玉米叶夹角基因ZmLA1.02的图位克隆及功能分析

Leaf angle is an important trait that affects high-density breeding in maize, and it is also highly correlated with plant type breeding and yield breeding. Then it is of great theoretical and practical significant to do the leaf angle gene clone work via map-based clone, function analysis and value analysis of breeding. In the previous study，the leaf angle gene ZmLA1.02 was located on the first chromosome within 600kb; this project is to do the fine mapping of ZmLA1.02 and identify the key candidate gene associated with leaf angle combined with resequencing. Then the gene structure, expression analysis also will be done, and candidate gene association analysis were used to verify gene function and develop functional markers. BC-MAS method will be used to evaluate the breeding effect of the ZmLA1.02 site. The results of the study not only cloned a new maize leaf angle gene ZmLA1.02, but also helped to analyze the genetic mechanism of leaf angle traits, and also provide important gene resources and effective molecular markers for maize plant type breeding and high-density breeding.

玉米叶夹角是影响高密度育种的一个重要性状，与株型育种及产量高度相关；图位克隆叶夹角基因并对其功能和育种价值进行分析具有重要的理论意义和实践意义。课题组前期工作中利用突变体将控制叶夹角的基因ZmLA1.02定位在了玉米第一染色体600kb范围内。本项目拟利用目标区段重组交换的跨叠系开展ZmLA1.02精细定位工作，结合目标区段重测序方法筛选并克隆控制叶夹角的关键候选基因，分析基因结构及其时空表达模式。采用候选基因关联分析方法，初步验证基因功能，并开发功能标记。采取BC-MAS方法将ZmLA1.02导入到优良玉米自交系中，评价该位点的育种效应。本研究结果不仅克隆到一个新的玉米叶夹角基因ZmLA1.02，有助于解析叶夹角性状的遗传机理，也为玉米株型育种及高密度育种提供重要的基因资源和有效的分子标记。

本项目以叶夹角突变体la1.02为材料，通过精细定位及图位克隆等策略，成功克隆了控制玉米叶夹角和叶片皱缩的关键基因，命名为ZmSRL5；遗传分析表明srl5受单个隐性核基因控制（3:1，χ2-test，p=0.78）。利用B73与srl5突变体杂交构建的4596个F2个体将srl5定位在1号染色体90 kb区间内，该区间包含两个候选基因Zm00001d028159和Zm00001d028160。测序分析表明Zm00001d028159在野生型和突变体中无序列差异；而在突变体中，Zm00001d028160的第一个内含子存在着3745 bp的插入。RNA-seq reads拼接结果以及RT-PCR结果都证实该片段的插入会导致转录的提前终止，即Zm00001d028160为关键候选基因，并命名为ZmSRL5。ZmSRL5编码含有4个跨膜结构域的CASP Like蛋白。.ZmSRL5的CRISPR/Cas9的两个片段缺失的基因敲除系KO1和KO2具有和srl5相似的表型，进一步验证了ZmSRL5为功能基因；时空表达分析表明，ZmSRL5属于组成型表达，且在发育的叶片中高表达，在成熟的叶片中低表达；亚细胞定位结果表明ZmSRL5蛋白定位于细胞质膜。 本项目精细定位并图位克隆了控制玉米叶夹角和叶片皱缩的关键基因，并通过遗传转化后代解析了其调控叶夹角的生物学功能。