玉米蜡质相关突变体glossy6的基因克隆及功能探析

发布时间：2018-08-27 16:42

【摘要】：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,对植物生存生长发育具有重要的生理及生态学功能,主要表现在植物抗旱性、抗病虫害及抗射线等生物及非生物胁迫的防护机制中,并在水生植物向陆生植物进化过程中起到了重要的意义。目前对植物表皮蜡质的研究,主要围绕其蜡质生物合成代谢以及组成成分的研究中,如玉米、高粱、大麦、水稻、模式生物拟南芥以及苔藓类植物中报道的蜡质相关的研究；而对其在抗生物非生物胁迫机制,以及参与蜡质生物合成代谢过程中基因功能的研究还不是很多；而克隆这些蜡质相关基因是解释整个蜡质生物合成及抗性的关键。近年来随着第二代测序技术的日臻成熟及重要作物基因组测序的完成,基因定位及克隆方法不断改进。本研究以玉米蜡质生物合成代谢相关突变体glossy6为试材,构建了F2代遗传分离群体；转录组测序BSR-Seq分析将glossy6定位在3号染色体113.5-146.7 Mb的区间内；对至少6个等位的glossy6的Mu转座子插入突变体的Seq-Walking全基因组范围Mu旁侧序列检测证明：glossy6, GRMZM2G139786(Chr3:127,513,712-127,514,561 bp),编码一个DUF(Domain of Unknown Function)538蛋白,该基因由850个碱基的一个外显子组成,编码224个氨基酸的蛋白。基于以上研究基础,本研究试图从glossy6参与的可能的pathway来对其功能进行解析,主要工作如下：1、glossy6和玉米中其他已经克隆的及未克隆的glossy突变体相似,均表现出明显的苗期叶片表皮蜡质缺乏,且glossy6较其他突变体表型更明显,表现为叶片翠绿发亮(而正常玉米叶片表面呈灰绿色,角质层明显覆盖有一层蜡质)；在常温下离体叶片失水速度较野生型快,证明其叶片通透性大于正常野生型。2、叶片表皮扫描电镜SEM观察表明glossy6突变体叶片表面蜡质晶体明显减少；投射扫描电镜TEM观察表明glossy6突变体细胞内部具有针状晶体存在,我们推测是由于细胞内微粒体合成的脂类物质未能正常运输到细胞外,进而导致叶片表皮蜡质减少(杜依聪工作)。3、基于BSR-Seq以及Seq-Walking的方法克隆了glossy6 (GRMZM2G139786),编码一个未知功能的DUF538蛋白,同源进化树分析表明GLOSSY6在玉米中有一个相似度较高的同源旁系基因,其他禾本科作物中同源性较高,而在拟南芥等双子叶植物中同源基因很少。4、基于蜡质积累表型结果,我们对glossy6突变体及其对应野生型苗期叶片表皮及角质、以及微粒体蜡质检测,GC-MS液相质谱分析这几部分组分中脂类物质的成分及含量发现：玉米苗期(出苗后6-7天)叶表皮蜡质主要以碳原子大于等于32的醛类和初级醇类组成；glossy6突变体相对于正常野生型苗期叶片表皮蜡质含量明显降低；主要表现为初级脂肪酸、初级烷烃、初级醛类、初级醇及蜡质前体成分的降低,尤其是初级醇类和醛类降低比较明显。微粒体蜡质检测结果表明突变体内蜡质较野生型积累,但积累程度远远低于表皮蜡质中的,因此可以肯定的说,gl6突变体较野生型表皮蜡质减少而从某种程度上说,可能是glossy6突变体蜡质运输到外面的少了,但也不排除其生物合成的蜡质也是降低的。5、RNA-Seq差异表达基因结果分析表明,glossy6和正常野生型中均表达的24,426个基因中,glossy6相对于正常野生型53个显著基因表现为显著下调表达(log2FC=-2; P value
[Abstract]:As the last self-defense line between plants and the external environment, plant epidermal wax layer has important physiological and ecological functions for plant survival and development. It is mainly manifested in plant drought resistance, disease and pest resistance, radiation resistance and other biological and abiotic stress protection mechanisms, and in the evolution of aquatic plants to terrestrial plants. Current studies on wax biosynthesis and its components in plant epidermis, such as maize, sorghum, barley, rice, model organism Arabidopsis and bryophytes, as well as the mechanisms of wax resistance to abiotic stress, and In recent years, with the development of second-generation sequencing technology and the completion of important crop genome sequencing, gene mapping and cloning methods have been improved. The F2 population was constructed from a mutant glossy 6 related to waxy biosynthesis and metabolism in maize; glossy 6 was located in the region of 113.5-146.7 Mb on chromosome 3 by transcriptome sequencing BSR-Seq analysis; Mu transposon insertion of mutant glossy 6 at least 6 alleles was sequenced in the Mu-Walking genome-wide side sequence. Detection showed that glossy 6, GRMZM2G139786 (Chr3:127,513,712-127,514,561 bp) encoded a DUF (Domain of Unknown Function) 538 protein, which was composed of an exon of 850 bases encoding a protein of 224 amino acids. Based on the above study, this study attempted to improve the function of glossy 6 through a possible pathway involving glossy 6. The main results were as follows: 1. Glossy 6 was similar to other cloned and uncloned glossy mutants in maize, and showed obvious wax deficiency in seedling leaf epidermis, and glossy 6 was more obvious than other mutant phenotypes, showing bright green leaves (while normal maize leaves showed gray green surface, cuticle covered with obvious). The dehydration rate of the isolated leaves was faster than that of the wild type at room temperature, indicating that the leaf permeability was higher than that of the normal wild type. It was speculated that the lipids synthesized by intracellular microsomes could not be transported out of the cell normally, which led to the reduction of leaf epidermal wax (Du Yicong works). 3 Glossy 6 (GRMZM2G139786) was cloned based on BSR-Seq and Seq-Walking methods, encoding an unknown function of DUF538 protein. Homologous evolutionary tree analysis showed that GLOSSY 6 was in maize. There is a homologous paragenic gene with high similarity. Other gramineous crops have high homology, but few homologous genes are found in dicotyledons such as Arabidopsis. Based on the results of wax accumulation phenotype, we detected the epidermis and keratin of glossy 6 mutant and its corresponding wild type seedlings, and microsomal wax, GC-MS liquid chromatography mass spectrometry. The results showed that the main components of leaf epidermal wax were aldehydes and primary alcohols with carbon atoms greater than or equal to 32 at seedling stage (6-7 days after seedling emergence); the content of leaf epidermal wax in glossy 6 mutant was significantly lower than that in normal wild type seedling stage; the main manifestations were primary fatty acids and primary alkanes. The decrease of hydrocarbons, primary aldehydes, primary alcohols and waxy precursors, especially primary alcohols and aldehydes, was more obvious. The results of microsomal wax test showed that the accumulation of wax in the mutant was much lower than that in the wild type, but the accumulation of wax in the epidermis was much lower than that in the epidermis wax. Therefore, it is certain that the gl6 mutant was less than that in the wild type epidermis wax and The results of RNA-Seq differential expression gene analysis showed that glossy 6 was significantly lower than that of the normal wild type in the expression of 24,426 genes. Expression (log2FC=-2; P value)
【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2016
【分类号】：Q943.2

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