一个水稻柱头外露率QTL: qSE4的图位克隆与功能初步研究

Based on our previous research on QTL mapping of rice stigma exsertion rate, a QTL: qSE4 which control double and total rice stigma exsertion rate were detected repeatly in F2 and F2:3 population in different plantting enviroments and the contribution rates were relatively high. In order to fine mapping and cloning of qSE4, the BC3F2, BC4F2 populations and nearly isogenic lines of qSE4 were constructed. qSE4 was fine mapped on a 408.99Kb interval on rice Chr.4. Bioinformatics analysis indicated that a gene encoded auxin response factor (LOC-OS04g43910) might be the target gene. Further the relative expression levels and genome sequencing of LOC-OS04g43910 in parents and nearly isogenic lines of qSE4 showed that LOC-OS04g43910 most likely to be the target gene of qSE4. Finally, knock out of LOC-OS04g43910 under parent DaS background by using Crisp/Cas9 was conducted, and the T0 transgene plants with homozygous mutations performed lower stigma exertion rate (30.7%, 26.4%) compare with that of the parent DaS (77.3%). .In the next step, the transgene T1 plants of gene complement and gene knock out of LOC-OS04g43910 were used to identify the genotypes and phenotypes. According to the results of genotypes and phenotypes identification, the target gene will be determined. On the bases of the target gene identification, the gene expressions under different development stages and environments, the subcell location of encoded protein and downstream genes identification regulated by target gene will be conducted to preliminary clear the function of target gene. Finally, the haplotype analysis of target gene among different rice varieties will be conducted to uncover the geographical distribution, origin, and the trace of evolution of target gene.

在前期重复检测到效应值较大的控制水稻双边和总柱头外露率的qSE4的基础上，构建了BC3F2，BC4F2群体及近等基因系。将qSE4精细定位于水稻第4染色体上408.99Kb区域。生物信息学分析、目标基因在亲本及近等基因系的表达量以及基因测序分析表明一个生长素响应因子LOC-OS04g43910很可能为目标基因。进一步，在亲本大S背景下，利用crisp/cas9技术将目标候选基因进行敲除，结果纯合的敲除转基因T0植株，其柱头总外露率（30.7%，26.4%）显著低于背景亲本大S（77.3%）。下一步考查互补与基因敲除转基因T1植株的基因型和表型，确证目的基因。进一步通过目的基因的时空表达模式，编码蛋白的亚细胞定位以及Chip-Seq目标基因所调控的下游基因鉴定，初步明确目标基因的功能。最后通过目标基因的单体型分析，初步明确目标基因的地理分布，起源以及进化规律。

针对目前杂交水稻不育系柱头外露率的调控机理不清晰；不育系，尤其是粳稻不育系的柱头外露率不高的问题。在前期重复检测到效应值较大的控制水稻双边和总柱头外露率QTL：qSE4的基础上，通过多代回交，自交构建了BC3F2，BC4F2群体及近等基因系。将qSE4精细定位于水稻第4染色体上408.99Kb区域。 生物信息学分析、目标基因在亲本及近等基因系的表达量以及基因测序分析表明一个生长素响应因子LOC-OS04g43910很可能为目标基因。进一步，在亲本大S背景下，利用crisp/cas9技术将目标候选基因进行敲除，结果纯合的敲除转基因T1植株，其柱头总外露率显著低于背景亲本大S。最终考查了互补与基因敲除转基因T1植株的基因型和表型，确证了目的基因。此外，通过qRT-PCR 分析了qSE4目的基因的不同组织部位，不同生长发育时期的时空表达模式；明确了目标基因编码蛋白的亚细胞定位；最后通过多组学分析，初步明晰了qSE4目的基因对不同植物激素的响应特征。研究结果有助于阐释水稻柱头外露率的调控机理，同时将目标基因导入其他水稻不育系，可改善其异交习性，提高制种产量，降低杂交水稻制种成本，助力杂交水稻持续健康发展。

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