细胞周期调控蛋白UVI4调控番茄果皮发育特征的分子机制

Tomato pericarp including exocarp, mesocarp and endocarp is the major part of fruit, which determines the fruit quality. Pericarp characteristics are formed during the fruit growth and development which is tightly associated with cell cycle progression. Endocycle which has S phase but no M phase widely exists in plant cells and is usually associated with cell expansion. This project is to clarify the relationship between the cell cycle regulator SlUVI4 and pericarp development, based on the analyses of SlUVI4 transcription, endocycle in pericarp cells, and physical and chemical characteristics of pericarps at the different stage of fruit development in several tomato varieties. Analyses on SlUVI4 overexpression lines and knockout mutants generated in this project will facilitate the understanding of SlUVI4 roles in the regulation of endocycle in pericarp cells and pericarp development. Protein interactions between SlUVI4 and APC/C (Anaphase-promoting complex/Cyclosome) components will be tested. Moreover, proteomic and transcriptome analyses will reveal the accumulation change of cell cycle related proteins and transcriptional change of genes associated with pericarp development in SlUVI4 overexpression lines and knockout mutants, respectively. Taken together, this project will help us understand the function of SlUVI4 in the regulation of endocycle through the interaction with APC/C and consequent transcription of pericarp development related genes and pericarp development. All these data could be used to improve the tomato fruit quality in the future.

果皮（含外果皮、中果皮和内果皮）是番茄果实的最主要部分，其特征也是决定果实品质的最主要因素。果皮特征取决于果实生长发育，而细胞周期对果实发育具有关键调控作用。核内周期是有DNA合成期而无分裂期的细胞周期形式，对细胞体积的增大具有重要作用。本项目将揭示细胞周期调控蛋白SlUVI4在果实发育不同时期和不同番茄品种中的表达变化、果皮细胞的核内周期程度以及果皮的物理化学特征，并在此基础上明确SlUVI4与果皮发育之间的相关性。同时，利用SlUVI4过表达植株和缺失突变体，探讨SlUVI4对果皮细胞核内周期以及果皮发育特征的调节，并通过蛋白质互作分析，明确SlUVI4与APC/C蛋白复合体之间的互作关系；此外，利用蛋白质组学和转录组学分别研究SlUVI4过表达与缺失对细胞周期相关蛋白积累的影响和对果皮发育相关基因转录的影响，明确SlUVI4影响果皮发育特征的调控网络，为番茄果实品质改良提供理论基础。

细胞周期是番茄果皮发育特征形成的基础，包括有丝分裂周期和核内周期，分别影响着细胞的分裂增殖和延伸增大，而细胞周期调控蛋白SlUVI4在有丝分裂周期向核内周期转换过程中起着非常重要的调控作用。摸清番茄SlUVI4同源基因与果皮糖酸代谢之间的联系，明确SlUVI4基因在番茄果皮细胞分裂与延伸的作用，阐明SlUVI4基因调控番茄果皮发育特征的分子机制，对改善番茄理化性质和培育高品质番茄品种具有重要价值。.本项目调查了番茄SlUVI4同源基因在不同番茄品种果实发育不同时期的表达变化，探讨了SlUVI4表达与番茄果皮物理化学特征之间的联系，利用基因编辑技术产生了SlUVI4敲除突变体，分析了SlUVI4基因对番茄种子萌发后下胚轴伸长生长的影响，研究了SlUVI4基因突变对果皮细胞分裂与延伸以及糖酸代谢的影响，发掘了受SlUVI4基因影响的细胞周期及糖酸代谢相关基因，筛选出了与SlUVI4互作的APC/C蛋白复合体激发子。.结果表明，番茄SlUVI4基因在各个组织中均有表达，在果实发育的不同阶段呈现先高后低的特征。补温与生长素处理均可以抑制番茄下胚轴中SlUVI4基因表达，促进下胚轴细胞伸长生长。以番茄栽培种Heinz1706为母本，利用基因编辑技术产生2个不同突变类型的sluvi4突变体，突变体失去了对补温和生长素的敏感性。sluvi4突变体果实大小、重量与野生型没有显著差别，但是果皮厚度与中果皮细胞大小明显高于野生型。流式细胞分析显示，番茄果实由绿熟期向红熟期过渡过程中，中果皮细胞核内周期显著增加，而sluvi4突变体的核内周期程度显著高于野生型。sluvi4突变体果实与野生型相比转色时间早且短，代谢组分析显示突变体的糖酸比及番茄红素含量接近野生型的2倍。转录组分析显示sluvi4突变体与野生型相比红熟期果实中果皮中5479个基因表达上调，1433个基因表达下调，其中与淀粉、糖代谢、类胡萝卜素合成、次生代谢物合成等相关的基因表达有明显表达差异。酵母双杂、双分子荧光互补及免疫共沉淀实验筛选鉴定了SlUVI4与APC/C激活子SlCCS52的互作，明确了SlUVI4通过影响APC/C活性来调控番茄果皮发育特征的分子机制，为高品质番茄育种奠定基础。