The Identification Of PbrbZIP Gene Family And Functional Study Of PbrbHLH169 Related To Stone Cell In Pear Fruit

Stone cells were a unique cell type in pear fruit and belonged to brachysclereid.The size,distribution and density of stone cells and the size of stone cell clusters were important factors affecting the taste and processing quality of pear fruit.The area of pear fruit cultivated in China was the first around the world,but the main pear varieties with high content of stone cells that caused a rough flesh texture and reduced fruit quality and economic value.The lignin and cellulose were the two major components in the secondary walls of stone cells,and their synthesis and accumulation were one of the key factors to cause stone cell formation.The researches on lignin and cellulose were mainly reported in the simulation of Arabidopsis thaliana and poplar,and there were few reports on the synthetic regulation mechanism of lignin and cellulose in the fruits.Therefore,in this study,we determined the content of stone cells,lignin and cellulose in 236 sand pear fruits,and screened transcription factors,which might be related to stone cells formation,by analyzing transcriptome database of different pear fruits at 50 DAF,verifying the function of these genes by molecular biology experiments.In order to provide theoretical basis for the molecular regulation of stone cells.The results were as follows:1.We had measured the content of stone cells,lignin and cellulose in fruit of 236 sand pears（Pyrus pyrifolia）.The results shown that the mean content of stone cells,lignin and cellulose in stone cells was 10.93%,29.73%and 18.03%,respectively.The phloroglucinol-hydrochloric acid staining results shown that the stone cell masses were mainly distributed in the core of fruit,and presented a radial shape extending from core to flesh.Additionally,the correlation between the content of stone cells and lignin and cellulose in different varieties was performed,the results shown that the content of lignin in stone cells was higher than that of cellulose,and the correlation was higher between stone cells and lignin than that between stone cells and cellulose,this result indicated that the lignin play more important role during the stone cells formation in pear fruit.Additionally,we also screened 11 transcription factors（including bZIP、bHLH、ERF、MYB and NAC）,which might be related to stone cells formation,by analyzing transcriptome database of different pear fruits at 50 DAF.QRT-PCR results shown that these genes expression has similar trend with the content of stone cells,indicating that these genes might participated in the stone cells formation.2.We had identified the 86 PbrbZIP transcription factors by analyzing the bZIP gene family from pear whole genome-wide database.86 PbrbZIP transcription factors were divided into 11 subfamilies.The analysis of gene structure and conserved domain further demonstrated the reliability of evolutionary classification.And the analysis of collinearity indicated that genome-wide replication events played a major role in the expansion of the bZIP family.Simultaneously,purified option was the primary force driving family evolution.We had screened 9 genes related to the synthesis of lignin in stone cells by genes co-expression and fluorescence quantitative experiments,it provided solid foundation for the further study about gene functional identification.3.The genetic function identification of PbrbHLH169 transcription factor related to lignin and cellulose synthesis in stone cells of pear fruit was performed.The results of subcellular localization showed that PbrbHLH169 belongs to nuclear protein.The relative expression levels of PbrbHLH169 in different developmental stages and different tissues of’Liyuanxiangli’ was analyzed,the result shown that the expression of PbrbHLH169 was highest in fruits at 35DAF and 49DAF and has relatively higher value in stem and leaf.At the same time,the expression of PbrbHLH169 in pear fruits with high and low-stone cells content shown the same trend as the changed in stone cells content.The transient overexpression of PbrbHLH169 caused significant changes of lignin and cellulose content in pear fruit.Compared with the no-load control,the lignin content in pear fruit increased by 45.45%and the cellulose content increased by 53.14%.Simultaneously,the expression levels of genes related to the synthesis of lignin and cellulose were significantly increased.Stable genetic transformation of Arabidopsis thaliana,T2 generation positive seedlings of transgenic Arabidopsis thaliana were obtained,the content of lignin in transgenic Arabidopsis thaliana stem significantly increased.