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  

Bioinformatics Analysis and Knockout Vector Construction of Soybean E3 Ubiquitin Ligase Gene GmHOS1a and GmHOS1b

作　　者：王昕 李聪 许志永 刘斌[1] 赵涛[1] 刘军[1] 李宏宇[1] WANG Xin;LI Cong;XU Zhi-yong;LIU Bin;ZHAO Tao;LIU Jun;LI Hong-yu(Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China;Shandong Dezhou Agricultual Product Quality Inspection Center,Dezhou 253000,China;Institute of Nanfan&Seed Industry,Guangdong Academy of Sciences,Guangzhou 510316,China;Heilongjiang JSBN Seed S&T.Co.,Ltd,Harbin 150028,China)

机构地区：[1]中国农业科学院作物科学研究所,北京100081 [2]山东省德州市农产品质量检测中心,山东德州253000 [3]广东省科学院南繁种业研究所,广东广州510316 [4]黑龙江省金色北农种业科技有限公司,黑龙江哈尔滨150028

出　　处：《大豆科学》2023年第2期165-174,共10页Soybean Science

基　　金：国家自然科学基金(32072091);湛江市领航计划项目(211207157080997)。

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。HIGH EXPRESSION OF OSMOTICALLLY RESPONSIVE GENES 1(HOS1) is an integration factor of several signaling pathways in plants, it plays important roles in processes such as growth and development, stress response, photomorphogenesis and flowering time regulation etc., but its function in soybean has not been revealed. By using reverse genetics, we cloned two HOS1 homologous genes in soybean, GmHOS1a and GmHOS1b, and analyzed their protein structures, expression patterns and functions by bioinformatics analysis. Through phylogenetic analysis, we found that GmHOS1a and GmHOS1b are highly conserved to Arabidopsis and rice HOS1 proteins, both of which have N-terminal ring finger domain and conserved motif ELYS-like. This suggests that GmHOS1a and GmHOS1b may have similar functions to Arabidopsis HOS1. However, subcellular localization showed that GmHOS1a and GmHOS1b proteins were only localized in the nucleus, which was different from that of Arabidopsis HOS1 protein. Therefore, we speculated that the action mode of GmHOS1a and GmHOS1b may be different from that of Arabidopsis. Further analysis showed that the expression patterns of GmHOS1a and GmHOS1b were similar, the highest expression of GmHOS1a and GmHOS1b exited in trifoliate leaves, followed by flowers and single leaves. The expression pattern of GmHOS1a and GmHOS1b were rhythmically. The expression of GmHOS1a increased after exposure to light and reached a peak before darkness. These results suggest that GmHOS1a and GmHOS1b in soybean may be involved in the regulation of circadian rhythm. To further explore the function of GmHOS1a and GmHOS1b, we constructed nine knockout vectors of GmHOS1a and GmHOS1b using CRISPR/Cas9 system, and screened out six efficient vectors through hair root experiments, which will be used in the future soybean genetic transformation. This study provides a theory basis for exploring the function of GmHOS1a and GmHOS1b, and provides vector materials for further genetic analysis.

关 键 词：大豆 GmHOS1s CRISPR/Cas9 E3泛素连接酶 亚细胞定位 生物钟 

分 类 号：S565.1[农业科学—作物学]