高色素基因SlCOL1调控番茄果实叶绿体发育的机理解析

The nutritional quality of tomato fruit is mainly determined by carotenoids, soluble solids, ascorbic acid and so on. High pigment genes play an important role in improving tomato fruit quality. We had found that the SlCOL1 gene affected the development of chloroplast of tomato fruit and leaf based on our transgenic functional analysis. Overexpression of the SlCOL1 would lead tomato fruit to high pigment phenotype. The immature fruit of SlCOL1 overexpressed transgenic lines would be dark green for more chlorophyll accumulation, and the red ripe fruit was dark red and the ascorbic acid content was significantly higher than the wild type. Otherwise, suppress the expression of SlCOL1 would decrease the accumulation of chlorophyll in the immature fruit, and the color of the fruit was lighter. Yeast two-hybrid had screened out that the nuclear transcription factor NF-YB/NF-YC and two myb transcription factors of the GARP family, GLK1 & GLK2 can interact with SlCOL1. Based on those previous studies, we will focus on the accumulation characteristics of the main nutrient components in the different development stage fruits of SlCOL1 transgenic lines. And yeast one- and two-hybrid, pull down, Co-IP, EMSA and ChIP-Sequence, and combined with transcriptome and metabolomics analysis will be performed to reveal the physiological and molecular mechanism of SlCOL1 in regulating the chloroplast development and nutrient accumulation in tomato fruit.

番茄果实营养品质主要由类胡萝卜素、可溶性固形物、抗坏血酸等决定，高色素基因对提高番茄果实品质具有重要作用。项目组前期通过转基因功能分析发现SlCOL1基因影响番茄叶片及果实叶绿体的发育，超量表达该基因导致番茄果实高色素表型，未成熟果实中叶绿素大量积累、果实深绿色，红熟期果实深红色，抗坏血酸含量显著高于对照；抑制该基因的表达则减少番茄未成熟果实中叶绿素的积累，果实颜色变浅。酵母双杂交结果显示SlCOL1与核转录因子NF-YB/NF-YC、GARP家族的两个Myb转录因子GLK1、GLK2等互作。本项目拟在前期研究基础上，开展SlCOL1转基因材料果实发育过程中各主要营养组分积累特性的研究，利用酵母单双杂、pull down、Co-IP、EMSA、ChIP-Seq和转基因功能验证等技术手段，结合转录组及代谢组学分析，初步揭示SlCOL1调控番茄叶绿体发育和营养物质积累的生理机理和分子机制。

GLK2是影响番茄果实叶绿素合成的关键基因，本项目首次研究发现，番茄高色素基因SlCOL1可以通过与GLK2直接互作，并通过影响GLK2蛋白的稳定性，调控番茄果实叶绿素的积累。通过对SlCOL1转基因纯合株系进行表型观察，发现超量株系果实绿熟期叶绿素大量积累，表现为深绿果实，叶绿素的含量极显著高于对照，红熟期果实抗坏血酸含量也显著高于对照；抑制该基因的表达则影响果实绿熟期叶绿素积累，果实颜色变浅，叶绿素的含量极显著低于对照。同时，项目组还发现，超量表达COL1株系表现出果实变小，花和果实数目减少，产量降低等表型，而RNA干扰抑制COL1表达株系恰好相反，果实变大，花及果实数目增加，增产。项目组利用酵母单杂交及GAL4/UAS方法，验证了SlCOL1直接结合SFT的基因启动子并抑制SFT基因的表达来影响花序发育，进而影响番茄产量。通过转基因功能验证，项目组确定了SlCOL1互作蛋白SlBBX24超量表达后同样可以促进番茄果实叶绿素的积累，在影响番茄果实品质方面发挥着同样重要的作用，进而为高色素育种提供新材料。项目执行以来，共培养研究生5人，其中博士研究生2人，硕士研究生3人；有1名硕士研究生进入到博士研究生阶段学习。共发表标注本项目资助的SCI论文6篇。