Analysis Of MLP Family Genes And Functional Research Of BoMLP423 In Brassica Oleracea

Self-incompatibility(SI)refers to the phenomenon of the hermaphrodite plants with normal function and mature male and female gametes cannot be fertilized when pollinated with self-pollen or cross-pollen with the same genotype.According to the regulation mechanism,the self-incompatibility can be divided into gametophytic self-incompatibility(GSI)and sporophytic self-incompatibility(SSI).Brassica is a typical sporophyte self-incompatible plants(SSI),and the research on their molecular regulate mechanism mainly focuses on the SI signal transducers which could inhibit self-pollen germination or pollen tube growth.Recent years,it makes a remarkable progress on the mechanism of sporophyte self-incompatibility signal transduction in Brassica oleracea,but the isolation and identification of SRK downstream factors also relatively deficiency.ARC1 and its homologous family genes have the function of E3 ligase,which degrade downstream substrates by ubiquitination.In Arabidopsis thaliana,At ARC1 is a pseudogenetic factor,when co-transferred SCR-SRK of Arabidopsis lyrata,the transgene plants showed a strong self-incompatibility.It demonstrated that ARC1 may be not the only downstream factor of SRK in self-incompatibility signal transduction.In this study,the selected highly self-incompatibility "A4" and "F1" were used as the materials,transcriptome and proteomic technologies were recruited to screen differential gene expression of pistils in the early self-pollination and cross-pollination process.We analyzed it from the aspects of bioinformatics,subcellular localization,prokaryotic expression,promoter activity analysis,q RT-PCR,yeast two-hybrid,T-DNA insertion and so on.The main results as follows:1.Gene identification and expression analysis of MLP family genes in Brassica oleracea(1)Through plant genome data and Pfam 32.0 database,we got 29 major latex family protein genes in BoMLP family.They were randomly distributed on 8chromosomes in B.oleracea,in which 6 chromosome contains 7 genes,3 and 5chromosome contain 1 gene.(2)All of the BoMLP family proteins contain a conserved Bet-v-1 domain,the length was 143-218 aa,relative molecular mass was 16.6 to 24.3 k Da,and the isoelectric points was 4.64 to 6.05.(3)There were 29 members of BoMLP in B.oleracea,and could be divided into I-III subfamilies according to the similarity of evolutionary relationship and sequence.Each subfamily genes were high homology and had a closely evolutionary relationship.Promoter sequence analysis results showed that most of the BoMLP promoter regions contained a cis-element which responses to defense or stress,including abscisic acid response element(ABRE),participation,dehydration,induced response element(MYB),gibberellin stress response element(WYKY),defense stress element(MYC),and auxin response element(ARF).Transcriptome profile analysis found that among the 29 BoMLP genes,only BoMLP3,BoMLP4,BoMLP6,BoMLP10,BoMLP11,BoMLP15,BoMLP16,BoMLP423 and BoMLP27 differentially expressed after self-pollination and cross-pollination.(4)The fluorescence quantitative PCR results showed that the m RNA expression levels of BoMLP3,BoMLP4,BoMLP6,BoMLP10,BoMLP11,BoMLP15,BoMLP16,BoMLP423 and BoMLP27 in the self-pollination and cross-pollination processes were consistent with the transcriptome.It hints that those BoMLP genes may involves in self-incompatible reactions in B.oleracea.2.BoMLP423 cloning and functional research in Brassica oleracea(1)After treated with self-pollen and cross-pollen to highly self-incompatible B.oleracea “A4”,we collected stigma and analyzed protein expression profiles at post-pollination 0 min and 60 min.One protein named BoMLP423 was identified,which was down-regulated expressed.Transcriptome data showed that the m RNA expression level of BoMLP423 was first down-regulated expressed at 0-15 min,then up-regulated expressed at 15-30 min,at last extremely down-regulated expressed at 30-60 min,however,it was not significantly changed m RNA expression level in cross-pollination at 0-60 min.Fluorescence quantitative PCR results showed that the m RNA expression levels of BoMLP423 showed a “down-up-down” pattern in the early self-pollination process,and almost not changed m RNA expression levels in the early cross-pollination process.Those results consistent with the transcriptome and proteomic results.(2)The full gene length of BoMLP423 was 989 bp,the coding region was 468 bp.It is a hydrophilic protein,contains two exons and one intron,encodes 155 amino acid residues,the relative molecular weight was 17 k Da,and theoretical isoelectric point was5.12.(3)BoMLP423 located in the cytoplasm.q RT-PCR results showed BoMLP423 was expressed in stamen,petal,sepal,leaf,stigma and bud in B.oleracea,which was highly expressed in stigma and bud.(4)BoMLP423 prokaryotic expression results showed that its protein was 17 k Da.(5)The promoter region of BoMLP423 contains much more cis-acting response elements,including drought induced response,Me JA response,auxin response,abscisic acid response,defense stress response and so on.(6)Yeast two hybrid results showed that the full length of BoMLP423 did not interact with SRK.We speculated that BoMLP423 may not directly participate in the SRK-ARC1-EXo70A1 signal transduction pathways.(7)At MLP423 is a BoMLP423 vertical homologous gene in Arabidopsis thaliana,mlp423 mutant showed short filaments and short pod length phenotype.Those results showed that BoMLP423 may participate in the self-incompatibility response in the stigma cytoplasm in B.oleracea.Our results will provide a new direction for further studying BoMLP423 function in self-incompatibility response.