Interactional And Functional Analysis Of The Roles Of MAPK4 With ARC1 In Pollination From Ornamental Kale

Plant mitogen-activated protein kinase（MAPK）is a serine-threonine protein kinase that plays important functions in plant abiotic stress,biotic stress,growth and development of plants.Plant self-incompatibility（SI）refers to the response mechanism by which plants reject self-pollen and accept alien pollen in order to maintain genetic diversity.Ornamental kale（Brassica oleracea var.acephala）,an important ornamental and edible plant in the north China,was used as the experimental material in this paper.The BoMAPK4 gene from the stigma of ornamental kale was isolated by RT-PCR and then its sequence was analyzed.The BoMAPK4 protein was obtained by prokaryotic expression and purification,and the polyclonal antibody was prepared for Western blot analysis of protein expression in various tissues of ornamental kale and protein expression changes after pollination.In order to analyze the protein interaction between BoMAPK4 and SI-related regulatory proteins SRK and ARC1,yeast two-hybrid system and Luciferase Complementation Assay（LCA）were performed.Finally,in order to analyze the role of BoMAPK4 in pollination,U0126 inhibition experiment and MAPK4 phosphorothioate antisense oligonucleotide inhibition（Anti-sense Oligonucleotide Inhibition,AS-ODN）experiments were carried out.The main research results obtained are as follows:（1）The open reading frame of ornamental kale BoMAPK4 is 1122 bp long,encoding 373amino acids,with serine/threonine domain,no signal peptide and no transmembrane structure.Besides,the phylogenetic tree analysis and sequence alignment found that the amino acid sequence identity of ornamental kale BoMAPK4 and Bn MAPK4（Brassica napus）,Br MAPK4（Brassica rapa）,At MAPK4（Arabidopsis thaliana）was 99.7%,99.5%,95.4%,indicating that BoMAPK4 is highly conserved across species.（2）The sequence of the BoMAPK4 coding region was constructed into the prokaryotic expression vector p ET-14b,and transformed into BL21（DE3）E.coli for prokaryotic expression through IPTG induction.The results of SDS-PAGE show that the recombinant protein of BoMAPK4 are specifically expressed at a molecular weight of about 45 k Da.High-purity recombinant BoMAPK4 protein was obtained by the method of affinity chromatography.（3）Polyclonal antibodies against BoMAPK4 were prepared by immunizing mice with BoMAPK4 protein and the specificity was good.The total protein was extracted from sepals,petals,anthers,stigma,style and ovary of ornamental kale.Western blot analysis showed that BoMAPK4 was less expressed in petals,anthers and ovaries,more expressed in sepals and styles,and the highest expression in stigma.The expression level of BoMAPK4 was analyzed on the stigma 60 min after compatible pollination（CP）and the stigma 60 min after self-incompatible pollination（SI）.The results showed that the expression of BoMAPK4 in the stigma of compatible pollination increased at 60 min,while the expression of BoMAPK4 in the stigma of incompatible pollination decreased.（4）The vectors p Myr-BoMAPK4,p GADT7-BoMAPK4,Nluc-SRK₉₁₀,Nluc-SRK_13b,Nluc-Bo ARC1 and Cluc-BoMAPK4 were constructed,and then the yeast two-hybrid experiment was carried out.The results showed that BoMAPK4 did not interact with SRK₉₁₀,SRK_13band Bo ARC1 in the cytoplasm,but with Bo ARC1 in the nucleus.LCA results observed the interaction of BoMAPK4 and Bo ARC1 fluorescence signal.（5）The stigma of self-incompatibility line(S_13-bS_13-b)of ornamental kale was inhibited with U0126.Self-incompatibility pollination and compatible pollination are then performed.Aniline blue staining results showed that the number of SI pollinated pollen grains and pollen tubes did not change significantly after adding U0126,but the number of compatible pollinated pollen grains and pollen tubes decreased significantly after adding U0126.（6）The Oligodeoxynucleotide（ODN）of ornamental kale MAPK4 was designed.The stigma of self-incompatibility line(S_13-bS_13-b)was incubated with S-ODN（control）and AS-ODN（MAPK4-inhibiting specific sequence）for 4 h.Then,the stigmas after two different incubation treatments were subjected to compatible pollination and self-incompatibility pollination,respectively.The results of aniline blue staining experiment showed that the number of pollen grain adhesion and pollen tube germination in the stigma incubated by AS-ODN decreased significantly during compatible pollination,but did not change significantly during incompatible pollination.