Functional Analysis Of The Brachypodium Distachyon FKF1 Gene And Its Application In Rice Germplasm Innovatio

Plant flowering is a key stage in plant growth and development,jointly controlled by many pathways.FKF1 is one of the important genes in the photoperiod regulation of plant flowering.In the past,many studies on the role of FKF1 in photoperiodic regulation of flowering were conducted in the model plant Arabidopsis thaliana.Compared with monocotyledonous grasses,there are differences in the mechanism of photoperiod regulation.As a gramineous model plant,Brachypodium distachyon,the explanation of its photoperiodic regulation mechanism has important reference value for the research of gramineous plants and can provide a feasible research direction for the photoperiodic adaptive breeding of gramineous plants.In this study,Brachypodium distachyon "Bd21" was used as material.To analyze the functional role of BdFKF1 in photoperiodic regulation of flowering,The expression patterns of the BdFKF1 gene were analyzed by RT-q PCR under different light treatments(long day: 16 h light/8 h dark,short day: 8 h light/16 h dark)and long day at different phenological stages(seedling stage,tillering stage,booting stage,heading stage).The BdFKF1-GFP fusion expression vector was constructed to observe the subcellular localization.The interaction between BdGI and BdCDF1 proteins and BdFKF1 was determined by the yeast two-hybrid technique and further verified by BiFC and Co-IP techniques.Yeast single hybridization indicated that BdFKF1 could not directly regulate the BdCO gene.To verify the biological function of BdFKF1,transcriptional sequencing of the BdFKF1 gene overexpression plants and mutant plants was performed using RNA-Sequencing technology.Finally,BdFKF1 transgenic rice plants were created through genetic transformation experiments,providing a theoretical basis for artificial regulation of rice flowering time and rice germplasm innovation.The main research results are as follows:(1)Analysis of expression pattern of BdFKF1 geneThe expression of the BdFKF1 gene is regulated by photoperiod and the circadian clock,and the expression level of the BdFKF1 gene fluctuates the most atIVthe booting stage under long sunshine.(2)Subcellular localization and interaction analysis of the BdFKF1 proteinSubcellular localization showed that the BdFKF1 protein was in the nucleus.Yeast two hybrid analysis showed that the BdFKF1 protein interacted with the BdGI protein and the BdCDF1 protein,and further confirmation of the interaction was obtained through BiFC and Co IP experiments.Yeast single hybridization experiments have shown that the BdFKF1 protein cannot bind to the promoter of the BdCO gene and cannot directly regulate the transcription of BdCO.(3)Study on regulation of flowering stage by BdFKF1 geneOver-expressed plants of BdFKF1 gene showed early flowering phenotype,while mutant plants of BdFKF1 gene showed late flowering phenotype.Transcriptome sequencing results showed that the mutation of the BdFKF1 gene had a great effect on transmembrane transport,cell membrane composition,and membrane formation,while the overexpression of the BdFKF1 gene mainly affected protein synthesis.BdFKF1 not only participates in the regulation of flower formation through the photoperiod pathway but also participates in various physiological processes such as plant hormone signal transduction and regulation of plant photomorphogenesis.This indicates that the pathway of BdFKF1 regulating flowering may not be a single linear pathway of GI-FKF1→BdCO→BdFT,but rather a complex and massive regulatory network.(4)Research on new germplasm creation of transgenic riceThrough Agrobacterium tumefaciens transformation,the BdFKF1 gene was transferred into rice.The photoperiod-related genes OsGI,OsCDF1,OsHd1,OsHd3 a,and OsEhd1 in rice were selected for a RT-q PCR experiment.Because these genes have circadian rhythm,13 time points within 48 h were selected for the experiment.The results showed that,compared with wild-type rice,the expression of those genes in BdFKF1 transgenic rice was affected.Among them,OsCDF1 and OsHd3 a were the most affected,with 9 of the 13 time points showing significant differences.Analysis of the circadian rhythm of gene expression showed that there was no significant change in the circadian rhythm of gene expression except for OsCDF1.Compared to wild-type rice,the circadian rhythm of transgenic rice fluctuated more;that is,the level of gene expression changed more in a day.The circadian rhythm of the OsCDF1 gene changes significantly.The expression peaks of the OsCDF1 gene in wild-type rice appear in ZT1 and ZT9,while in transgenic rice,the expression peaks of the OsCDF1 gene appear in ZT1,ZT7,and ZT13.