大蒜体细胞胚发生的分子调控网络构建和关键基因功能分析

Garlic is the commercial vegetable species which were propagated by the vegetative propagation. Virus accumulation and degeneration of garlic species are serious, and it is difficult for the improvement and innovation of germplasm resources. Convention tissue culture has the disadvantage of low propagation coefficiency, low quality of plantlets, high hyperhydric rate and difficult to subculture. Somatic embryogenesis can solve the problems above. However, the somatic embryogenesis system of garlic is not stable and its molecular mechanism is still unclear. Based on our previous study, culture condition was optimized to establish the stable somatic embryogenesis system. Morphological and anatomical characteristics of different tissue and cell during somatic embryogenesis were analyzed to construct precise detection system. The third-generation sequencing method of full-length transcriptome was applied to get the garlic genome of the transcriptomic level. The second-generation sequencing method was applied to get the specific mRNA, lncRNA, and miRNA under the different stage of somatic embryogenesis. The molecular regulatory network of whole RNA level will be constructed. The transgenic technology of garlic will be applied to analyze the function and regulatory mode of key genes of somatic embryogenesis. This project not only can enrich the theory of plant somatic embryogenesis but also have great application value on the recovery of garlic degeneration, the industry of virus-free and rapid propagation and genetic improvement of garlic.

大蒜作为具有重要经济价值的无性繁殖蔬菜，病毒积累和种性退化严重，遗传改良和种质创新困难。常规大蒜组培技术繁殖系数低、种苗质量差、玻璃化率高、继代培养难。体细胞胚途径能克服上述不足，然而大蒜体细胞胚发生体系尚不稳定，且分子调控机理仍为空白。在我们前期大蒜体细胞胚发生研究的基础上，本项目通过优化培养条件，建立体细胞胚稳定发生体系；通过分析体细胞胚不同阶段组织和细胞的形态与解剖特征，构建大蒜体细胞胚的精确鉴定体系；利用第三代全长转录组测序技术获得大蒜转录组水平的基因组，结合第二代测序技术获得体细胞胚发生不同阶段的特异mRNA、lncRNA和miRNA，进而构建全转录水平的分子调控网络；通过大蒜转基因等技术分析关键基因在体细胞胚发生过程中的功能，明确其调控模式。本项目研究结果不仅对丰富植物体细胞胚发生机理有重要理论意义，而且对大蒜种性恢复、脱毒快繁产业化和品种改良有重要应用价值。

大蒜是重要的食用和药用蔬菜，在栽培过程中不能形成种子，靠鳞茎进行无性繁殖，而无性繁殖过程中存在着繁殖系数低、病毒积累、种性退化等问题。体细胞胚发生途径可克服大蒜无性繁殖过程中存在的不良问题。目前为止，对体胚发生的分子机理研究较少，且体胚发生过程中lncRNA-miRNA-mRNA联合作用机制的研究还未报道。本项目通过优化培养条件发现，大蒜花序轴、鳞茎盘和发芽叶基胚性愈伤组织诱导最适宜的2,4-D浓度分别是3.0、2.0、2.0-3.0 mg/L；在2,4-D浓度为1.0-2.0 mg/L时适宜胚性愈伤组织的保持和增殖；在2,4-D 0.25 mg/L +6-BA 0.5 mg/L条件下，体胚的萌发系数最高；通过形态和组织观察，精确鉴定了体细胞胚发生的外植体阶段EX、愈伤组织阶段CA、胚性愈伤组织阶段EC和体胚阶段SE，并构建了这4个重要阶段的mRNA、lncRNA和miRNA文库。在mRNA文库中，共获得125348条Unigene，通过RT-qPCR验证和激素含量测定发现， ICA1d、IBA和cZ的积累有助于促进愈伤组织的形成，高水平的生长素及其类似物和低水平的IP、tZ有利于早期胚性愈伤组织的形成，而低水平的生长素及其类似物、CTK和ABA更利于球形胚的形成；在lncRNA和miRNA文库中，分别鉴定到14680个lncRNA和219个miRNA，分析发现，44个lncRNA可以作为34个miRNA的前体，1511个lncRNA被预测为144个miRNA可能的靶点，45个lncRNA可以作为29个miRNA的eTM。通过构建以miRNA为核心的ceRNA网络发现，144个miRNA可能与1511个lncRNA、12208个mRNA结合。在体胚发生相邻阶段的DE lncRNA - DE miRNA - DE mRNA网络中，通过KEGG富集相邻阶段DE mRNA发现，体胚发生过程中生长素相关的ceRNA网络(lncRNAs-miR393s-TIR)可能在体细胞胚胎发生的整个阶段发挥作用。通过RT-qPCR进一步验证，lncRNA125175-miR393h-TIR2网络可能在体细胞胚胎发生中起主要作用。我们的研究结果极大丰富了植物体细胞胚发生相关知识，理论上填补了体细胞胚发生ceRNA调控网络的空白，为研究ceRNA网络在大蒜体细胞胚发生中的作用奠定了基础。