烟草 NtPHYB1 感知低温信号调控种子萌发的功能与分子机制

In sowing season, temperature is the main environmental factor inhibitting seed germination in north and southwest areas of China. It is great important for crop production through improvement of germination traits under low temperature. Our previous study have confirmed that NtPHYB1 involved in temperature-mediated seed germination in tobacco, however, the function and molecular mechanism for which remains to be elucidated. In this project, the function of NtPHYB1 during seed germination under low temperature will be studied through integrating germination phenotypes of NtPHYB1 transgenic seed, temporal and spatial expression pattern of NtPHYB1 and its protein, and the results of subcellular localization for NtPHYB1. Yeast two hybrid (Y2H) will be used for screening the interacting protein of NtPHYB1, and Bimolecular Fluorescence Complementation and Coimmunoprecipitation technique will be used to confirm the results. Then the overexpressing and knock out vector for the gene of NtPHYB1interacting protein will be constructed and introduced into tobacco. Taken the transgenic seeds as the materials, germination phenotypes and contents of planthormones ABA and GA for which were analyzed, the results will clarify the interaction between NtPHYB1 and its interacting protein that regulat seed germination under low temperature through change the ratio of ABA/GA. This study will provide innovative knowledge for PHYB participating in temperature-regulated signal pathway during seed germination, and provide new ideas for molecular breeding of crops to resist low temperature.

低温是我国北方和西南地区播种季节限制种子萌发的主要环境因子，改良种子低温萌发性状对于作物生产具有重要的意义。前期我们已经发现烟草NtPHYB1可以感知低温信号调控种子萌发，但其功能和分子机制仍有待阐明。本项目将结合低温环境下NtPHYB1转基因种子萌发表型，NtPHYB1时空表达谱，NtPHYB1亚细胞定位结果，研究NtPHYB1抗低温萌发的功能。通过酵母双杂交实验筛选NtPHYB1的互作蛋白，利用双分子荧光互补和免疫共沉淀实验进行验证，构建候选蛋白基因的超量表达和敲除载体并转化烟草。以NtPHYB1及互作蛋白的转基因种子为材料，在梯度温度下观测种子萌发表型，测定种子植物激素ABA和GA含量，分析NtPHYB1感知低温信号通过其互作蛋白作用于ABA/GA调控种子萌发的分子机制。该研究将为PHYB参与的种子萌发的温度信号通路提供创新性知识积累，为作物抗低温分子育种提供新的思路。

低温是我国北方和西南地区播种季节限制种子萌发的主要环境因子，改良种子低温萌发性状对于作物生产具有重要的意义。前期我们已经发现烟草NtPHYB1可以感知低温信号调控种子萌发，但其功能和分子机制仍有待阐明。本项目以野生型种子作为对照，完成了低温环境下NtPHYB1过表达和缺陷种子萌发表型鉴定，发现野生型种子在15℃下萌发率显著高于NtPHYB1过表达种子。以低温敏感型（SX-2）、低温抗性（中烟 90）和 K326（中间型）为实验材料，利用实时定量 RT-PCR 方法研究 NtPHYB1基因在种子萌发不同发育阶段的表达特征，结果发现在SX-2种子萌发过程中NtPHYB1表达呈现随时间递增而下降的特征，而中烟90和K326种子在不同低温处理下NTPHYB1表达量未能呈现出有序规律。以K326为实验材料，完成NtPHYB1 蛋白在烟草细胞中的定位，将其定位于细胞核，以上结果有助于理解低温NtPHYB1调控种子萌发的功能。在此基础上，完成酵母双杂交CDNA文库构建和诱饵载体构建，筛选到23个候选蛋白，通过双分子荧光互补和免疫共沉淀实验对其中2个蛋白SCE1-like和IAA8-like与NtPHYB1的互作情况进行验证，发现其分别存在强和弱的互作。构建了SCE1-like和IAA8-like的超量表达和敲除载体并转化烟草，获得转基因种子材料，在低温条件研究其萌发表型，结果发现SCE1-like负调控烟草种子萌发。进一步研究表明NtPHYB1调控烟草种子萌发是通过改变ABA/GA的比值。该研究将为PHYB参与的种子萌发的温度信号通路提供创新性知识积累，为作物抗低温分子育种提供新的思路。此外，本研究共发表论文 9 篇，其中 SCI 论文 5 篇，获得发明专利 2 项，已培养研究生2名，正在培养研究生4名。