Functional Analysis Of PheFKF1 Involved In Flowering Regulation In Phyllostachys Edulis

The flowering mechanism study of bamboo is a heat topic and difficult problem in biological research.Bamboo is a perennial disposable flowering plant with unique flowering characteristics.There are no obvious signs before most bamboos flowering,and large areas of bamboos usually die after flowering,resulting in serious economic losses.The study of the molecular regulation mechanism of bamboos flowering is of high theoretical significance and potential economic value.F-box gene family is one of the largest gene families in plants which is widely involved in plant growth and development.FKF1(flavin-binding,KELCH REPEAT,F-box 1)is mainly involved in plant flowering regulation and plays an important role in photoperiod pathway and gibberellin pathway.In our paper,the FKF1 homologous gene was obtained by homologous comparison based on the bamboo genome database and named as PheFKF1.The bioinformatics analysis and cloning and functional research of key genes were carried out systematically,providing a basis for exploring the flowering mechanism of bamboo.The main results are as follows:1.Bioinformatics analysis of PheFKF1 gene family in moso bambooA total of 5 homologous genes belonging to ZTL/FKF1/LKP2 gene family were identified in the genome of moso bamboo by homology comparison and screening of conserve domains.They were named as PheFKF1,Phe ZTL1-1,Phe ZTL1-2,Phe ZTL1-2 and Phe ZTL2-2 respectively.Domain analysis showed that LOV,F-box and Kelch repeats conserved domains were found in all five genes.Phylogenetic tree analysis showed that FKF1 gene was conserved among above species.In addition,the analysis of promoter action elements showed that the promoter regions of the five genes contained different hormone response elements in addition to the core elements,such as conservative light response elements and promoter enhancement regions.Among which,the promoter of PheFKF1 contained GA response elements,suggesting that it might be involved in the GA response pathway.2.Expression analysis of PheFKF1 in moso bambooThe expression level of PheFKF1 was the highest in the leaves at the early flowering stage of moso bamboo,which was much higher than that of non-flowering samples,suggesting that PheFKF1 might play a certain role in the flowering initiation process of moso bamboo.Analysis of circadian rhythm expression patterns showed that the expression of PheFKF1 was highest at 14: 00 PM and 18: 00 PM,indicating a significant circadian rhythm.Subcellular localization results showed that PheFKF1 was mainly localized in the nucleus.Transcriptional activation assay confirmed that PheFKF1 was a transcription repressor.3.Functional analysis of PheFKF1Overexpression of PheFKF1 can reduce bolting time and reduced the rosette number in Arabidopsis,and 35S::PheFKF1 can restore the late flowering phenotype of fkf1 mutant,which proves that PheFKF1 can indeed regulate flowering.According to q RT-PCR results,the expression levels of CO,FT,SOC1,FLC and other genes related to flowering regulation were significantly changed in PheFKF1 overexpressed plants,which means that PheFKF1 may be involved in flowering regulation by regulating the expression of these genes.In order to analyze the relationship between PheFKF1 and Gibberellins(GAs)flowering regulation pathway,Arabidopsis were sprayed with GA3.It was found that the bolting time of all Arabidopsis plants was advanced,and the PheFKF1 overexpressed lines had the highest sensitivity to GA,followed by the wild type,and the fkf1 mutant had the lowest sensitivity.It is speculated that PheFKF1 may play an important role in Gibberellin regulates flowering pathways4.Study on regulation mechanism of PheFKF1In order to further analyze the mechanism of PheFKF1,the interacting proteins Phe Hd1,Phe ID1 and Phe DELLA were screened by yeast two-hybrid and Bi FC experiments.It was speculated that PheFKF1 could regulate downstream genes by forming protein complexes and thus participate in several regulation of flowering pathway of bamboo.In summary: In this paper,PheFKF1 was obtained by comparison based on bamboo database,and its bioinformatics analysis and gene function verification were conducted systematically,and it was speculated that it might be involved in the flowering pathway of Bamboo.Overexpression of PheFKF1 can induce early bolting in Arabidopsis thaliana and increase the sensitivity to GA.Meanwhile,PheFKF1 could interact with Phe ID1,Phe Hd1 and DELLA proteins,suggesting that PheFKF1 may be involved in multiple pathways to regulate bamboo flowering.