天女木兰MsARF5基因在后熟种子胚胎发育中的功能及其作用机制

Magnolia sieboldii is a kind of rare and endangered wild plants resources tree species which focus on the protection of ornamental, aromatic and medicinal value in oneself, It has broad development prospects. This tree is given priority to with seed propagation, incomplete development of embryos lead to seed germination and seedling emergence difficult, greatly limiting the development and application processes. So far, during after-ripening process embryo development mechanism remains unclear. Based on seed transcriptome sequencing data, depth of excavation embryo development differentially expressed genes, comparing the NCBI database and refer to the results of previous research applicants found that Auxin response factor (ARF) significantly upregulated expression with embryo development. Therefore, this project intends to start from MsARF5 gene. Through cloning, gene function, expression mechanism and MsARF5 control goals target genes, the applicant sought to determine the biological function of genes MsARF5, prove its role in the after-ripening seed embryo development and reveal the molecular mechanism of MsARF5 regulation embryo development. This research provides a scientific reference of after-ripening embryo development.

天女木兰是我国重点保护的集观赏、芳香和药用价值于一身的珍稀濒危野生木本植物资源，具有广阔的开发应用前景。该树种以播种繁殖为主，种子具深休眠特性，种胚发育不完全致使种子萌发、出苗困难，极大限制了开发应用进程。至今，对天女木兰种子后熟期间种胚发育调控机制仍不清楚。申请者在天女木兰种子转录组测序数据基础上，深度挖掘种胚发育相关的差异基因，通过NCBI数据库比对并参考项目组前期研究结果，发现生长素响应因子ARF5随种胚发育显著上调表达。因此，本项目拟从天女木兰MsARF5基因入手，通过基因克隆、基因功能、表达调控机制以及MsARF5调控的目标靶基因等方面开展研究，旨在明确MsARF5基因的生物学功能、探明其在后熟种子胚胎发育中的作用，揭示MsARF5基因调控种子胚胎发育的分子机理，为具有后熟特性种子的种胚发育调控提供科学的借鉴和参考。

天女木兰是我国国家重点3级保护野生植物，集观赏、芳香和药用价值于一身，具有广阔的开发应用前景。针对天女木兰种子种胚发育不完全、种子萌发和出苗困难，种子后熟调控机制不清楚等问题，基于前期天女木兰种子无参转录组数据，运用基因克隆、SMARTer RACE 侧翼序列扩增、实时荧光定量PCR、酶联免疫、拟南芥基因功能验证、染色体步移及第三代全长转录组测序等技术，对天女木兰MsARF5基因分子结构、时空表达、基因功能、启动子活性及目标靶基因挖掘与筛选等内容进行测试与分析。研究结果显示MsARF5基因全长2958bp，985aa，具较强亲水性，无跨膜结构；确定了适于不同组织器官内参基因3个（SrBP1，60S，B5），适于种子吸胀和层积过程内参基因4个（rps4、SrBP1、UBQ和ACT7）；明确MsARF5基因时空表达模式，该基因在种子吸胀12h和层积30d、60d表达量最高，该阶段发生了以生长素调控的后熟相关生理变化；验证了拟南芥中MsARF5基因的过表达会促进植株维管组织发育，促使苗高、地径、叶片宽度等生长指标显著增加；挖掘MsARF5调控的互作蛋白及靶基因9个，其中IAA17、BRX、AUX1等呈正相关，IAA1、IAA19、PIN3与之表达呈负相关，基因间相互作用调控种子完成后熟过程。本项目揭示了MsARF5基因调控种子胚胎发育的分子机理，为具有后熟特性种子的种胚发育调控提供科学的借鉴和参考。