甜瓜掌状裂叶pll基因的分子调控机制及功能验证

With classical genetics, numerous leaf development regulators have been identified, but the picture is far from complete. So far, the molecular mechanism of lobed leaf morphogenesis in melon has not been reported. The mutant with palmately lobed leaves named bm7 is an excellent material for the research of molecular mechanism of lobed leaf morphogenesis in melon. Previous studies showed that the palmately lobed leaf trait in melon was controlled by a single recessive gene named PLL, and the gene was located within the candidate region containing only one gene which is similar to the ANT transcription factor by map-based cloning. Recently, Single-base insert frameshift mutations were detected in bm7 by high-throughput sequencing, which is confirmed the effectiveness of the early genetic mapping results. This study takes bm7 as the main material to investigate the molecular mechanism of palmately lobed leaf morphogenesis in melon. First, the function of PLL will be verified in melon by the method of overexpression and interference. Second, spatiotemporal expression pattern of PLL will be analyzed by the method of real-time fluorescence quantitative PCR and reporter gene GUS. Third, downstream genes regulated and controlled by PLL will be detected by the strategy combining electrophoresismobility shift assay and RNA-seq. What’s more, the function of these downstream genes will be confirmed by CRISPR / Cas9 gene editing technology, which will add new content to the regulatory networks of leaf morphogenesis. And cell morphology of bm7 will be observed by scanning electron microscope. Based on these results, we can draw out the molecular mechanism of palmately lobed leaf morphogenesis in melon. At the same time, the palmately lobed leaf parental backbone materials will be obtained, through recessive gene PLL will be infiltrated into backbone materials of Xinjiang melon by the marker-assisted breeding technology. This study will lay the foundation for the application of palmately lobed leaf gene, and it has high theory and great application potential.

在植物叶片发育研究中还未见甜瓜裂叶形态建成分子机制的报道。我们获得的甜瓜掌状裂叶突变体bm7为开展这方面研究提供了良好的材料。前期研究获知甜瓜掌状裂叶性状受隐性单基因PLL控制，通过图位克隆技术已将PLL基因定位于只含有一个类似于ANT转录因子的候选区域内。通过高通量测序证实bm7中隐性PLL发生单碱基插入移码突变，进一步验证了前期遗传定位结果。基于此，本项目拟利用甜瓜及模式植物烟草进行PLL基因功能互补实验验证其功能；获知PLL基因时空表达规律；用凝胶阻滞和转录组测序技术确定PLL调控的下游基因, 用CRISPR/Cas9技术对筛选出下游基因进行功能验证，完善裂叶建成基因调控网络；通过电镜观察从细胞层面揭示甜瓜掌状裂叶形态建成的原因。同时，利用分子标记辅助育种技术，将该隐性基因渗入到骨干材料，获得用于新品种选育的骨干亲本和适于杂交制种厚皮甜瓜裂叶母本系，为裂叶基因的应用奠定基础。

本项目的主要研究内容及成果为：1.六个甜瓜裂叶候选基因的序列比较及生信分析：克隆了候选区域的六个甜瓜裂叶候选基因，CDS区序列比对表明，仅有MELO3C010784在两个供试甜瓜品种中存在大片段序列差异。六个候选基因所表达的蛋白的生信分析结果表明：MELO3C010784基因在bm7品种中片段缺失突变导致了其中一个AP2结构域丧失功能，可能是两个供试甜瓜品种叶型差异的主要原因。候选基因互作蛋白功能分析表明，MELO3C010784参与叶片形状的调控，因此，MELO3C010784最有可能是甜瓜裂叶相关的主效基因；2.pll基因的亚细胞定位研究：构建了两个供试品种pll基因的GFP亚细胞定位载体，注射烟草叶片，激光共聚焦显微观察显示pll基因定位于细胞膜和细胞核中；3.pll基因的差异表达分析结果表明，pll基因在JS和bm7品种中存在显著性差异，在bm7中上调表达；4.转录组学分析：差异表达基因的GO富集分析表明，bm7可能通过细胞分裂途径调控叶型差异。KEGG气泡图表明与植物激素信号转导相关的基因显著丰富，其中大多数是生长素应答基因，可能通过生长素调节叶片发育；5.pll基因启动子克隆及其生物信息学分析：克隆获得甜瓜两个供试品种pll基因启动子，将启动子的全长及5’端缺失截短片段与 GUS 基因融合，通过农杆菌介导的GUS瞬时表达系统转化烟草，组织化学染色结果表明，Cmpll基因的启动子不是导致裂叶的主要原因；6.pll基因功能验证：将pll基因构建的过表达载体采用叶盘转化法转化烟草，获得烟草阳性植株；建立了两个供试甜瓜品种的再生体系，为甜瓜的过表达遗传转化奠定基础。构建了pll基因和PDS基因的沉默表达载体，通过农杆菌介导的子叶注射法，注射PDS基因的甜瓜叶片出现部分白化表型，pll基因沉默表达后甜瓜叶片出现部分裂叶表型，表明pll基因的表达量与叶型之间具有关联性，利用分子标记辅助育种技术、高代回交再自交等手段，将pll基因渗入等甜瓜骨干材料，获得了用于新品种选育的骨干亲本和适于杂交制种厚皮甜瓜裂叶母本系。本项目的研究成果为揭示甜瓜及植物裂叶形态建成、叶型发育及裂叶相关基因的开发和利用奠定良好的基础。