Functional Analysis Of Differentially Expressed Protein PbTM9SF3 In 'Jin Zhui' And 'Ya Li'

The pear cultivar is a gametophytic self-incompatibility?GSI?fruit tree which blongs to Rosaceae,Pyrus species.While‘Jin Zhui'cultivar,belong to self-compatible?SC?,is a spontaneous bud mutant of‘Ya Li'cultivar.Previous research has shown that mutant non-S?named modifier factors?factor is responsible for‘Jin Zhui'cultivar.However,the specific modifier factors and its molecular mechanism in‘Jin Zhui'cultivar are still obscure.In this study,therefore,‘Ya Li'and‘Jin Zhui'were used as materials.The gene associated with SI,PbTM9SF3,was screened by Proteomics Sequencing Technology.Firstly,the bioinformatics of PbTM9SF3 was systematically analyzed,and its expression was further analyzed by qRT-PCR technology.Secondly,this subcellular localization of PbTM9SF3 was peformed using particle bombardment method.Finally,the function of PbTM9SF3 was studied by yeast interaction experiment.The main results are as follows:1.A total of 489 differentially expressed proteins?DEPs?were identified by iTRAQ technology,of which 149 were up-regulated and 340 were down-regulated.Bioinformatics analysis showed that most of them were mainly involved in metabolic pathways.The full-length of PbTM9SF3,with 1782 bp,was successfully cloned from pollen of‘Jin Zhui'cultivar.The PbTM9SF3,named EMP70,is nine transmembrane9 superfamily proteins?TM9SF?.The analysis result of protein domain of PbTM9SF3gene suggested that it is grouped in one large non-cytoplasmic region and nine transmembrane regions.It is presumed that this protein is likely to be used as a channel and transporter for small molecules.The sequence of PbTM9SF3 is highly conserved in evolution according to amino acid sequence alignment.Phylogenetic tree analysis supported that PbTM9SF3 and MdTM9SF3 was clustered into one branch,and the homology of amino acid sequence was 98%.2.The RT-PCR result showed that the expression of PbTM9SF3 in pollen of‘Jin Zhui'cultivar was significantly higher than in leaf and style of‘Jin Zhui'cultivar.In addition,the expression of PbTM9SF3 in pollen of‘Jin Zhui'cultivar was significantly higher than in pollen of‘Ya Li'cultivar,which indicated the change of self-compatibility of‘Jin Zhui'cultivar was related to PbTM9SF3.3.The PEZS-NL vector which is controlled with CAMV 35s promoter,containing a GFP reporter gene and a PbTM9SF3,was transferred into onion epidermal cells by particle bombardment method.Green fluorescent protein?GFP?was found in the plasma membrane under the fluorescence microscope,indicating that this gene is a membrane protein.4.The PbTM9SF3,PbS₂₁-RNase and PbS₃₄-RNase was cloned from‘Jin Zhui'cultivar.To investigate the function of PbTM9SF3 in SC,the interaction between PbTM9SF3 with PbS₂₁-RNase,PbS₃₄-RNase was verified by yeast two hybrid.The results proved that co-transformed yeast strains with AD-PbTM9SF3,BD-PbTM9SF3could not grow nomally,indicating there is no interaction between PbTM9SF3 and two S-RNase in yeast interaction.