二穗短柄草BdFKF1基因表达、亚细胞定位及蛋白互作分析

The Expression,Subcellular Localization and Protein Interaction of BdFKF1 Gene in Brachypodium distachyon

FKF1在光周期途径中是响应蓝光节律表达的基因,在光周期途径中有重要的作用。为探索二穗短柄草BdFKF1基因的表达模式、亚细胞定位和蛋白互作关系,本研究通过RT-qPCR分析BdFKF1基因在长日照、短日照、连续光照、连续黑暗、昼夜颠倒和长日照下昼夜节律与光周期的调控;构建绿色荧光蛋白融合表达载体转化烟草观察亚细胞定位;同时运用酵母双杂筛选结合初步鉴定2个互作蛋白BdCDF1和BdGI,并用双分子荧光互补(BiFC)和免疫共沉淀法(Co-Immunoprecipitation)进行验证。RT-qPCR分析表明BdFKF1基因在长日照、短日照、连续光照、连续黑暗、昼夜颠倒和长日照下不同发育阶段都保持一定的昼夜节律并受光周期的调控。亚细胞定位显示BdFKF1基因编码的蛋白位于细胞核中;利用X-α-Gal检测到BdFKF1可能与BdCDF1和BdGI有潜在的互作关系,BiFC方法进行验证结果显示BdFKF1与BdCDF1有互作关系,BdFKF1与BdGI互作关系不明显。免疫共沉淀法检测结果表明Bd FKF1与BdCDF1和BdGI都有互作关系,以期为后续该基因的功能验证打下基础。

FKF1 is a gene expressed in response to the blue light rhythm in the photoperiod pathway,and plays an important role in the photoperiod pathway.In order to explore the expression pattern,subcellular localization and protein interaction of the BdFKF1 gene of Brachypodium distachyon.In this study,RT-qPCR was used to analyze the regulation of the circadian rhythm and photoperiod of the Bd FKF1 gene under long-day,short-day,continuous light,continuous darkness,day-night inversion,and long-day light.In order to study the expression pattern,subcellular localization and protein interaction relationship of the Bd FKF1 gene of Brachypodium distachyon,the green fluorescent protein fusion expression vector was constructed to transform tobacco observation sub cell localization.At the same time,the combination of yeast two-hybrid screening and X-α-Gal detection was used to initially identify the two interacting proteins BdCDF1 and BdGI,and verify them with Bi-molecular Fluorescence Complementation(BiFC) and Co-Immunoprecipitation(CoIP).RT-qPCR analysis showed that the BdFKF1 gene maintains a certain circadian rhythm and is regulated by the light cycle in different developmental stages under long-day,short-day,continuous light,continuous darkness,day-night inversion,and long-day light.The subcellular location shows that the protein encoded by the BdFKF1 gene is located in the nucleus.The X-α-Gal detects that BdFKF1 may have a potential interaction relationship with BdCDF1 and BdGI.The verification result of the BiFC method shows that BdFKF1 and BdCDF1 interact with each other.The Bd GI interaction is not obvious.The results of Co-immunoprecipitation showed that BDFKF1 interacted with BDCDF1 and BDGI.Real-time fluorescent quantification PCR was used to analyze the expression pattern of Bd FKF1 under different light treatments,hoping to lay the foundation for the subsequent functional verification of the gene in the study.