Identification,expression Analysis Of Fw2.2-like Gene Family In Pear And Functional Characterization Of Some Members

The tomato fw2.2(fruit weight-2.2)gene was the first gene associated with a quantitative trait to be identified and cloned,controlling 30% of fruit size variation.fw2.2-like,a homolog of fw2.2,has also been shown to play an important role in the growth and development of many plants.Pear is one of the world’s major fresh fruits with abundant fruit size variation.The sequencing of the whole pear genome has been completed,but the study on the fw2.2-like gene family in the whole pear genome has not been reported.In this paper,we take Korla fragrant pear as the research object,Korla fragrant pear large-fruited bud variant‘Zaomeixiang’ pear,large-fruited type ‘Yali’ pear ’ and small-fruited wild ‘Duli’ pear were used as controls,and the pear fw2.2-like gene family members were screened and identified in the pear whole genome data,and their molecular characteristics and functions were initially investigated using bioinformatics.Real-time fluorescence quantitative q RT-PCR was used to explore the expression patterns of pear fw2.2-like gene family members in fruits of different pear germplasm,to analyze the association between relative gene expression,cell number and fruit size development,to predict the candidate fw2.2-like proteins involved in the cell division process of pear fruits,and to perform subcellular localization and functional validation.The main results were as follows:(1)The results of the developmental dynamics of pear fruit cytokinesis showed that the single fruit weight and volume growth rate of the four different pear germplasm were similar during fruit cytokinesis,and the growth rate was the fastest 10 to 20 days after bloom,and in general,the fruit weight and volume growth rate of the four different pear germplasm in different periods basically showed ‘Yali’ pear >‘Zaomeixiang’ pear > Korla fragrant pear > ‘Duli’ pear.Paraffin section observation found that: ‘Duli’ pear fruit cell division speed was slow,limit its fruit cell number growth,cause the fruit was smaller than ’kulle balsam pear’,‘Zaomeixiang’ pear and ‘Yali’ pear.The most vigorous and critical period of fruit cell division of’kulle balsam pear’,‘Zaomeixiang’ pear and ‘Yali’ pear were from 10 DAFB to 20 DAFB,and the fruit of‘Yali’ pear the longest cell division period.(2)Fourteen pear fw2.2-like genes were identified from ’Dangshan’ pear genomic data,and all pear fw2.2-like proteins contained one PLAC8 structural domain.Pear fw2.2-like proteins had amino acid lengths ranging from 178 to 415 AA and molecular weights of 16.99 to 47.51 k Da,all of those were unstable hydrophilic proteins.Gene structure analysis showed that the pear fw2.2-like gene family members consisted of three to seven exons and the gene structure was highly conserved in evolution.Pear fw2.2-like gene family members were localized on nine different chromosomes,named PbFWL1 to PbFWL14,and covariance analysis indicated that the pear fw2.2-like genes were amplified by gene duplication during evolution,but the amplification was slow and very conserved during evolution.GO annotation analysis of PbFWLs proteins suggested that PbFWLs may regulate some biological processes in pear by binding to other proteins.Evolutionary tree analysis showed that PbFWLs proteins could be classified into five groups,with PbFWL1 and PbFWL2 being the closest to fw2.2 and PbFWL5 being the closest to Pf CNR1,speculating that PbFWL1,PbFWL2 and PbFWL5 may be involved in regulating the size of pear fruits and other organs.NCBI-SRA database database has been used to download transcriptional data from 7 tissues of ’Dangshan’ pear and analyze the expression pattern of PbFWLs gene in pear.The results show that PbFWLs gene has tissue specificity,and some genes have high expression in young tissues,but almost no expression in mature tissues.These results suggest that PbFWLs gene may play an important role in early tissue development.(3)To ensure the reliability of gene expression analysis,this study systematically evaluated the stability of eight internal reference genes in pear fruit gene expression analysis,and the results showed that Actin1 was most stably expressed in the floral receptacle at flowering stage,and TUB2 was most stably expressed in fruit and pulp gene expression analysis at the cell division stage of pear fruit.To investigate the effect of pear fw2.2-like genes on fruit size,real-time fluorescence quantitative PCR was used to analyze the expression of all members of the pear fw2.2-like gene family in pear fruit cytokinesis of four different genotypes.Due to the highly similar sequences of PbFWL12 and PbFWL13,it was not possible to design primers to separate the two sequences,and the final 13 PbFWLs expression levels were monitored and the expression levels of PbFWLs were correlated with receptacle(fruit)cell numbers for analysis.The results showed that the expression levels of 13 members of the pear fw2.2-like gene family did not correlate with the number of receptacle cells,and only the expression of PbFWL1/2/5 in 10 to 40 days after flowering showed a negative correlation between small-fruited pears(‘Duli’ pear)was greater than that of medium-fruited pears(Korla fragrant pear and ‘Zaomeixiang’ pear)than that of large-fruited pears(‘Yali’pear),which was negatively correlated with the number of pear fruit cells,suggesting that PbFWL1/2/5may had a positive effect on pear cell division by regulating the rate of cell division in pear fruit at the young fruit stage ultimately affects fruit size.The fluorescence in situ hybridization analysis of PbFWL1/2/5genes revealed that the in-situ hybridization hybridization signal was gradually weakened until it disappeared along the equatorial axis at the maximum in the heart of pear fruit,indicating that PbFWL1/2/5genes might be involved in the regulation of division of fruit flesh cells in the heart and near the heart.(4)To investigate some of the molecular mechanisms of PbFWL1/2/5 genes regulating cell division,subcellular localization analysis was performed on them,and the results showed that PbFWL1,PbFWL2 and PbFWL5 were membrane proteins.The analysis of promoter cis-acting elements and protein interactions in different pear germplasm using bioinformatics methods showed that: the differences in promoter cis-acting elements of PbFWL1/2/5 genes may be the reason for the differential expression of the corresponding genes in different pear germplasm;PbFWL2 may be interacts with growth hormone response factor,pear G protein γ subunit protein and LEA,mentioned the above view needs to be experimentally verified.(5)To verify the functions of PbFWL1/2/5 genes,we constructed p CAMBIA1303-PbFWLs plant overexpression vectors and overexpressed them in Arabidopsis and Micro-Tom tomatoes,respectively,and found that: the underground parts of the transgenic Arabidopsis plants were significantly smaller than the control plants in the T2 generation compared with the control,while the The above-ground part of the tissues and organs were also slightly smaller than the control plants;the organs of the transgenic tomato T0 generation plants did not show the same tendency to be smaller and the phenotypic traits were unstable as in Arabidopsis compared with the control.