Identification Of Pear Sugar Transporter Genes And PbtMT4 And PbSWEET Genes Function Analysis

The synthesis and accumulation of sugar is an important factor for the flavor of pear fruit,which can directly affects the degree of consumers' favor to fruit,and also is a common scientific problem in breeding and basic theoretical research.Because of the complex metabolic pathway of sugar,and also relate to the long distance transport,trans-membrane transport and accumulation.It is the result of coordinated regulation of multiple genes.So it is difficult to analysis the mechanism of sugar metabolic.Additionally,more researches have been focused on the synthesis of sugar components of fruits,and few researches focus on the mechanism of sugar transport.Therefore,this research proposed through genome,transcriptome,proteome and transgenic technology analysis,focus on the sugar transporter identification and gene function in pear fruit,the results are as follows:1.In our study,a total of 1,810 proteins were identified during pear fruit development.The association analysis of proteins and transcripts expression revealed that there were 1,724,1,722,and 1,718 associated proteins identified in stages 15 DAFB,80 DAFB,and 145 DAFB,respectively.The correlation coefficients of the overall differentially expressed proteins and transcripts were significant,except 15 DAFB-VS-80 DAFB.Finally,a total of 35 important differentially expressed proteins related to fruit quality,including sugar,stone cells,and aroma synthesis,were identified in pear,among of them,3 differentially expressed proteins related to the sugar accumulation.2.A total of 75 ST genes were identified in the pear genome based on systematic analysis.Furthermore,all ST genes identified were grouped into eight subfamilies according to conserved domains and phylogenetic analysis.Analysis of cis-regulatory element sequences of all ST genes identified the MYBCOREATCYCB1 promoter in sucrose transporter(SUT)and monosaccharide transporter(MST)genes of Chinese white pear,while in grape,it is exclusively found in SUT subfamily members,indicating divergent transcriptional regulation in different species.Gene duplication event analysis indicated that whole genome duplication(WGD)and segmental duplication play key roles in ST gene amplification,followed by tandem duplication.Estimation of positive selection at codon sites of ST paralog pairs indicated that all plastidic glucose transporters(pGlcT)subfamily members have evolved under positive selection.In addition,the evolutionary history of ST gene duplications indicated that the ST genes have experienced significant expansion in the whole ST gene family after the second WGD,especially after apple and Chinese white pear divergence.According to the global RNA-seq results of Chinese white pear fruit development,gene expression profiling showed the expression of 53 STs.Combined with qRT-PCR analysis,two polyol/monosaccharide transporters(PLTs)and three tonoplast monosaccharide transporters(tMTs)members were identified as candidate genes,which may play important roles for sugar accumulation during Chinese white pear fruit development and ripening.Finally,we cloned and analyzed the function of PbtMT4 gene,the subcellular localization result show that it was located on the tonoplast membrane,and play important role for plant growth and development.In addition,the high expression of PbtMT4 can significant improve the expression of other PbtMTs,indicating that the PbtMTs may regulated by sugar contents.3.18 PbSWEET transporters identified in pear,and were divided into four clades based on the phylogenetic tree.Conserved motifs and six exons of the PbSWEET transporters were found in six species(Pear,Apple,Peach,Wooland strawberry,Japanese apricot and Arabidopsis).PbSWEET transporters contained seven TMSs that evolved from an internal duplication of an ancestral 3-TMSs unit,connected by the fourth TMS.This is the first direct evidence identifying internal repeats through bioinformatics analysis.Whole genome duplication(WGD)or segmental duplication and dispersed duplication represent the main driving forces for SWEET family evolution in six species,with former duplications more important in pear.Gene expression results suggested that PbSWEET15 and PbSWEET17 have no expression in any tissues,suggesting that they were pseudogene and that 62.5%of PbSWEET duplicate gene pairs have functional divergence.Additionally,PbSWEET6,PbSWEET7,and PbSWEET14 were found to play important roles in sucrose efflux from leaves,and the high expression of PbSWEET1 and PbSWEET2 might contribute to unloading sucrose from the phloem in the stem.PbSWEET5,PbSWEET9,and PbSWEET10 might contribute to pollen development.Finally,the function of PbSWEET genes were analyzed in yeast mutant(EBY4000),and found that PbSWEET10 and PbSWEET11 can growth on SD medium which containing 2%glucose,indicating that the PbSWEET10 and PbSWEET11 can transport the glucose.Subcellular localization analysis indicates that most of PbSWEETs were localized on the plasm membrane,except PbSWEET3,which located on the tonoplast membrane,indicating that they were functional on the plasma membrane.