分析QBph4.2两等位基因在不同组织（特别是叶鞘的韧皮部）接虫前后的相对表达水平，从等位基因时空表达模式的差异来阐明QBph4.2的表达是否参与了宿主-褐飞虱的互作，从而初步揭示抗性的分子机制。此外，本研究通过评价QBph4.2在珍汕97、9311背景下的近等基因系的基因效应，将为育种家们提供新的抗性基因及其功能标记，并有助于褐飞虱的防控。

褐飞虱是水稻产区最重要的害虫之一。利用水稻抗性基因进行抗虫育种是应对褐飞虱危害的重要策略。而不断发掘新的抗性基因不仅为明晰抗性机制提供理论基础，同时也为抗虫育种提供了丰富的基因资源。本项目研究内容是以澳洲野生稻导入系与9311衍生的QBph4.2 近等基因系F2分离群体为材料，对本QTL 进一步精细定位，发掘了QBph4.2连锁的功能性分子标记，并通过分子标记辅助选择和回交育种策略，成功将QBph4.2导入到优良恢复系9311中，应用于抗虫育种。利用20000个NIL-F2株系将QBph4.2精细定位在110 kb的区段。此外，我们还利用近等基因系材料初步研究了QBph4.2介导的抗虫机制。我们分别对QBph4.2的近等基因系阳性和阴性材料（NIL+和NIL-）进行了苗期和成株期抗性鉴定、褐飞虱蜜露分泌量测定、褐飞虱存活率测定、拒虫性以及耐受性测定，结果发现QBph4.2能显著提高植株的苗期和成株期抗性、抑制褐飞虱蜜露分泌量和降低褐飞虱存活率，但是在拒虫性和耐受性上没有显著差异，表明QBph4.2可能是通过抗生性机制对褐飞虱产生抗性的。我们利用QBph4.2的近等基因系检测了SA和JA信号通路上的一些基因的表达模式。结果表明褐飞虱取食后QBph4.2基因可能激活了JA依赖的抗性途径。通过与QBph4.2共分离的标记XC4-27对其进行正向选择，并利用其两侧标记RM261和HJ16进行负向选择降低连锁累赘，然后通过与9311连续回交4次，并结合水稻6K育种芯片进行背景检测，最后得到了导入QBph4.2及其连锁片段小于400 kb，且背景回复率99.2%的9311高世代回交系。根据苗期抗性和褐飞虱蜜露分泌量鉴定结果，以及田间农艺性状的考察，最后获得了褐飞虱抗性明显改良且产量等主要农艺性状与9311相仿的改良家系。由于一些不可控的原因，我们最终没有确定QBph4.2的候选基因，也就没有完成遗传转化功能互补验证，即没有克隆到该基因。此外，我们利用一些经费开展了其它研究工作。包括褐飞虱抗性基因的全基因组关联分析，以及其它褐飞虱抗性基因的定位。