海岛棉显性无腺体基因Gl2e的克隆及功能验证

Cottonseed is rich in protein, fat, carbohydrate, cellulose and mineral elements and so on, yet it is underutilized due to the presence of gossypol, a binaphthyl terpene in pigmented glands throughout nearly whole plant and is toxic to human and non-ruminant animals. The content of secondary metabolite is closely related to the quantity of storage organ just as gossypol and pigment gland. Although the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been investigated at molecular level. What kind of regulatory relations between them? These are the urgent need to explore and resolve important issues. Gl2e was found the only one dominant glandless gene of cotton which control pigment gland. In the preliminary work,the Gl2e gene was fine-mapped using a large scale segregating population and a BAC clone with Gl2e gene was contained by genomics,bioinformatics methods and techniques. The BAC was full-length sequenced and analyzed with the function of genes annotated. In this study, we will further study of these ORFs and identify the real candidate Gl2e gene by characteristics of gene expression and transgenic method. Cloning and functional identification of Gl2e can help us further study the molecular mechanism of pigment gland, relations between gossypol and pigment gland, gene network of gland regulation. On the other hand, it will also provide a significant candidate gene for cultivating cotton variety with high content gossypol in plant vegetative tissue instead of existing in cotton seed or very low content gossypol by gene engineering.

棉酚具有一定的毒性，其存在极大地限制了棉籽中的丰富的棉籽蛋白及油脂资源的有效利用。棉酚又与腺体的存在密切相关。然而腺体发生形成的分子机制是什么？腺体发育与棉酚之间究竟是一种什么样的调控关系？这些都是亟待探索和解决的重要问题。Gl2e基因是目前发现的唯一一个控制腺体有无的显性单基因。在前期研究中，我们运用基因组学、生物信息学结合功能基因组学研究方法和技术，针对该控制棉酚腺体性状的关键基因Gl2e，构建了大规模的遗传分离群体，进行了基因的精细定位,获得了包含Gl2e基因的BAC克隆并获得了BAC全长序列。在此基础上，本研究将进一步分析本研究我们将对候选ORF进行深入研究，通过基因表达分析、染色体定位，最终克隆出Gl2e基因并进行功能验证。本研究为揭示腺体形成的分子机理，最终创制棉籽中棉酚含量低而植株其他部位棉酚含量高的新材料，为棉花低酚育种提供理论与材料基础。

棉酚具有一定的毒性，其存在极大地限制了棉籽中的丰富的棉籽蛋白及油脂资源的有效利用。腺体是棉酚的主要存储器官。棉酚又与腺体的存在密切相关。然而腺体发生形成的分子机制是什么？腺体发育与棉酚之间究竟是一种什么样的调控关系？这些都是亟待探索和解决的重要问题。Gl2e基因是目前发现的唯一一个控制腺体有无的显性单基因。在本研究中，利用显性无腺体突变体海1，我们成功克隆了一个控制腺体形成的关键基因Gl2e,该基因属于bHLH类转录因子。病毒介导的沉默实验证明，当Gl2e基因的表达受到抑制后，在新生组织茎和叶片中腺体消失。序列分析发现，在单节隐性突变体材料中，gl2和gl3发生了单碱基的插入导致蛋白翻译提前终止。有腺体材料和VIGS干扰后的无腺体材料的转录组差异分析发现，次生代谢异戊二烯代谢途径中有相当多的基因在干扰后下调表达，棉酚合成受阻。下调表达基因显著富集在次生代谢、茉莉酸信号和WRKY转录因子家族。棉酚合成途径中的基因如WRKYs, TPSs等在干扰叶片中显著下调表达，表明腺体与棉酚生物合成之间存在密切联系。我们推测，虽然腺体的形成与棉酚的生物合成是两条独立的生物学途径，但是，他们之间也存在一定的分子联系：腺体形成基因Gl2e可能通过作用于茉莉酸途径中的JAZ或WRKYs，调控腺体细胞的分化；另一方面Gl2e作用于GaWRKY1调控TPS基因的表达，调控棉酚的生物合成。腺体形成关键基因的克隆将为植株有腺体种子无腺体棉花新材料的培育提供重要的候选基因。