HW基因调控番茄果实蜡质合成的分子机理研究

Waxes are main components of tomato fruit epidermis, which play important roles in tomato fruit appearance quality and storage property. However, the molecular mechanisms regulating the biosynthesis of tomato fruit waxes still remain unknown. We previously identified hw mutant with Wo gene (published in PNAS) contained background LA3186 through EMS, which caused increased wax content. We mapped HW gene to an approximately 110-kb fragment on chromosome 10. We will isolate this gene and characterize the regulatory roles in wax synthesis based on tomato genome annotation and sequence variations between hw mutant and its background material. And then, we will conduct Y2H and ChIP-seq to screen interacting proteins and downstream targets, respectively. Combining with transgenic analysis, wax determination and RNA-seq, we intend to unfold the molecular mechanisms of tomato fruit waxes biosynthesis mediated by HW gene. In addition, we found that overexpression of Wo gene caused increased waxes in tomato fruit and its expression level was up-regulated in hw mutant, suggesting that Wo gene may be regulated by HW gene. The relationship between these two genes will be clarified using molecular biological approaches. It is well worthwhile to improve the fruit appearance quality and storage property in tomato.

蜡质是番茄果实表皮的重要组成部分，对果实外观品质和采后贮藏特性具有重要影响，但是其调控机理仍不清楚。本课题组利用EMS诱变含有Wo基因（已在PNAS上发表）的材料LA3186，筛选到一个果实蜡质增加的突变体hw。目前成功将HW基因定位于第10号染色体约110Kb的物理区间。本项目在此基础上，利用番茄基因组注释信息并结合突变体和对照材料之间的序列变异，分离HW基因并鉴定其蜡质调控功能。采用Y2H和ChIP-seq分别筛选该基因的互作蛋白和下游靶基因。利用转基因分析、蜡质测定、转录组分析等手段，解析HW基因调控番茄果实蜡质合成的分子机理。另外，本课题组发现超量表达Wo基因导致番茄果实蜡质显著增加，而且确定Wo基因在hw突变体中上调表达，说明Wo基因可能受HW基因的调控。采用分子生物学手段研究二者之间的互作关系。本研究对于揭示番茄果实蜡质合成的分子机理以及改良果实外观品质和贮藏特性具有重要意义。

番茄的贮藏、保鲜是重要的生产性问题。番茄果实角质发育和蜡质形成是影响水分散失速率的关键因素。因此，解析番茄果实角质发育和蜡质形成的调控机制具有非常重要的意义。本项目研究发现番茄多毛突变体LA3186叶片和果实中蜡质含量显著高于对照，并且蜡质合成途径相关基因CER6、PAS2等表达量上调。Wo超量株系中果实和叶片中的蜡质含量与对照相比均显著提高。Wo基因可以通过结合到CER6启动子中的L1-box来调控其表达。以上结果表明茸毛基因Wo直接结合到蜡质合成基因CER6的启动子上调控番茄蜡质合成。同时，我们发现Wo与番茄MYB转录因子SlMYB31之间存在蛋白互作。SlMYB31超量转基因株系果实和叶片中的蜡质含量均显著高于对照，而干涉株系中则与之相反，蜡质显著降低。此外，SlMYB31干涉植株果实的表型发生明显改变，与对照相比亮度要高很多，并且极易失水。Wo与SlMYB31的双突变Wo-OE/SlMYB31-RNAi和SlMYB31-OE/Wo-RNAi的蜡质含量要分别低于Wo-OE和SlMYB31-OE植株。以上结果说明Wo与SlMYB31协同调控番茄蜡质的积累。