二穗短柄草BdCO基因表达与生物学功能分析  

Analysis of BdCO gene expression and biological function in Brachypodium distachyon

作　　者：路雪萍 童伟杨 舒健虹[2] 马培杰[2] 赵德刚 王小利[2] LU Xueping;TONG Weiyang;SHU Jianhong;MA Peijie;ZHAO Degang;WANG Xiaoli(Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Guizhou Key Lab of Agro-Bioengineering,Institute of Agro-Bioengineering/College of Life Sciences,Guizhou University,Guiyang 550025,China;Guizhou Institute of Prataculture,Guizhou Academy of Agricultural Sciences,Guiyang 550006,China)

机构地区：[1]贵州大学生命科学学院/农业生物工程研究院,山地植物资源保护与种质创新教育部重点实验室,贵州省农业生物工程重点实验室,贵阳550025 [2]贵州省农业科学院草业研究所,贵阳550006

出　　处：《植物生理学报》2022年第5期889-903,共15页Plant Physiology Journal

基　　金：贵州省高层次创新型人才培养项目[黔科合平台人才(2018)5634-2和黔科合人才(2016)4003];贵州省科技计划项目[黔科合平台人才(2020)5005]。

摘　　要：CONSTANS(CO)基因在光周期途径中发挥重要调控作用。本研究旨在探究CO基因在二穗短柄草(Brachypodium distachyon)中的生物学功能。通过实时荧光定量PCR分析表明Bd CO基因在长日照、短日照、连续光照、连续黑暗和不同发育阶段都呈现明显昼夜节律并受光周期调控。将构建的目的蛋白与绿色萤光蛋白融合表达载体注射入本氏烟草(Nicotiana benthamiana)中,发现BdCO定位于细胞核,预示该蛋白在细胞核中发挥作用。利用酵母单杂交技术筛选出与BdCO启动子结合的关键转录因子BdGI,通过双荧光素酶检测进一步证实BdGI对BdCO转录有激活作用。采用酵母双杂交、双分子荧光互补和免疫共沉淀三种方法证明了BdCO蛋白与BdFKF1蛋白、BdZTL蛋白分别在体内和体外存在互作关系。综上所述,BdCO基因表达受生物钟和光周期双重调控,该基因编码蛋白可能在细胞核内行使功能。CONSTANS(CO)plays an important regulatory role in the photoperiodic pathway.This study aims to explore the biological function of the CO gene in Brachypodium distachyon.Real-time fluorescence quantitative PCR analysis showed that the Bd CO gene maintained a certain circadian rhythm and was regulated by photoperiod at different developmental stages,including long day,short day,continuous illumination,and continuous darkness.Then,the constructed target protein was fused with the green fluorescent protein expression vector,and was injected into Nicotiana benthamiana.The expression analysis result suggested that Bd CO is located in the nucleus.This indicates that the protein plays a role in the nucleus.The key transcription factor BdGI that binds to the Bd CO promoter was screened by yeast one-hybrid assay,and the activation of BdGI on Bd CO transcription was further verified by a dual-luciferase assay.The results of the yeast two-hybrid assay,bimolecular fluorescence complementation(BiFC)analysis,and co-immunoprecipitation(Co-IP)assay indicate that BdCO interacted with BdFKF1 and BdZTL in vivo and in vitro.In summary,the expression of the BdCO gene is regulated by the circadian clock and photoperiod,and the protein encoded by the BdCO gene may perform its function in the nucleus.

关 键 词：二穗短柄草 CO基因 表达分析 蛋白互作 

分 类 号：Q943.2[生物学—植物学]