Genome Characterization And Preliminary Study Of Transcription Factor NF-Y Family Related To Dormancy Release Of Pyrus Calleryana Decne Seeds

Pear is a temperate fruit widely cultivated in China,which has important economic value.As one of the oldest pear species,Pyrus calleyana dence is often used as grafted rootstock because of its disease-resistant.However,the seeds of Pyrus calleyana dence have dormancy,which requires a long time of stratification to release dormancy.It has a certain restriction on production.At present,the molecular mechanism of pear seed dormancy release is not clear.Nuclear factor Y（NF-Y）is a kind of transcription factor widely existing in eukaryotes.It has been reported in model plants that it can participate in important physiological processes such as embryo and seed development,flowering time and stress response,but it is rarely reported in the release of pear seed dormancy.In this paper,seeds of Pyrus calleyana dence were used as experimental materials to analyze the NF-Y family,its expression pattern and function.The main results are as follows:1.The genome of Pyrus betulaefolia and Pyrus bretschneideri are integrated and studied by bio informatics strategy.57 NF-Y family members were identified in Pyrus betulaefolia,including 13 PbNF-YA,27 PbNF-YB and 17 PbNF-YC;53 NF-Y family members were identified in Pyrus bretschneideri,including 16 PbrNF-YA,22 PbrNF-YB and 15 PbrNF-YC.The number of amino acids in each member ranges from 50-845,the molecular weight ranges from 6247.04-89255.32 Da,and the number of aliphatic amino acids ranges from 43.85-99.53.From the theoretical isoelectric point value,most of them are neutral and alkaline,and NF-YA and NF-YC are unstable proteins,only a few NF-YB members are stable proteins.The hydrophilic indexes are negative,indicating that NF-Y family members are hydrophilic proteins.They are unevenly distributed on chromosomes.These differences imply the diversity of biological functions of pear NF-Y family members.From the gene structure,motif analysis and cluster analysis of model plant Arabidopsis thaliana,it is found that these family members are conservative.The analysis of protein conserved domain showed that all members of NF-YA subfamily contained CBFB_NFYA domain,NF-YB subfamily members and NFYC subfamily members all contain CBFD_NFYB_HMF domain.The three-dimensional protein structure modeling of NF-Y subfamily members,it is found that the structure is highly similar,indicating that these subfamily members are highly conservative.2.Preliminarily identify the members of PcNF-Y gene family that mediates ABA to participate in the release of seed dormancy of Pyrus calleyana dence.The ABA content of embryo and seed coat continued to decline during seed stratification.The ABA content in seed coat was very high during dormancy,but decreased sharply after 14 days of stratification,and then decreased slowly.The content of ABA in seed embryo did not change significantly in the early stage,but decreased to a very low state when dormancy was released.Based on the genomes of Pyrus betulaefolia and Pyrus bretschneideri and the results of early transcriptome dates,16 differentially expressed NF-Y members genes（PcNF-YA1-8,PcNF-YB1-3 and PcNF-YC1-5）and genes of ABA metabolism and signal transduction pathway（PcCYP707A13,PcPYL1-2 and PcPP2C1-3）were screened.Gene expression analysis showed that most members of PcNF-Ys family and PcPYL1/2 in seed embryo and seed coat were downregulated during dormancy release,while PcCYP707A2,PcCYP707A3 and PcPP2C2 were up-regulated.The double luciferase experiment confirmed that PcNF-YA7 activated the activity of PcPYL1 promoter,and PcNF-YA5 had significant inhibitory effects on PcCYP707A3 promoter,PcNF-YA6 on PcPP2C2 promoter and PcNF-YC1 on PcCYP707A3 promoter.However,the results of yeast one-hybrid experiment showed that the related PcNFYs members could not directly bind to these promoters.3.Through target prediction and analysis of miR169 expression in seed coat and seed embryo during dormancy release,it is found that PcmiR169 was negatively correlated with the expression of PcNF-YA.It is speculated that PcmiR169 degraded PcNF-YA members during dormancy release,further regulates the dormancy release of Pyrus calleryana Decne seeds.In conclusion,the transcription factor NF-Y is involved in the transcriptional regulation of the promoter of ABA in metabolism and signal transduction pathway during the release of seeds dormancy,but the mediating mechanism needs to be further analyzed and determined.