元江普通野生稻抽穗扬花期耐热性QTL(qHTH5)的精细定位及克隆

Owing to the rich genetic diversity of wild relatives , wild relatives of rice are precious germplasm resources. In an earlier study, we have detected the QTL(qHTH5) for heat tolerance at the heading stage on chromosome 5 from Oryza rufipogon Griff.（Yuanjiang wild rice Hehuatang NO.3）in near-isogenic line,where O. sativa ssp. Indica (Shuhui 527) was used as the recipient parent, and located in 95kb interval.This study will make further fine mapping, and will screen wild rice BAC library for BAC clone sequencing and analysis. We will conduct transcriptome analysis for spikelets of QTL-NIL and recurrent parent under high temperature stress at the heading stage（0, 1, 2, 3d）, to determine differentially expressed genes. Sharing QTL analysis will be performed according to the results of pQTL and eQTL mapping in a small F2 population, and will further analyse candidate gene of the qHTH5 combining the gene sequencing of wild relative and cultivated rice with gene expression measurement. We validate the function of qHTH5 through functional complementation analysis, over expression and RNA interference. At the same time, we also intend to explain its molecular mechanism preliminarily through the expression pattern and bioinformatics analysis, so as to promote the development of rice heat tolerance breeding in China.

野生稻具有丰富的遗传多样性，是水稻育种的宝贵种质资源。在前期研究中，我们利用一个以蜀恢527为背景的的近等基因系，在5号染色体短臂上鉴定了一个来自于元江普通野生稻荷花塘3号的抽穗扬花期耐热性QTL(qHTH5)，并将其初步精细定位在95kb之内。本研究拟进一步精细限定QTL区间，并筛选元江野生稻BAC文库，对目的BAC克隆测序及序列分析；用RNA-seq技术将回交得到的单QTL近等基因系与轮回亲本在抽穗扬花期模拟高温胁迫下的小穗（0, 1, 2, 3d）进行转录组分析，获得差异表达基因；然后利用一个F2小群体进行pQTL与eQTL作图，并进行共享QTL分析，并结合测定野生稻/栽培稻序列和表达量分析等手段进一步确定qHTH5候选基因。通过功能互补、过量表达和RNA干涉验证qHTH5基因的功能。同时通过表达模式和生物信息学分析初步解释其分子机理，以期促进我国水稻耐热育种的发展。

水稻(Oryza sativa）生殖发育期是对高温胁迫最敏感阶段, 尤其在扬花期高温胁迫将直接导致影响花粉管伸长和正常散粉，导致不能受精而形成空、秕粒，降低结实率。本项目通过图位克隆策略克隆了元江野生稻孕穗期耐热性主效QTLqHTF5，基因遗传转化实验证明过表达转基因系扬花期耐高温性显著增强,具有调控水稻扬花期耐高温特性的功能,同时qHTF5 RNAi转基因株系扬花期的的耐热性比对照Sasanishiki显著降低。通过功能预测发现, HTF5编码一个脯氨酸合成酶共转录细菌同源蛋白（proline synthetase co-transcribed bacterial homolog protein），该基因具有磷酸吡哆醛（pyridoxal 5’-phosphate, PLP）结合功能。采用HPLC技术对转基因株系和受体亲本Sasanishiki花粉中高温胁迫下的游离PLP含量分析，发现qHTF5超量表达转基因株系能够有效降低高温胁迫条件下小穗花粉细胞中游离PLP（一种活性醛）的过量积累对花粉细胞的毒害作用。qHTF5 RNAi株系游离PLP的含量最高，结实率比Sasanishiki显著降低。对超量表达转基因株系、RNAi株系和受体亲本Sasanishiki高温胁迫处理后与对照正常温度处理下的小穗花粉中H2O2、MDA相对含量、SOD和POD活性比较分析,证明qHTF5HHT3超量表达有效调节了小穗花粉高温胁迫下的ROS动态平衡水平。研究结果证实了qHTF5的自然变异能够增强水稻扬花期花粉耐高温特性, 而且为培育水稻耐高温新品种提供了重要基因资源。