玉米蜡质相关突变体glossy6功能及耐旱机制的研究

Epicuticular waxes are essential to most of the aerial plants since it directly exchange with the outside environment. Especially protects the plants from the water loss, UV irradiation as well as the bacterial, fungi and insects. Here we found and cloned the waxes related maize glossy6(gl6), which show drought sensitivity under the drought stress. Further SEM (Electronic scanning electron microscopy) and TEM (transmission scanning electron microscope) analysis, combine with the epidermal wax analysis in gl6, indicated that the waxes content is significantly decreased in gl6 mutant. gl6, encodes a DUF538 protein, was a new wax gene which has not been studied yet. Based on above research, we are aiming to study the function of gl6 by complementary assay in Arabidopsis, RNAi in normal maize inbred line, as well we construct the Mu insertional additional allele for gl6. Besides that, we expected to study the spatio-temporal expression pattern of gl6 via subcellular position, GUS staining and RNA-Seq analysis. Meanwell, we will conduct the Y2H, BIFC and other related technology to study the interacted protein of GL6. Based on above work, we expected to lay the foundation of the waxes biosynthesis pathway as well as the drought resistance mechanism in maize and other crops.

表皮蜡质是绝大多数陆生植物和外界环境交流的屏障，对植物自身有着保护作用，尤其是防止非气孔性水分散失及病虫害、紫外射线等伤害。本研究发现并克隆了一个玉米苗期蜡质显著减少的突变体glossy6(gl6)，在干旱条件下该突变体较正常野生型玉米敏感。经表皮蜡质的电子扫描及透射扫描观察，结合突变体表皮蜡质的初步检测，证明该突变体较野生型叶片表皮蜡质明显减少。gl6编码一个新的未知功能的DUF538蛋白。本项目拟在现有基础上，对目标基因进行转基因互补验证，并构建等位的玉米Mu转座突变体及RNAi干扰突变体对其进行验证。通过亚细胞定位、共定位以及以GUS染色、RNA-Seq分析研究其时空表达特异性；通过酵母双杂交、BIFC等技术研究其互作蛋白并研究其功能。通过以上工作，为研究玉米叶片表皮蜡质积累，以及glossy6在玉米苗期抗旱机制中的作用奠定一定理论基础。

植物表皮蜡质层作为植物与外界环境交替的最后一道自我防护的防线，在水生植物向陆生植物进化过程中起到了重要的意义，蜡质的存在提高了植物的保水能力及对其他生物非生物胁迫的耐受力。关于拟南芥等模式植物表皮蜡质调控、蜡质生物合成及胞外转运相关的基因已有很多研究及报道。玉米中已发现的和玉米叶表皮蜡质相关的突变体超过35个，但被克隆并深入研究其分子生物学功能的却是少数。但往往单子叶和双子叶植物间同源基因功能有所差异，且起作用的器官也不尽相同，因此研究玉米中叶片蜡质合成及代谢机理，有助于深入了解玉米抵御外界生物及非生物胁迫机制，并为抗逆育种奠定一定的理论依据。本研究发现了一个在玉米苗期控制叶表皮蜡质代谢相关的突变体glossy6(gl6)。对突变体叶表皮细胞膜透性、叶片保水及水分散失速率、叶绿体浸提率、蒸腾、干旱及复水实验表明突变体较野生型叶片保水性降低，蒸腾、水分散失较快，干旱较敏感。蜡质含量及成分分析表明Gl6基因的突变导致超长链脂肪酸（VLCFA）以及其衍生物的含量减少。通过BSR-Seq及Seq-Walking两种高通量测序分析方法克隆了Gl6（GRMZM2G139786），编码一个DUF(Domain of Unknown Function)538蛋白，基于Gl6可能参与蜡质代谢途径的功能，进行了亚细胞定位及共定位，发现该基因在内质网、囊泡膜、高尔基体、细胞膜等各个亚细胞器，通过影响玉米蜡质的生物合成、胞内转运、胞外运输等各个环节从而影响表皮蜡质的分泌和积累。