薄皮甜瓜CmLOX10和CmLOX18基因在香气物质合成中的功能解析

Lipoxygenase (LOX) belongs to multigene family and plays important roles in biosynthesis of volatile compounds. The applicant have searched for 18 LOXs from the melon genome database, but the function of each gene is unknown. The purpose of the project is to complete the bioinformatics analysis according to the CmLOX10 and CmLOX18 genes cloned from oriental sweet melon and verify the functional in vitro by constructing the prokaryotic expression and subcellular localization vector of the two genes. Then the overexpression vector of the two genes which are constructed through the Gateway technology are transferred into tomato to verify the functions. Volatile compounds in leaves and fruits,the two genes and related genes involved in volatiles synthesis pathway expression are measured in transgenic tomatoes to identify the function in biosynthesis of volatile compounds of the two genes. Eventually it will lay the foundation for the expression regulation of the two genes that the promoters of the two genes are cloned and followed by constructing the expression vector containing reporter gene(GUS) and transferring into melon for transient expression to identify whether the two promoters of the two genes response to fruit ripening . When the functions of the two LOXs in biosynthesis of volatile compounds are clear, it can provide a certain theoretical basis for potential using LOX genes and improve melon varieties through genetic engineering.

脂氧合酶（LOX）在植物中以多基因家族存在，在香气合成中具有重要作用。申请者从甜瓜基因组库中搜索到18个LOX，但各个成员的功能未知。本项目拟在"玉美人"薄皮甜瓜中克隆到CmLOX10和CmLOX18两个基因全长及其生物信息学分析的基础上，拟构建两个基因的原核表达载体和亚细胞定位载体进行体外功能验证。利用Gateway技术构建两个基因超表达载体并将其转入"中蔬六号"番茄中以获得稳定表达的转基因植株；测定转基因植株叶片和果实中香气成分、两个基因及香气合成途径上关键基因的表达量，以鉴定两个基因在参与香气形成中的作用；最后通过克隆两个基因的启动子，构建和报告基因（GUS)的表达载体并在甜瓜果实中瞬时表达，检测两个基因启动子活性及对果实成熟的响应，为两个基因的进一步表达调控奠定基础。明确两个LOX基因在香气物质合成中的作用，为潜在利用相应基因或者通过基因工程改良甜瓜品种提供了一定理论依据。

脂氧合酶（lipoxygenase, LOX）在植物中以多基因家族存在，广泛参与生长发育、果实成熟衰老及香气物质合成等。本项目根据从薄皮甜瓜中克隆的CmLOX10和CmLOX18基因全长，分别对CmLOX10和CmLOX18进行了原核和真核表达进而获得重组蛋白，确定了CmLOX10最适合反应pH为5.0，最适反应温度为45℃，CmLOX18的最适合反应pH为4.5，最适反应温度为25℃，两个蛋白均以亚油酸（LA）为最适底物，利用正相色谱法，结合9-氢过氧化物和13-氢过氧化物两个标准品，明确了两个重组蛋白均是13-LOX。通过瞬时转化烟草和拟南芥原生质体，分别明确了CmLOX10定位在细胞膜上，而CmLOX18定位在非叶绿体的细胞器中。分别构建了两个基因的遗传转化载体，并分别转化了番茄和甜瓜，初步验证了两个基因在果实香气物质合成中的功能，尤其是CmLOX18可能参与了C6香气物质的合成，但是与C5物质的生成无关。分别克隆了两个基因的启动子，并分别瞬时转化烟草，明确了两个基因对非生物胁迫、激素和信号物质的响应。.发表相关论文5篇，其中SCI收录4篇，中文权威期刊1篇。培养博士生3名，硕士生2名。.