棉花GhACNAT基因调控花药发育的分子机理研究

The anther development and pollen formation is the core for studying plant fertility, is a complex process from stamen primordium differentiation, anther and filament forming, anther crazing and mature pollen grains releasing, where many genes in signal transduction and metabolic pathway are involved in. The abnormal development of anther and pollen, the changes of physiological and biochemical in the process caused pollen abortion and sterility. The deficiency of biosynthesis or transduction of JA (Jasmonic Acid) inhibits the development of stamens, which affects the fertility of the male, and caused abortive fertility. The gene GhACNAT relating to anther development and pollen releasing has been identified from the male sterile mutant MS1 of Gossypium hirsutum L. The expression levels of GhACNAT gene in reproductive organs (bract, petals, stigmas and stamens) were significantly higher than the vegetative organs (roots, stems, and leaves) and fibers of different developmental stages by Real-Time PCR analysis and virus inducing gene silence (VIGS). GhACNAT gene kept significant high-level expression in flower organs of sepals, petals, stamens and stigmas. Here, we suppose that GhACNAT gene perhaps participates in JA biosynthesis and signal transduction to regulate anther development, especially pollensac opening and related genes expression in the process. In this project, three types of transgenic cotton plants will be developed, including overexpression, RNAi and knockout by CRISPR/Cas9 of GhACNAT gene. The function of GhACNAT gene will be exhibited by the comparison of phenotype and anatomy of anther in transgenic plants with wild type, including the ultrastructure, cell morphology and structure changes of the anthers, and then their fertility. Further, we will analyze the expression level of genes involved in JA biosynthesis and signal transduction, and detect the content of endogenous JA in reproductive organs between the transgenic cotton plants and wild type plants. According to the tissue specific GUS staining and GFP subcellular localization, the GhACNAT characterization is able to be further exhibited. After all, the molecular mechanism of GhACNAT gene regulating anther development and pollen formation must be exposed through this study. While the signal transduction and metabolic pathway concerning GhACNAT are revealed, the transgenic cotton plants with changed fertility are able to be developed by molecular design breeding for creation of controllable fertility lines and crossbreeding.

花药发育和花粉形成是植物育性研究的核心，从雄蕊原基分化，花粉囊和花丝形成，花粉囊开裂及成熟花粉粒释放是一个复杂的过程，有许多信号传递和代谢途径基因参与，其异常发育或其中生理生化物质改变可能造成败育。茉莉酸生物合成或感受缺陷会抑制雄蕊发育，影响雄性生育能力甚至导致不育。我们已经从陆地棉中分离一个参与花药发育的基因GhACNAT，初步研究其在花器官中特异表达，可能通过参与茉莉酸生物合成代谢和传导来调控花药发育和花粉囊开裂及相关基因的表达。本项目通过培育过量、抑制表达和基因敲出的转基因棉花并与野生型进行比较研究，分析该蛋白在花药发育时期的功能，观察其超微结构、细胞形态和育性。分析该基因参与的茉莉酸合成代谢途径中关键基因表达变化，检测内源茉莉酸含量，结合该基因组织特异性GUS染色和GFP亚细胞定位等，揭示GhACNAT基因调控棉花花药发育和花粉形成的分子机理，为棉花杂交育种提供理论和物质基础。

陆地棉(Gossypium hirsutum L.)雄性不育系突变体ms1，和野生型C312相比，花粉囊不开裂，没有花粉或微量没有活力的花粉。从雄性不育突变体ms1和陆地棉野生型C312（ms1的背景植株）的花药发育早期、中期和晚期三个不同发育时期差异表达基因中，经生物信息学分析和基因表达验证，筛选了差异表达显著的基因GhACNAT，GhACNAT属于酰基转移酶(EC 2.3.1.)家族基因，该酶家族主要催化酰基的转移，参与脂质的合成与代谢。GhACNAT基因的表达模式分析发现该基因在陆地棉XC20和C312的雄蕊、柱头等生殖器官中特异表达，且在突变体ms1花药中的表达量明显低于C312；拟南芥中启动子分析表明发现GhACNAT基因根尖分生组织，叶片和花瓣中气孔和保卫细胞，刚形成的合子胚中，在发育中花粉粒中均有表达；推测该基因在棉花花药发育过程中起着重要作用。通过农杆菌介导转基因，获得了GhACNAT-RNAi植株和基因编辑的mGhACNAT植株，其中GhACNAT基因RNAi转基因棉花株系营养器官生长无明显异常，而生殖器官出现了花粉囊不开裂、花丝变短、柱头开裂扭曲、部分花粉失活、棉铃大小形态异常等表型，通过扫描电镜观察，发现这部分花药皱缩，中空，没有内涵物，而且表面刺毛突出消失或畸形。GhACNAT-RNAi植株与对照株CK生殖器官基因表达差异，其中脂质，糖类和氨基酸的代谢过程关键酶基因表达显著改变，GhACNAT-RNAi植株中脂质的合成与代谢和茉莉酸合成相关的一些基因表达水平发生了显著的降低，经GC-MS测定的茉莉酸含量及其显著降低。验证试验中经外源MeJA（茉莉酸甲酯）处理，GhACNAT-RNAi植株中花粉囊不开裂性状得以恢复正常开裂和花粉粒的释放。结果表明：GhACNAT基因是通过控制脂质和茉莉酸的合成调控小孢子发育从而来影响棉花的育性。已经获得的GhACNAT-RNAi棉花转基因败育彻底株系，现在开展田间育种和农艺性状聚合等研究。