| Flowering plants have developed various mechanisms for sexual reproduction,consisting of several sequential processes from pollination to fertilization.In reproduction,pollination is the first and most critical step and many hermaphrodite flowering plants have evolved self-incompatibility(SI)systems to avoid inbreeding and promote outcrosses to generate genetic diversity.Based on genetic mechanisms,SI can be divide into sporophytic and gametophytic systems.The Brassicaceae is a typical sporophytic SI family in which,a single S-locus controls the S haplotypes.Indeed,this family has served as a model plant for the study of intracellular signal transduction associated with SI plants.After the completion of annotated genomes for humans and other animal and plant species,research programs advanced from genetic into the genomic and post-genomic era.Analysis of proteins has long been an important field to biological processes.The advent of(two dimensional electrophoresis,2-DE)allowed thousands of protein spots to be separated on one gel.With the availability of genomics databases,and advances in mass spectrometry(MS)and peptide mass fingerprinting(PMF),identification of protein spots,separated by 2-DE,afforded a proteomics platform to investigate a wide range of biological processes.In addition,employing yeast two-hybrid and pull-down assays to assess protein-protein interaction,in vitro and in vivo,offers an efficient and effective way to study protein function.In this study,we used 2-DE to separated differentially expressed pistil proteins after self-and cross-pollination,at different time points,to identify differentially expressed proteins(DEPs)by MS.Expression levels of m RNA for the DEPs identified from self-pollinated pistils and those up-regulated in cross-pollinated pistils were next analyzed in floral organs and leaves.In some cases,q RT-PCR was employed to assess the expression level,in the pistil,after either self-or cross-pollination.Pull-down assays and electrophoretic mobility-shift assays were also used to detect pistil proteins in B.oleracea that interacted with CML27,SRBP1 RNA binding ability assay,two newly identified SI genes.Yeast two-hybrid assays were conducted to detect the interaction between a new SI factor Bo ROH1 and Exo70A1.The main outcomes from this work are as follows:(1)Isolation and identification of differentially expressed pistil proteins Morphological observations and affinity index methods were used to confirm self-incompatible of plant material A4 and cross-compatible with F1 plants.Optimization of the parameters for 2-DE,including IPG strip,sample loading,isoelectric focusing conditions,to achieve a suitable 2-DE system to separate B.oleracea pistil proteins.PDQuest software was used to analyze the 2-DE images of pistil proteins revealed that,in the self-pollination process,the 1 h time point was where the major changes in protein profiles took place.A comparison of 2-DE protein maps from self-pollination 0 min and 1 h identified 26 DEPs;here,6 were specifically expressed,and 16 and 4 were up-and down-regulated,respectively,at 1 h post self-pollination.Analysis of self-and cross-pollination 1 h 2-DE maps,26 proteins were identified to be differentially expressed,in which,2 and 1 specifically expressed in self-or cross-pollinated pistil 2-DE maps,and 6 up-regulated expressed and 17 down-regulated expressed in self-1 h 2-DE map.In total,we successfully identified 44 protein spots,and Gene ontology and KEGG analysis revealed an array of proteins that we grouped into the following eight biological processes: stress response and defense(15,34%),protein metabolism(7,16%),carbohydrate and energy metabolism(6,14%),regulation of translation(5,11%),pollen tube development(4,9%),transport(4,9%),cell cytoskeletal(2,4.5%)and unknown protein(1,2.5%).In total,2,870 and 3,209 differentially expressed B.oleracea genes(DEGs)were identified in the 0 min versus 30 min and 0 min versus 60 min group,respectively.Collectively,GO and KEGG analyses allowed annotation of 2,738 and 815 DEGs,respectively;with 95% and 28% DEGs being present in 0 min versus 30 min;for the 0 min versus 60 min DEPs,2904 and 961 DEGs were annotation,respectively,with 96% and 32% DEGs.Through our analysis of the combined proteomics and transcriptomicse data,we propose that CML27,SRBP1 and ROH1 are involved in self-incompatibility signal transduction.(2)Molecular cloning and expression analysis of DEPs in B.oleracea Analysis of self-pollinated pistil specifically expressed proteins C2H2,VPS29,annexin2 and cross-pollinated 1 h pistil up-regulated expressed proteins CML27,myosin,tubulin and BPS1 m RNA expression levels in floral organs,leaf,self-and cross-pollination process.RT-PCR assays indicted that expression of C2H2,annexin2,VPS29 and tubulin were up-regulated in pistil versus pollen,whereas myosin and BPS1 similarly were expressed in both pistil and pollen;CML27 up-regulated in pollen versus pistil.Our q RT-PCR assays indicated that in both self-and cross-pollination process,C2H2 and CML27 display “up-down expression pattern”;myosin up-regulated;Tubulin and BPS1 display “down-up expression pattern”.VPS29 display “up-down expression pattern” in self-pollination process and down regulated expressed in cross-pollination process;Annexin2 down regulated expressed in self-pollination process and display “down-up expression pattern” in self-pollination process.(3)Pistil expression of SI related novel gene CML27 and identification of its interacting proteins CML27 was expressed in and purified from E.coli and then co-incubated with self-pollinated B.oleracea pistil proteins,followed by protein separation on SDS-PAGE gels.One band,specifically located between 130 k Da and 180 k Da was excised from the gel and interrogated by LC-MS-MS(Q-TOF).Some 19 different proteins were identified in this band,and Gene ontology and KEGG analysis revealed an array of proteins grouped in the following categories: stress and defense response(2,10.5%),regulation of translation(5,26%),gene silencing(2,10.5%),protein metabolism(4,21%),Ca2+ binding(2,10.5%),chromosome organization(2,10.5%),vesicle mediated transport(1,5.5%)and SI related protein(1,5.5%).Five identified proteins were related to Ca2+(AGP31,EF-2,EF-1α,PLDα1 and PLDα2),one related to vesicle mediated transport(coatomer subunit alpha-2-like)and one known SI related gene(SLG).All of these proteins implicate CML27 being involved in the SI response,with a likely function in increased Ca2+ concentration in pistil papilla cells leading to self-pollen rejection.(4)Molecular cloning of SI novel gene SRBP1 and function analysis The amino acids of SRBP1A/2A 98% identity with SRBP1 in A.thaliana.Bioinformation analysis found the N-terminal of SRBP1 and SRBP1A/2A contain a conserved RNA recognition domain,and C-terminal is a glycin rich domain.In transgenetic A.thaliana plants,GUS expressed in root tip,shoot apex,lateral root,vascular in leaf,pistil and anther,and the expression level of GUS depends on development stage.Electrophoretic mobility-shift assays found prokaryotic expressed SRBP1 proteins binds to long-RNA Cm GAIP,not mi RNA.Purified SRBP1 protein from N.benthaminan specifically binds to mi RNA166.Both of them hint SRBP1 can binds to long RNA and mi RNA,the post-modification of SRBP1 may plays a key role in its mi RNAs binding ability.(5)Expression and interaction between SI novel gene ROH1 and Exo70A1 We demonstrate that the ROH1 gene is a single-exon gene encoding a 398-amino-acid protein in B.oleracea.In addition,ROH1 is expressed in stems,pistils,anthers,young roots and leave.Notably,expression levels varied in different tissues.After pollination,the m RNA expression level of ROH1 changed in an “up-down-up pattern” and peaked at 1 h post-pollination.The m RNA expression level of Exo70A1 tracked that of ROH1 implying that they are involved in reproductive development.Yeast two-hybrid assays established an interaction between ROH1 and Exo70A1 in B.oleracea. |
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