玉米皱小叶基因的遗传机理及应用研究

Leaf plays a significant role in photosynthesis pathway, and provides most of the organic nutrition for maize growth and development via photosynthesis. Maize leaf size decides its plant shape, and has a decisive effect on creating ideal plant shape. It also affects the plant density so that yield per unit area is closely related to it. Thus, the discovery of small wrinkled leaf in maize has an important significance in studying the mechanism of leaf development and in creating maize ideal plant shape. In this project, segregation populations with different backgrounds were constructed based on the newly discovered small wrinkled leaf mutant (small wrinkled leaf 1, swl1). Then phenotyping data from field, bulked segregation analysis(BSA) and map-based cloning were employed to identify and clone the gene which induced the phenotype of small wrinkled leaf. After that, gene function will be studied via histocytological analysis, physiological and biochemical study and molecular biological methods. Furthermore, analytical approaches of multi-omics and related biological methods will be applied in dissecting the molecular mechanism of leaf development in maize. Finally, we will utilize the strategy of genome-wide association study (GWAS) of candidate genes to find out the excellent alias or haplotype of the key gene that regulated leaf shape in maize, and to develop useful molecular markers for maize breeding. The achievements of this project will lay a theoretical foundation for the improvement of leaf shape in maize, and offer theoretical direction for maize breeding practices.

叶片是玉米进行光合作用的主要器官，通过光合作用积累玉米生长所需要的营养物质。玉米叶片大小是决定植株形态和创制理想株叶型的决定性因素，且影响种植密度，与单位面积产量密切相关。玉米皱小叶突变体的发现对解析玉米叶片发育机理，创制理想株型玉米材料具有重要意义。本项目对一个新的玉米皱小叶突变体swl1（small wrinkled leaf1）构建不同背景材料的分离群体，并结合田间表型分析、混合分组分析（BSA）及图位克隆的手段，对叶片皱小发育变异的基因进行克隆和鉴定；通过结合组织细胞学观察、生理生化分析、分子生物学分析等多学科交叉手段，对基因的生物学功能开展研究；结合多组学分析手段以及相关分子生物学方法，解析玉米叶型发育的分子机理；利用候选基因的关联分析策略对玉米叶型调控关键基因的优异等位位点或单倍型进行挖掘并开发标记。为玉米株叶型改良奠定理论基础，为玉米育种实践提供理论指导。

表皮蜡质层位于表皮细胞之外，分为蜡质和角质层，在降低非气孔蒸腾、抵御外界伤害、防止病虫侵害、抵抗紫外线等方面发挥重要的作用。蜡质合成、转运异常影响叶片的形态及大小。因而，克隆与蜡质合成、转运相关基因，并对其功能进行解析，具有重大的理论意义和应用价值。利用EMS诱变玉米自交系RP125的花粉，获得了一份表型稳定遗传的玉米皱小叶突变体swl1，本研究对该突变体的农艺性状进行了考察，对突变体叶片进行了组织细胞学观察，与玉米自交系B73构建了F2及BC1F1遗传分离群体，结合图位克隆，转录组学以及代谢组学对其分子机理进行解析。取得研究结果如下：.（1）、swl1为玉米皱小叶突变体，叶片皱缩，伸展不完全，叶面积极显著减小。对蜡质含量测定发现，突变体叶片中脂肪酸、初级醇及烷烃的含量高于野生型，但花丝中结果相反。叶片组织细胞学观察表明突变体叶片上表皮细胞发育畸形，排列不规则。（2）、采用图位克隆策略，将目标基因定位在indel19和indel14之间，物理距离为134 kb的区段内。该区段共有5个候选基因，设计特异性引物并克隆，测序比对发现在Zm00001d017251的最后一个外显子上存在一个T到A的突变，编码的氨基酸中缬氨酸变为天门冬氨酸，突变导致SWL1蛋白失去正常功能。我们发现材料swl2与swl1具有相似表型，双突植株具有同样表型，扩增后发现swl2存在一个G到A的突变。（3）、表达谱分析发现swl1的表达属于组成型表达。对蛋白含量检测发现突变体蛋白含量下降，说明突变影响了SWL1蛋白的生物合成，造成蜡质合成生物途径受阻。（4）、转录组结果表明，共检测到569个差异表达基因，其中上调表达175个，下调表达394个。富集分析表明，差异基因主要富集在脂肪酸生物合成、植物激素信号转导等通路，说明突变基因影响了脂肪酸的生物合成以及激素信号的转导。（5）、代谢组结果中筛选到179种差异代谢物，差异代谢物富集分析发现主要富集在类固醇生物合成，倍半萜和三萜的生物合成以及类胡萝卜素的生物合成。说明蜡质合成与这些生物合成途径密切相关。