大葱蜡质合成关键酶基因KCS的克隆及功能分析

Allium fistulosum is one of the traditional vegetables and condiments in China. We found a glossy mutant glbg which has a little cuticular wax on epidermal cells in our field, it has strong assistant to thrips, so, it is not only an excellent anti-thrips germplasm resource, but also a best study material for synthesis mechanism of wax. By metabolism and transcriptome analysis of cuticular wax in Allium fistulosum in earlier studies, we found that the waxy components in mutant have changed, the key genes KCS which control wax ingredients has changed its sequence and expression levels, so we speculated that the KCS gene was the key factor which led to epidermal cells less covered by wax in mutant. The material was named as Allium fistulosum mutant glbg. We researched the metabolic properties of waxy in morphology and physiology biochemistry. KCS gene function was confirmed by yeast heterologous expression systems and Arabidopsis mutants complementary technologies; KCS gene transcription factors was researched by yeast one-hybrid technology; KCS gene regulation systems was researched by combining existing transcriptome analysis; F2 genetic population was constructed by Allium fistulosum wild-type and mutant green hybrids; development of molecular markers according to KCS gene sequence variability to confirmed linkage between KCS genotype and waxy phenotype. This accomplishment of this project will elucidate the molecular mechanism of KCS gene regulation of wax, reveal the formation mechanism of less wax mutant glbg, provide a theoretical basis and technical support for wax metabolic research and anti-thrips breeding on Allium fistulosum.

大葱是我国传统的蔬菜和调味品之一。本课题组发现一株少蜡突变体glbg。该材料既是优异的抗蓟马种质资源，又是研究蜡质合成机制的最佳材料。前期开展的表皮蜡质代谢组和转录组分析，发现突变体中蜡质组分变异，控制蜡质成分的关键基因KCS基因序列和表达水平均发生改变，推测KCS是导致突变主要原因。本项目以glbg突变体为试材，结合形态学、生理生化学对蜡质代谢特性进行研究，利用酵母异源表达体系及拟南芥突变体互补验证KCS基因的功能，通过酵母单杂技术挖掘调控KCS基因的转录因子，结合已有的转录组分析结果，建立KCS基因的调控网络；根据KCS基因的序列变异开发设计分子标记，利用野生大葱和突变体构建F2遗传群体，验证KCS基因型与蜡质表型的连锁关系。本项目的完成将阐明KCS基因调控蜡质合成的分子机制，揭示大葱glbg突变体形成机理，为葱类蔬菜蜡质代谢研究及抗蓟马育种提供理论依据和技术支持。

大葱是我国传统的蔬菜和调味品之一。蓟马是危害大葱最严重害虫之一。表皮蜡质与抗蓟马性相关，该课题的研究具重要的理论意义和应用价值。.本项目主要研究内容：以glbg突变体为材料，观察glbg的形态学特征、表皮结构；分析glbg叶片生理、生化特性；克隆glbg相关的候选基因KCS基因，并利用F2群体验证；构建KCS基因表达载体并转化酵母，分析其在脂肪酸合成过程中的作用；构建植物表达载体转拟南芥突变体，分析KCS基因在蜡质合成过程中的作用，并进行亚细胞定位；利用酵母单杂技术分离KCS基因的转录因子，并分析大葱蜡质合成调控机制。.通过课题的实施，取得了如下重要结果：遗传分析显示GLBG表型为单基因隐性突变导致；扫描电镜和透射电镜结果显示，GLBG叶表皮蜡质层比BG薄，蜡质晶体厚度小、积累少，蜡质运输途径正常；GLBD和BG叶表皮蜡质的含量与组分差异显著，大葱的主要蜡质成分三十一烷-16-酮GLBD极显著减少；GLBG与BG叶绿素含量无显著差异，气孔密度、气孔导度；非气孔性失水率、蒸腾速率无显著性差异；LBG的净光合速率极显著高于BG。BG和GLBG叶片中蜡质合成相关基因的表达中，KCS6基因存在差异显著两者核苷酸序列在282、931、959、 1106和1114bp处发生SNP。氨基酸序列比较结果显示它们之间存在302、311、360和363位氨基酸存在不同；利用F2代群体100个单析，验证显KCS基因在有蜡和少蜡性状在CDS的第1106和1114位存在SNP；AfKCS6基因亚细胞定位于内质网上；酵母异源表达AfKCS6基因结果显示，GLBG与野生型之间有差异，但不差异不显著。AfKCS6基因能互补拟南芥KCS突变体；构建AfKCS6基因RNAi载体，转化大葱，获得转化植物，叶表皮蜡质减少；酵母双杂交技术双分子荧光互补实验LUC实验证实：AfKCS6与AfCER2-like1存在互作关系；克隆CER2-like1基因，且GLBG在该基因开放阅读框第528位缺失C，导致移码突变。.本项目的完成将阐明glbg突变体形成的分子机制，为葱类蔬菜抗蓟马育种提供理论依据和技术支持；对其它相关作物的蜡质分子设计遗传改良和遗传操纵也具有重要的理论和实践意义，也将为探明蜡质代谢调控机制提供参考依据。