玉米籽粒发育相关基因OS1的功能研究

Maize kernel is mainly composed of embryo and endosperm，where endosperm accounted for about 85% of the entire kernel weight, is the main source of food for human and livestock. os1 is a spontaneous mutant that shows smaller embryo and opaque endosperm on the top. We have cloned the gene by using the fine mapping strategy through large BC1F1 population derived from os1 and Mo17, as well as candidate gene analysis and functional complementary test. OS1 gene is a transcription factor containing RWP-RK conserved domain and the protein encoded by the gene is localized in the nucleus, which was confirmed by subcellular localization. This project will study on the molecular mechanism of the OS1 gene during the kernel development based on research results. The expression patterns of OS1 gene in the specific cell types of the embryo and endosperm will be determined by transformation of the constructions of OS1 promoter with GUS and GFP fusion vector; the candidate target genes regulated by OS1 will be cloned through the transcriptome sequencing and Chip assay, and the DNA sequence of targets gene promoter will be determined by gel EMSA assay. The functional analysis of OS1 gene in regulating the embryo development and endosperm components will shed light on the genetic improvement of maize kernel.

玉米籽粒主要由胚和胚乳组成，其中胚乳占整个籽粒重量的85%左右，是人类和家畜的主要食物来源。os1是一个玉米自然诱发突变体，表现为胚变小和胚乳顶端不透明。课题组前期利用os1与Mo17构建作图群体，通过精细定位、候选基因分析和功能互补试验，成功克隆了该基因，OS1基因为一个编码含有RWP-RK保守结构域的转录因子，亚细胞定位结果表明该基因编码的蛋白定位在细胞核中。本项目将在现有研究基础上，通过构建OS1::promoter与GUS和GFP编码框表达载体，利用转基因技术明确OS1基因在胚和胚乳的特定细胞中的表达情况；通过对制备OS1基因抗体及Chip试验，挖掘OS1基因调控的候选靶基因，然后通过凝胶阻滞实验确定OS1基因与靶基因启动子作用的DNA序列；结合OS1靶标基因的功能分析，阐明OS1基因在调控籽粒胚和胚乳发育过程中的功能，旨在为玉米籽粒性状的遗传改良奠定基础。

本项目对RWP-RK转录因子基因OS1进行了深入的分子调控机理分析，结果表明，该基因的突变造成了籽粒胚变小，同时胚乳变成opaque，深入的表型分析结果显示，突变体籽粒中胚和胚乳的养分含量发生了变化，主要变现为胚中的油分含量降低，胚乳中的氨基酸组分含量降低，而淀粉含量升高，主要原因是胚体积减小，细胞学观察表明，糊粉层细胞排列在突变体中出现了不规则，而且紧贴着糊粉层细胞的亚糊粉层细胞在突变体中消失，这可能是胚乳出现opaque或者白色胚乳的主要原因。组织切片结果表明，从第6天的原胚期开始，突变体中胚的发育就出现了一定程度的延迟，随着时间的推移，胚大小变化趋于明显。转录组分析表明，养分运输和再分配的基因表达发生了明显下调，这些基因的表达主要集中在BETL层细胞。综上，OS1基因的分子调控机理为通过调控养分养分转运基因或者印迹基因的表达，进而调控玉米籽粒养分在胚和胚乳中的运输和再分配。