黄瓜脂氧合酶基因CsLOX02和CsLOX08在C9醛类香气合成中的功能解析

C9 aldehydes are the main aroma compounds of cucumber. LOX is the first key enzyme in the formation of aldehydes volatiles in cucumber fruit. We found that CsLOX02 and CsLOX08 had close relationship with the formation of C9 aldehydes in the cucumber, and it was assumed that they were very important in the formation of C9 aldehydes. Up to now, we didn’t know the mechanism which regulated the formation of C9 aldehydes. The relationship of expression mode of CsLOXs (which express especially in the fruit) and enzyme activities, the content of C9 aldehydes were analyzed using No.26 and 14-1 which have the big difference in the aroma volatiles. The enzyme activity and characteristics of LOXs recombinants were identified using the prokaryotic expression of CsLOX02 and CsLOX08, and the overexpression vector, RNAi vector will be constructed using the gateway technology and then they will be transformated to the cucumber inbred line 14-1 and double mutant of Arabidopsis which were lack of 9-LOX enzyme activity to identified the role of CsLOX02 and CsLOX08 and the response of LOXs to the treatment of low night temperature, the ABA and JA will be probed. The expression of LOXs, the accumulation of JA of the transformation plants were surveyed in order to analyze the molecular mechanism of CsLOX02 and CsLOX08 gene which participated in the aldehyde synthesis of cucumber.

C9醛是构成黄瓜香气的重要成分，脂氧合酶(LOX)是黄瓜醛类香气形成的第一个关键酶。前期研究发现，CsLOX02和CsLOX08与黄瓜果实C9醛类合成关系密切，推测其在C9醛类合成具有重要作用。但目前对其调控C9醛类合成的机理还不清楚。本项目拟以华北型No.26和水果型黄瓜14-1为试材，通过分析果实中表达的CsLOXs在不同发育时期的表达特征、酶活性及与C9醛类香气含量的关系、应用原核表达产物共同温浴策略研究重组酶活性及产物特征、构建CsLOX02和CsLOX08基因过表达和抑制表达载体并分别对14-1和9-LOX缺失的拟南芥双突变体进行转化，研究其在C9醛类香气合成中的作用；并在野生型和转化株中分析损伤及外源激素等对基因表达及C9醛类香气产生的调控，阐明CsLOX02和CsLOX08在黄瓜C9醛类合成中的功能，为利用LOX基因改良黄瓜尤其是水果型黄瓜的风味提供科学依据。

黄瓜果实中C9醛类物质具有花香味，C6醛类具有青草味，这两类物质含量及其比例尤其是C9醛的含量对黄瓜果实的风味具有重要的影响。脂氧合酶(LOX)是黄瓜醛类香气形成的第一个关键酶，实验表明黄瓜中13型脂氧合酶基因对C9醛的形成非常重要。项目通过分析果实发育进程中脂氧合酶基因的表达模式、酶活性变化特征及其与C9醛类香气含量的关系，PCA分析结果表明9-LOXs基因中CsLOX02及CsLOX08和C9醛生成关系密切。研究了果实及幼苗中MeJA、损伤及SA处理对CsLOXs家族基因表达和LOX酶活性的影响。不同处理及不同部位的CsLOXs家族基因成员对MeJA、损伤及SA处理的响应不同。研究分析了不同温度处理对果实香气及脂氧合酶基因表达的影响。黄瓜果实在4℃的香气总量比在25℃香气总量低且香气高峰出现的晚；25℃条件下两材料中CsLOX08、CsLOX19、CsLOX17及CsLOX09不受诱导，CsLOX22和CsLOX23上调；4℃条件下两材料中CsLOX08、CsLOX16、CsLOX19、CsLOX20、CsLOX17和CsLOX09都上调；但基因表达情况因种质材料而异；构建了原核表达载体并获得了重组蛋白，进行了CsLOX02等基因在黄瓜中的过表达和抑制表达研究，获得了阳性株；建立和优化了通过体细胞胚途径对黄瓜遗传转化的方法；建立了黄瓜胚性愈伤组织保存和恢复生长的方法；为提高和改进黄瓜的遗传转化方法奠定了技术基础，为阐明CsLOXs在黄瓜醛类香气合成中的功能及黄瓜果实风味的改良提供科学依据。