大豆E3泛素连接酶基因GmHOS1a和GmHOS1b生物信息学分析和敲除载体构建
- 摘要:
- HIGH EXPRESSION OF OSMOTICALLLY RESPONSIVE GENES 1（HOS1）是植物中多种信号途径的整合因子，在植物发育、胁迫反应、光形态建成和开花调控中发挥至关重要的作用，是很有潜力的作物工程育种目标基因，但其在大豆中的功能尚未被揭示。为了揭示其在大豆中的功能，本研究利用反向遗传学，成功克隆了两个大豆HOS1基因，GmHOS1a和GmHOS1b，并通过生物信息学技术，对其蛋白结构、表达模式和功能进行了分析。进化树和结构域分析表明，GmHOS1a和GmHOS1b具有N端的环指结构域和与ELYS高度相似的保守基序，与拟南芥和水稻HOS1蛋白高度同源。亚细胞定位结果显示，GmHOS1a和GmHOS1b定位在细胞核中。Quantitative Real-time PCR（qRT-PCR）结果表明，GmHOS1a和GmHOS1b的基因表达模式相似，均在三出复叶中高度表达。48 h光周期RNA-sequencing（RNA-seq）和qRT-PCR分析表明，GmHOS1a和GmHOS1b基因的表达具有生物钟昼夜节律性，其中GmHOS1a的表达量显著高于GmHOS1b，GmHOS1a见光后表达量升高，黑暗前表达量达到峰值。为进一步探索GmHOS1a和GmHOS1b蛋白的功能，利用CRISPR/Cas9系统构建了9个GmHOS1a和GmHOS1b基因敲除载体，通过发根检测实验，筛选出6个高效工作载体，可供后续遗传转化实验使用。本研究为阐明该基因的功能和作用机理奠定了理论基础，并提供了可供遗传转化的载体材料。
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备注/Memo
国家自然科学基金（32072091）；湛江市领航计划项目（211207157080997）。