Bioinformatics Analysis Of Wheat FT/TFL1 Gene Family And Functional Identification Of TaFT3

Wheat(Triticum aestivum L.)is grown widely in the world and is an important source of energy and protein for people.Heading date is a critical determinant of regional adaptation for crops and it has a significant impact on crop yield.Therefore,cloning flowering genes and studying their mechanisms can serve as a theoretical basis for wheat breeding.FT(FLOWERING LOCUS T)/ TFL1(TERMINAL FLOWER 1)gene family coding Phosphatidyl Ethanolamine Binding Protein(PEBP)plays an important role in the regulation of flowering time and plant type,and is a key factor in regulating the transition from vegetative stage to reproductive stage.The identification of FT/TFL1 gene family in wheat would be helpful to elucidate the molecular mechanism underlying flowering control in wheat.The aim of this study is to make a comprehensive study on the molecular characterization,phylogenetic relationship and expression profiling of common wheat FT/TFL1 gene family using the new released wheat genome database(IWGSC Ref Seq v2.0),providing new evidence for further understanding the structure,evolution and function of plant expansion genes.Experiments of Chinese Spring under different photoperiods showed that LD condition could promote the development of young spike after crossing the double-ridge stage,but the molecular mechanism is still unclear,and the role of FT/TFL1 family genes in this process is rarely reported and remains to be further explored.In this study,RNA-seq was used to analyze the differentially expressed genes under LD and SD conditions during the transition period of the double-ridge stage,and the FT/TFL1 gene family member TaFT3 was discovered.Overexpression vector and CRISPR/Cas9 knockout vector were constructed to verify their function in wheat.The main research results are as follows:(1)The FT/TFL1 gene family was identified.Based on TaFT1 /VRN3 and Ta TFL1 genes in wheat,75 FT/TFL1 genes were identified in wheat by bioinformatics method.Through phylogenetic analysis,the FT/TFL1 family genes were divided into three subfamilies: FT&TSF,TFL1&BFT&CEN-like and MFT.At the same time,the members of the FT/TFL1 gene family members were analyzed by time tree according to the classification of bryophytes,Pteridophytes,gymnosperms and angiosperms.(2)The molecular characteristics,phylogenetic relationships and expression profiles of FT/TFL1 gene family members were studied.The gene length,exon number,protein amino acid number,molecular weight and theoretical electric points of FT/TFL1 family were analyzed and summarized by bioinformatics.The gene structure and protein sequence of FT/TFL1 gene were highly conserved by analysis of gene structure and protein conserved domain.Collinear analysis confirmed that the FT/TFL1 gene family expanded by gene replication events.Tissue specific expression analysis demonstrated that FT/TFL1 gene families were differentially expressed in different stages of wheat development and tissues.The analysis of cis-acting elements showed that FT/TFL1 gene may be regulated by photoperiod and hormones in wheat.(3)In order to explore the reason why LD promote young spike development,wheat leaves were collected after the diurnal nodes of LD and SD conditions during the transition period of double-ridge stage,and the expression patterns of FT/TFL1 family genes were analyzed by RNA-Seq.Results showed that the expression levels of TaFT3 was significantly different under SD and LD conditions,and the expression levels under SD conditions was higher than that under LD conditions,and TaFT3 has obvious circadian rhythm.(4)The spatio-temporal expression pattern of TaFT3 was explored.Subcellular localization experiments showed that TaFT-A3/B3/D3 was localized in the nucleus,cytoplasm and cell membrane.Experiment showed that TaFT3 gene could respond to vernalization,and the expression level was significantly increased after vernalization.The expression level of TaFT3 in SD was significantly higher than that in LD condition,indicating that TaFT3 could respond to SD condition after vernalization.Tissue specific expression showed that TaFT3 had the highest expression in leaf and spike primordium,and the expression in spike primordium was slightly higher than that in leaf.The specific expression analysis of spike primordium development stages showed that the expression of TaFT3 was the highest when spike primordium transitioned from the double-ridge stage to the glume primordium differentiation stage.(5)Overexpression of TaFT-A3/B3/D3 in Fielder and overexpression of TaFT-B3 in Chinese Spring showed early flowering phenotype under LD and SD conditions.By dissecting the young spike of overexpressed strains in Fielder,it was found that TaFT3 could promote the development of young spike at different photoperiods,thereby advancing the flowering time,and the promotion effect was stronger under SD conditions.The expression of wheat flowering related genes TaFT1/VRN3(VERNALIZATION3),VRN1(VERNALIZATION1)in TaFT3 overexpression plants was up-regulated,and the expression of VRN2(VERNALIZATION2)was down-regulated,indicating that the regulation of young spike development by TaFT3 was related to VRN1,VRN2 and VRN3 in wheat.Statistical analysis of agronomic characters showed that phenotype of overexpressed plants was not significantly different from that of wild type,and the spike yield was not affected.(6)The function of TaFT3 was further verified by CRISPR/Cas9 gene knockout technology,and the transcription levels of TaFT1,VRN1 and VRN2 were measured,and the experimental results showed that ft3-a/ft3-b double mutant had slow growth at seedling stage and had a late-flowering phenotype,and the phenotype of ft3-d single mutant was not significantly different from that of the wild type.The expression of TaFT3 and VRN1 was significantly down-regulated,TaFT1 was almost not expressed,and VRN2 was significantly upregulated in the double mutant of ft3a/ft3b.