Function Identifications Of Photoperiod Related Genes PbCOL8,PbTFL1 And PbELF3b Regulating Flower Bud Differentiation In Pear

China is the world's leading country in pear production.Pear has a long history of cultivation and is extremely rich in germplasm resources.The flower bud differentiation is the decisive factor of pear production.Pear flower bud could be classified as the classic summer-autumn differentiation type.Induced by shorting day length after summer solstice,pear trees stop the growth of branches and flower buds complete physiological and morphological differentiation in following time.It has been reported that nutrients,mineral elements,endogenous hormones and environmental factors such as light and temperature affect the differentiation of flower buds.However,the photoperiod signal pathways and signal transduction networks in pear flower bud differentiation is still unclear.In this study,'Dangshansuli' and 'Korla pear' were used as main materials,combined with genomics,genetics,molecular biology and cell biology,to identify key genes involved in flower bud differentiation in the photoperiod pathway of pear.The main results are as follows:1.By transcriptome research,the photoperiod response genes in pear were comprehensively analyzed.Pear seedlings grown under long-day(16 h light/8 h dark)and short-day(8 h light/16 h dark)conditions for 30 days were sampled for transcriptome sequencing.Analysis of transcriptome data revealed that about 27.2%of the genes expressed in the whole genome of pear show rhythmic expression patterns.Among them,6078 genes were rhythmic under long-day conditions,8141 genes were rhythmic under short-day conditions,and 2570 genes were rhythmic under different photoperiod conditions.Through homologous alignment and transcriptome data analysis,circadian clock core oscillators were identified in pear,and PbLCL,PbPRR,PbELF3,PbELF4,PbLUX and PbGI were found to be the core genes of pear clock system.Under short-day conditions,the peak time of most circadian clock genes was advanced,and the expression of PbTOC1b in PbPRR family was significantly up-regulated.These results lay a foundation for the study of pear photoperiod signal transduction regulating flower bud differentiation.2.By genome-wide analysis and functional research of CO-Like(COL)homologous genes in pear,it was demostrated that PbCOL8 is an important factor in photoperiodic regulation of flowering time.Fifteen PbCOL family members were identified in pear genome and were classified into three groups according to the phylogenetic analysis.PbCOL family members showed tissue specific expression pattern,however all PbCOL genes expression could be detected in leaves.Under 12 h light/12 h dark and continuous light conditions,PbCOL1,PbCOL8,PbCOL10 and PbCOL12 showed robust rhythms.Combined with transcriptome data analysis and RT-qPCR validation,PbCOL1,PbCOL8 and PbCOL12 responded to the induction of photoperiod.Transgenic experiments showed that the flowering signal activator FT and SOC1 genes were strongly inhibited and the flowering time was significantly delayed in PbCOL8 transgenic lines,indicating that PbCOL8 could regulate flower bud differentiation by affecting floral integrator genes.3.SOC1 is an important flowering activator.Eight SOC1 homologous genes were identified in pear genome and named as PbSOC1a-h.The promoter region of each gene contained multiple cis-regulatory elements related to plant reproductive development,including the key binding site CArG-box.PbSOC1-Like genes had a similar tissue expression pattern and were highly expressed in leaves,leaf buds and flower buds.During the differentiation and development of flower buds,the expression patterns of PbSOC1-Like genes were dynamically changed.Among them,PbSOC1a,PbSOC1d and PbSOC1g showed stable circadian rhythms and responded to different photoperiod conditions.TFL1 is an important flowering inhibitor.The genes with the highest similarity to TFL1 were PbTFL1a and PbTFL1b.Tissue specific expression pattern and photoperiod response pattern indicated that they were similar with TFL1.PbTFL1a and PbTFL1b proteins were localized to nucleus.Transgenic experiments showed that PbTFL1a and PbTFL1b can significantly delay the flowering time by inhibiting the floral meristem identity genes LFY and AP1.PbTFL1a and PbTFL1b had a conserved inhibitory effect on flower bud differentiation.These results suggested that pear flowering activators PbSOC1-Like and flowering inhibitors PbTFL1a and PbTFL1b play important roles in flower bud differentiation.4.'Korla pear' flower bud formation is difficult,so its cultivation area is limited.It is reported that flower bud differentiation disorder is due to abnormal nutrients and hormones.In this study,it is found that alternative transcriptional start sites mutation of PbELF3b gene affected flower bud differentiation of‘Korla pear'.By observing the internal structure of flower bud differentiation processes,it was found that the flower buds of 'Korla pear'began to turn black and gradually became necrotic from August in Nanjing.ELF3 is an important factor in the photoperiod pathway regulating flower bud differentiation.Analysis of PbELF3b sequences in different pear varieties revealed that there was 58bp intron deletion in the 'Korla pear' PbELF3b compared to 'Dangshansuli' PbELF3b,and named as PbELF3bD and PbELF3bN respectively.Transgenic experiments showed that the PbELF3b show conservative function of inhibiting flowering.Moreover,the PbELF3bD could enhance the inhibitory effect and result in an extremely late flowering.RACE technology was used to identify a new transcriptional mode in PbELF3b,named PbELF3b-?.The 58bp deletion of PbELF3bD was located in the upstream of the PbELF3b-? coding reigon,resulting in a significant decrease expression level of PbELF3b-? in 'Korla pear'.The results of subcellular localization showed that PbELF3b-? and PbELF3b-? were located in nucleus and cytoplasm,respectively.AtELF3-? was also cloned in Arabidopsis,indicating that alternative transcription start sites of ELF3 gene could be a universal regulatory mechanism.Transgenic experiments showed that AtELF3-? delayed flowering,but AtELF3-? promoted flowering.Yeast two-hybrid results showed that AtELF3-? and AtELF3-? can form heterodimers.In conclusion,this study confirmed that PbELF3b encodes PbELF3b-? and PbELF3b-? concurrently through alternative transcription starting sites,in which PbELF3b-? inhibits flower bud differentiation,while PbELF3b-? promotes flower bud differentiation.The intron deletion mutation of PbELF3b gene led to the decrease of PbELF3b-? expression in 'Korla pear',which may be an important reason for the disorder in flower bud differentiation.In summary,this study identified photoperiod related genes based on the whole genome of pear,and elucidated the functions of PbCOL8,PbSOC1-Like,PbTFL1a and PbTFL1b in flower bud differentiation.This study clarified the molecular mechanism of PbELF3b regulating flower bud differentiation through alternative transcriptions.These results provide theoretical basis and genetic resources for molecular breeding of pear flowering traits.