榛子授粉控制子房分化起始的IAA信号转导机制研究

Hazelnut has a good reputation as king of nuts, and has important economic value. Normal ovary development is the base of nuts yield formation. The differentiation of hazel ovary initiates after pollination, and the growth of most ovary stops during early developmental stage. The unique mechanism of hazel ovary initiation regulated by pollination almost remains unknown. Our previous study results indicated that IAA signal transduction was involved in the regulation of ovary development in hazelnut. Female inflorescence or young fruit during different ovary developmental stages will be used as study materials in present study, and IAA and its polar transport inhibitor treatments will be carried out. After development process observation of pollen tube, pollen tube cavity and ovary, accompanied by IAA temporal and spatial localization, IAA’s regulation function during ovary development will be verified from pros and cons sides. Then, auxin response factors CoARFs clone, sequence comparison, evolutionary tree construction and their temporal and spatial expression will be performed to screen key CoARFs which are involved in the regulation of ovary differentiation and development. CoARFs’ instantaneous infection, subcellular localization, promoter methylation and genetic transformation of over expression/ inhibition expression analysis will be carried out to confirm their function during different ovary developmental stage. Chromatin Immunoprecipitation and High-Throughput Sequencing will be used to obtain CoARFs’ key target sequences, and important IAA downstream genes which regulate ovary development will be identified. A new auxin signal pathway controlling hazelnut ovary development will be constructed in present study.

榛子有“坚果之王”的美誉，有重要的经济价值。正常子房发育是产量形成的基础，榛子子房在授粉后才开始分化发育，且在早期多数发育停滞，这种独特的子房分化发育机制还不明确。经初步研究，榛子子房分化发育习性与IAA信号调控相关。本项目以榛子雌花/子房为材料，进行IAA及其运输抑制剂处理，通过花粉管、花粉管腔、子房发育进程的显微观察及IAA时空定位等分析，正反验证IAA在子房发育进程中的作用；进而开展IAA响应因子CoARFs家族成员的克隆、比对及进化树构建、时空表达特征研究，筛选调控子房分化发育的关键CoARFs，并进行瞬时侵染、亚细胞定位及过表达/抑制表达遗传转化等研究，验证其在子房发育调控中的功能；最后，通过染色体免疫共沉淀-高通量测序等研究获得关键CoARFs的靶序列信息，鉴定IAA信号通路下游直接调控子房发育的重要基因。本研究有望建立IAA调控榛子子房发育的新信号通路。

榛子有“坚果之王”的美誉，有重要的经济价值。正常子房发育是产量形成的基础，榛子子房在授粉后才开始分化发育，且在早期多数发育停滞，这种独特的子房分化发育机制还不明确。将子房发育分为未授粉雌花期（S）、子房形成初期（F）、胚珠分化期（T）和胚珠生长期（FO）等4个时期，IAA的HPLC-MS/MS分析结果表明授粉后生长素水平升高，IAA的免疫组化分析表明生长素在雌花序和子房的生长中心富集，这表明榛子子房分化发育习性与IAA信号调控相关。对早期雌花序进行了IAA及IAA运输抑制剂TIBA处理，证实榛子子房的分化被IAA促进，而TIBA抑制子房的发育分化；采用Hi-C辅助基因组组装策略，通过单分子实时测序、组装和纠错，获得了总长度为343Mb、scaffold N50为32.88mb的高质量染色体级平榛基因组，并进一步构建了包含基因组、基因编码序列、蛋白质序列和各种注释信息的HazelOmics在线数据库（HOD），在基因组水平上鉴定到14个ARF基因家族成员，差异转录组分析共找到8个ARF基因家族成员在转录水平发生表达，对不同发育阶段差异表达的关键基因ChARF3进行了基因克隆、抗体制备及免疫组织化学分析，在珠被、胚乳、子叶、胚根等部位均检测到ChARF3的表达，为ChARF3参与胚珠的发育调控提供了分子证据；进一步，通过染色体免疫共沉淀-高通量测序等研究获得ChARF3的靶序列信息，发现靶基因主要在ko01110（Biosynthesis of secondary metabolites，次级代谢产物生物合成），ko04075（Plant hormone signal transduction，植物激素信号转导），ko00940（Phenylpropanoid biosynthesis，苯丙烷生物合成）等KEGG路径上发生显著性富集，认为ChARF3通过与下游MYB、NAC、bZIP、bHLH、MADS、AP2等转录因子相结合，启动榛子子房发育过程。本研究阐明了榛子授粉控制子房分化起始的IAA信号转导机制，为榛子的丰产栽培及育种提供了重要依据。