The Mechanism About Pear Pollen LRR-EXT And CRPs In Self-Incompatibility

Self-incompatibility(SI)is a mechanism for regulating the genetic divergence of plant kindom.Recent reports on 'Jinzhui'(Pyrus bretschneideri)which is a self-compatibility mutational cultivar of 'Yali'(Pyrus bretschneideri)suggest that the self-compatibility comes from the mutation of pollen and might be a non-S factor.However,no reports could find this non-S factor and explain the mutation mechanism.By high-throughput sequencing including RNA-seq and proteomics,following experiments were conducted:1.Screend out differential expressed genes between 'Jinzhui' and 'Yali' pollen and between self-pollinated pistils and non-self pollinated pistils in 'Jinzhui' Baesd on mature pollen of 'Jinzhui' and 'Yali',the differential expressed genes were analyzed.Comparing with 'Yali',there are 3168 up regulated genes and 3148 down regulated genes.After the further screening out,we found the 15 Cysteine rich peptides(CRPs)were differential expressed.Except Pbr014631.1(lipid transfer),other CRPs were all down regulated in 'Jinzhui'.We speculated that these genes might be candidate non-S factor in pear SI.Combined with the proteomics results,we found that a leucine rich protein(Pbr000552.2)is also up regulated in 'Jinzhui' pollen.We pollinated the styles of 'Jinzhui'by 'Jinzhui' and 'Yali' pollen and collected styles after 24h pollination,48h pollination and 72h pollination.Then these styles were performed RNA-seq to explore the differential expressed genes.Comparing the dyamic self-compaptible styles after pollination,37 heat shock proteins are differential expressed.However,there are only 8 differential expressed heat shock proteins in incompatible styles.Comparing compatible styles and incompatible styles after 24h pollination,there 45 up regulated heat shock proteins in compatible styles.These heat shock proteins could be clustered into three groups.Our high-throughput sequecing reveal some CRPs and heat shock proteins play important roles in SI.2.Screened out several differential expressed proteins between pollen proteins in'Jinzhui' and 'Yali'.Baesd on the 'Jinzhui' and 'Yali' pollen,we analyzed differential expressed proteins.We totally got 418252 spectrum,23 192 peptides and 6387 proteins.Comparing with 'Yali',there 140 up regulated proteins,125 down regulated proteins.Among these differential expressed proteins,we found some proteins involved in the S-RNase transporting(Pbr017310.2,Pbr026379.1)that belong to ABC transport family.The up regulation of these proteins might induce the breakdown of SI.Cullin-1-like isoform X1(Pbr011232.1),SKP1(Pbr025966.1)might participate the forming of SCF(Skpl-Cullin-F-box)which was a candidate complex for degradation of S-RNase.The up regulation of these proteins might lead to the degration of S-RNase in 'Jinzhui'.Some other candidate differential expressed proteins were also diged out,such as leciune rich proteins,heat-shock proteins,C-2 domain proteins and arabinogalactan-proteins.3.The candidate differential expressed genes were performed the evolutionary CRPs are gaining recognition as regulators of cell-cell communication in plants.We identified 9556 CRPs in 12 plant species and analysed their evolutionary patterns.In most angiosperm plants,whole genome duplication and segmental duplication are the major factors driving the expansion of CRPs family member genes,especially signal peptides.About 30%of the CRPs genes were found clustered on the chromosomes,except in maize(Zea mays).Considerable collinearities between CRPs genes between or within species reveal several syntenic regions on the chromosomes.Different subfamilies display diverse evolutionary rates,suggesting that these subfamilies are subjected to different selective pressures.CRPs in different duplication models also show contrasting evolutionary rates,although the underlying mechanism is unclear because of the complexity of gene evolution.The 1281 positively selected genes identified are probably generated within a certain period of time.While most of these belonged to maize and sorghum(Sorghum bicolor),new CRPs functions would also be expected.Up-regulation of 10 CRPs was observed in self-pollinated pear pistils and pollen tubes under self S-RNase treatments in vitro.The expression divergence between different CRPs gene duplication types suggests that different duplication mechanisms affected the fate of the duplicated CRPs.During the process of screening out differential expressed genes in 'Jinzhui',many extensins(EXTs)were found to be differential expressed.We performed a binformatic analysis on EXTs of pear,apple,strawberry,peach and plum.With the character of SPn motif,we screened the protein database.Then SPADA is used to complement the unannotated proteins.Total 515 sequences are gained and 10%are acquired from SPADA.The major duplication types of EXTs is WGD type.Many EXTs duplicated into non-EXTs and many non-EXTs duplicated into EXTs.However,this high frengent transition is not influnced by SPn motif.EXTs from WGD get a faster evolutionary rate than EXTs from single gene duplication.The explosive divergent time for EXTs is earlier than the divergence of rosaceae plants.The expressed EXTs in styles are more than EXTs expressed in pollen.The expression divergence of EXTs is more correlated with Ks than Ka.With the transcriptome of 'Jinzhui' and 'Yali'pollinated styles,we also find two EXTs that might paticipte in SI.4.With high throughtput sequencing,LRR-EXT is screened out for our further experiment.LRR-EXT is specific expressed in pear pollen and the expression level in'Jinzhui' pollen is higher than that in 'Yali'.The LRR-EXTs show a polymorphism in pear and are presented by SPPPPVH.JZ-LRR-EXT lacks 696 bases comparing with YL-LRR-EXT.And there is no intact SPPPPVH in JZ-LRR-EXT for the lacking seuqences.We used recombinant LRR-EXT to pretreat the pollen and then treat with S-RNase.Results suggest that JZ-LRR-EXT could reduce the toxicity of S-RNase while there was no use for that in YL-LRR-EXT,We also found that after the treatment of S-RNase,the inhibition of ROS(Reactive Oxygen Species)in the tip of 'Yali' pollen tubes pretreated with JZ-LRR-EXT is much more retarded.We speculated that the loss of intact extensin for JZ-LRR-EXT leads to the self-compatible for 'Jinzhui' pollen.However,the mechanism and genetic researc need to be performed.