一个具有育种潜力的水稻株高QTL qPH6的遗传解析和功能研究

As one of the most important crops worldwide, rice (Oryza sativa L.) provides 35-60% of the world's dietary calories and is consumed by more than three billion people. In the 1950s, world rice production has dramatically increased due to a reduction in plant height resulting from the use of the semi-dwarf gene sd1. The reduced plant height enhances lodging resistance, and improves harvest index and biomass production. However, the frequent use of sd1 gene might cause genetic vulnerability. Now, an appropriate increase in plant height on the basis of semi-dwarf varieties has been an important strategy of rice high-yield breeding. Characterization of genes, especially QTL (Quantitative Trait Loci), associated with rice plant height is urgent to break the ceiling of yield potential. In our previous work, we identified a novel QTL, qPH6, for plant height in the backcross introgression population derived from japonica c.v. Balilla and indica c.v. Dular. qPH6 has no pleiotropic effect on other agronomic traits including panicle number, grain number, grain weight and grain yield. So qPH6 is a valuable locus for breeding and can be used in plant architecture improvement. Fine mapping was carried out with a total of 2378 recessive individuals from F2 and F3 generations of Balilla and C33084, and the gene was finally narrowed into a 41-kb region containing three annotated genes. Two of them, LOC_Os06g44010 and LOC_Os06g44030, were considered as the candidates of qPH6 according to the sequencing analysis. In this project, complementary test will be used to confirm the candidate genes. The biological function of the qPH6 related to plant height will be validated by the performance of transgenic lines of RNAi and overexpression of qPH6. The genetic regulatory network of qPH6 involved in rice plant height will be revealed by transcriptome and proteomics. We will further analyze the genetic effects of qPH6 by construction of nearly isogenic line and assess the breeding value. The allelic variation of qPH6 in various varieties will also be uncovered. After the completion of the project, the molecular mechanism and breeding value will be clearly elucidated. About 2-3 high-level papers will be published.

理想株型培育是水稻品种高产育种的基础。株高是株型最重要的因子之一。目前籼稻矮秆基因主要是sd1，粳稻主要利用的是数量性状位点(QTL)。sd1的过度利用潜伏着由遗传单一而带来的风险，而控制株高的QTL研究尚不够深入。因此，挖掘具有重要利用价值的株高基因特别是QTL显得十分迫切。本课题组在以前的研究中从粳稻骨干亲本Balilla中鉴定出一个新的水稻株高QTLqPH6，不具育种负效应，具有重要的育种价值。本项目拟在精细定位和确定候选基因的基础上，利用互补试验进行候选基因的验证；构建过表达株系和RNAi株系，研究qPH6的生物学功能；通过转录组学和蛋白质组学，揭示qPH6调控水稻株高的分子机理和遗传调控网络；利用近等基因系明确该基因对各农艺性状的遗传效应，评价其对水稻品种改良的价值；通过测序明确qPH6在不同水稻高产品种中的遗传变异，为开展水稻株型的分子改良提供遗传和资源信息。

在以前的研究中，从Balilla和Dular为亲本的高世代回交导入群体中鉴定出一个新的水稻株高QTL qPH6。. 本项目将qPH6基因精细定位在BAC克隆AP003517上标记S6-334和S6-10之间41-Kb的物理距离内，共有3个开放阅读框，分别是Os06g44010、Os06g44020和Os06g44030，分别编码一个转录因子OsWRKY28、一个未知蛋白和一个WD40结构域的蛋白。通过互补试验和RNAi分析，验证了Os06g44030就是qPH6基因。与Dular相比，Balilla的qph6等位基因存在26个单核苷酸变异(SNPs)，其中SNP9和SNP10分别位于第4和第5外显子，都造成了氨基酸变异，导致其功能改变。qPH6基因是组成型表达，在根、茎秆、叶、叶鞘、花、种子及幼穗中均有表达，其中茎秆和叶鞘中表达量最高。近等基因系表型分析表明，qph6可显著降低株高，但对单株产量没有明显影响。qPH6近等基因系NIL(qPH6)表现为ABA不敏感，而NIL(qph6)对ABA较敏感。但ABA处理qPH6基因的RNAi株系，发现ABA处理后其发芽率与野生型无显著差异，结果表明qPH6基因只对水稻株高有影响，而对ABA敏感性无影响，株高和ABA敏感性是由两个不同基因控制的。. 在精细定位区间，还有一个WRKY转录因子基因OsWRKY28，包含了3个外显子和2个内含子，编码337个氨基酸。通过测序比对发现，Balilla和Dular编码的氨基酸并没有差异，而在启动子区存在8个SNPs和1个InDel，这些差异可能会影响OsWRKY28的转录，进而影响NIL (qPH6)和NIL (qph6)对ABA处理的敏感性。RNAi实验证明，降低OsWRKY28基因的表达，发现植株对ABA和SA的敏感性提高，而互补和过表达则降低了植株对ABA和SA的敏感性。结果表明OsWRKY28负调控ABA和SA的敏感性。. 综合以上结果，我们成功克隆了控制水稻株高的基因qPH6，来源于Balilla的qph6可显著降低株高，但对单株产量没有明显影响，在水稻抗倒伏育种中有潜在的育种价值。而同样位于候选基因区段内的另一个基因OsWRKY28对株高没有影响，但其等位变异是造成近等基因系对ABA敏感性的原因。