玉米雄性不育基因IPE1克隆与功能分析

发布时间：2018-05-02 06:12

本文选题：玉米 + 花药角质层　；参考：《中国农业大学》2017年博士论文

【摘要】：玉米(Zea mays)是一种重要的粮食作物和经济作物。雄性不育系在玉米杂交种子生产中具有重要应用价值。花药角质层和花粉外壁是花粉形成的两类保护性结构。在拟南芥(Arabidopsis thaliana)和水稻(Oryza sativa)中,部分控制花药角质层和花粉外壁发育的基因已被克隆。然而,在玉米中仅有少数几个基因被克隆,且它们的分子机制尚不清楚。通过筛选实验室Mutator突变体库,我们获得了一份雄性不育系-ipe1,应用图位克隆和分子生物学等方法,对IPE1基因进行了克隆并初步阐明了它调控玉米育性的分子机制,主要研究结果如下:(1)表型分析表明ipe1仅花药发育异常,其余器官发育无显著变化;与野生型相比,散粉期ipe1花药不能从颖壳露出,变小,萎蔫;碘-碘化钾染色证明ipe1花药无成熟花粉粒形成。(2)细胞学分析发现ipe1花药角质层和乌氏体形成受阻;突变体减数分裂是正常的,而柱状层发育异常导致孢粉素随机堆积和花粉粒外壁不规则,从而使小孢子从单核中期开始逐步降解;绒毡层相对于野生型降解提前。(3)通过图位克隆和等位性测验的方法,我们克隆了 IPE1基因,可能编码一种葡萄糖-甲醇-胆碱氧化还原酶。RT-PCR,qRT-PCR和原位杂交实验显示IPE1基因特异地在幼嫩的雄穗中表达,且在四分体和单核小孢子前期的花药绒毡层中表达量高。(4)生化分析显示突变体ipe1花药脂肪酸显著减少;与之相对应地是,RNA-Seq数据分析说明部分参与脂肪酸合成和延长、蜡质合成和类黄酮代谢的基因的表达被改变。暗示着IPE1通过参与脂类物质的合成,从而调控花药角质层和花粉外壁的形成。(5)表型分析和表达分析证明:IPE1,MS26,MS45协同调控玉米花药发育。IPE1和MS26参与花药角质层和花粉外壁的形成,MS45参与花药角质层的成熟和花粉外壁的增厚。(6)进化树和拟南芥T-DNA插入系分析显示IPE1同源基因在雄性生殖器官发育中具有部分保守的功能。综上所述,IPE1是ω-羟基脂肪酸氧化途径中关键调节因子,与MS26和MS45协同调控玉米花药角质层和花粉外壁发育。我们的研究结果不仅为玉米雄性不育化制种提供了重要的基因资源;同时也丰富了玉米脂类物质合成的遗传网络。
[Abstract]:Zea mays is an important food and cash crop. Male sterile lines have important application value in maize hybrid seed production. Anther cuticle and pollen outer wall are two protective structures of pollen formation. In Arabidopsis thaliana (Arabidopsis) and Oryza sativa (Oryza sativa), some genes controlling the development of anther cuticle and pollen outer wall have been cloned. However, only a few genes have been cloned in maize, and their molecular mechanisms are unclear. A male sterile line, -ipe1, was obtained by screening the library of Mutator mutants in laboratory. The IPE1 gene was cloned by map cloning and molecular biology, and the molecular mechanism of its regulation of maize fertility was preliminarily clarified. The main results were as follows: (1) phenotypic analysis showed that ipe1 had only abnormal anther development, while the other organs had no significant changes, compared with wild type, ipe1 anther could not emerge from glume, become smaller and wilt. Iodine-potassium iodide staining showed that ipe1 anthers had no mature pollen grain formation. (2) Cytology analysis showed that the formation of keratinocytes and urnytes in ipe1 anthers was blocked, and meiosis of ipe1 anthers was normal. The abnormal development of columnar layer resulted in random accumulation of sporopollen and irregular outer wall of pollen grains, which led to the gradual degradation of microspore from the middle stage of mononuclear stage, and the tapetum was degraded earlier than wild type by means of map cloning and allelic test. We cloned the IPE1 gene, which may encode a glucose-methanol-cholinergic redox enzyme. RT-PCRnqRT-PCR and in situ hybridization experiments showed that IPE1 gene was specifically expressed in young male ears. Biochemical analysis in anther tapetum of tetrad and premononuclear microspore showed that ipe1 anther fatty acids were significantly decreased, whereas RNA-Seq data showed that they were partly involved in fatty acid synthesis and prolongation. The expression of waxy synthesis and flavonoid metabolism genes was altered. Suggesting that IPE1 is involved in the synthesis of lipids, Thus the phenotypic analysis and expression analysis on the regulation of anther keratinocytes and pollen outer wall showed that 1: IPE1 and MS26 MS45 were involved in the development of maize anther. IPE1 and MS26 were involved in the formation of anther cuticle and pollen outer wall. MS45 was involved in the maturation of anther cuticle. Phylogenetic tree and Arabidopsis T-DNA insertion line analysis showed that IPE1 homologous gene had a partially conserved function in male reproductive organ development. In conclusion, IPE1 is a key regulatory factor in 蠅 -hydroxyl fatty acid oxidation pathway, and coordinated with MS26 and MS45 to regulate the development of horniness layer and pollen outer wall of maize anther. Our results not only provide important genetic resources for maize male sterility seed production, but also enrich the genetic network of lipid synthesis in maize.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S513

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