The Study On S-locus Receptor Kinase Interaction Proteins In Brassica Oleracea

Self-incompatibility(SI)is a common genetic mechanism in flowering plants that allow plants to promote inter-generic hybridization,prevent inbreeding and maintain genetic diversity.Brassica crops have typical sporophytic self-incompatibility(SSI)characteristics.The hotspot of the international research about SSI molecular mechanism mainly focus on the synergistic effect of SI signal transduction components to inhibit self-pollen germination or pollen tube growth.Early,it was generally believed the SI signaling pathway is initiated only upon the landing of a self-pollen grain on the dry stigma.The pollen S-locus cys rich(SCR/SP11)specifically recognized and combined with extracellular domain of S receptor kinase(SRK),which caused transformation of SRK conformation,and then released the THL1/2 that combined with the kinase domain of SRK,to activate the kinase activity of SRK.Then the activated SRK with MLPK occurred interaction in an instant,and the SI signal was transferred to the stigma papilla intracellular.In the same time,ARC1 combined with SRK,and was phosphorylated,leading to cytoplasmic localization.Subsequently,being an E3 ubiquitin ligase,the activated ARC1 promoted ubiquitination degradation of compatibility factors Exo70A1 in the pistil,resulting in the failure of pollen germination and tube growth.However,it was still not clear that how the ubiquitination of Exo70A1 was achieved and what was the core region to constitute SRK-ARC1-Exo70A1 complementary interface determining interaction relationship,as well as the key amino acid residue composition in interaction region.And it was uncertain whether ARC1 was the sole substrate for SRK.Although significant advances have been made in understanding the Brassica self-incompatibility response during recent years,the researches about the isolation and identification of SRK downstream components in self-incompatibility signaling were behindhand.Since ARC1 was identified function as downstream targets of the SRK protein in 1998,it was until in 2009 that Exo70A1 was found ubiquitinated by ARC1 and degradated in SI pathway.However,antisense suppression of the ARC1 m RNA level in the self-incompatible Brassica napusa and Arabidopsis lyrate only leaded to a partial breakdown of self-incompatibility,and increasing Exo70A1 expression in self-incompatible B.napusa partially overcame the self pollen rejection.These results implied that the proposed SRK-ARC1-Exo70A1 pathway only presented one branch of the complex SI signaling network.The receptor kinases signal transduction pathways were usually composed of a number of signal components and signal branches.For self-incompatibility signal transduction process,current international generally considered that there were other unknown signal components involved in downstream regulation.In Brassica and Arabidopsis ecotypes,the different signal branch had different effect on signal transduction,so the focus of current work was to isolate and identify these unknown signal proteins and investigate their functions.Based on this,the self-incompatibility homozygous lines "A4" of Brassica oleracea L.was used as material in this study.The coding sequence of SRK kinase domain,ARC1 and Exo70A1 were amplified.The core interaction domains of SRK-ARC1-Exo70A1 were identified by a series of bioinformatic analysis,yeast two-hybrid screening and pull-down assays.Based on the transcriptome sequencing analysis,we screened some PUB proteins that interacted with SRK.Meanwhile,a novel MLPK homologous gene MLPKn1 was identified.The expression profile and functional analysis was conducted through bioinformatic analysis,q RT-PCR,transient expression and transgenic technology.The main results obtained were as follows:(1)The determination of core interaction domains between SRK-ARC1 and ARC1-Exo70A1The cDNA fragment of SRK kinase domain,ARC1 and Exo70A1 was amplified from the stigma of Brassica oleracea using molecular cloning technology.The sequence analysis showed the kinase domain of SRK consisted of three subdomains: DUF3660,Tyrkc and DUF3403.ARC1 contained four domains as follows: leucine zipper,coiled-coil domain,U-box motif and ARM domain.Exo70A1 was also divided into four subdomains: leucine zipper,hypervariable region,SUMO modification motif and typical pfam Exo70 domain.And on this basis,the 21 truncated fragments containing different functional domains were individually amplified from Brassica oleracea.Then the encoding sequences of SRK kinase domain with its truncation and the full length Exo70A1 with its truncation were separately subcloned into the p GADT7 vector to generate the AD recombinant plasmids.The encoding sequence of ARC1 with its truncation were respectively constructed into p GBKT7 vector to generate the BD recombinant bait plasmids.Recombinant plasmids were respectively co-transformed into the strain AH109,and then were planted on SD/-Leu-Trp-His-Ade/X-a-gal/25 m M 3-AT nutritional media to detect the growth and color change.The ?-galactosidase assay was conducted to detect interaction strength.The results showed,with the extension of the amino acid sequence,a greater ?-galactosidase activity was induced,in which the combinations of SRK kinase domain with ARM showed relatively high levels of ?-galactosidase activity(15.98),indicating the kinase domain of SRK and the C-terminal ARM of ARC1 was the core interaction domains of SRK-ARC1.No interactions were detected for either truncating SRK functional domains or ARM of ARC1.Meanwhile,the combinations of ARC1 contained the leucine zipper with coiled-coil structure and Exo70A1 contained the hypervariable region and SUMO modification motif exhibited the highest levels of ?-galactosidase activity in all combinations of ARC1-Exo70A1(25.07),which was the core domain mediated the interaction between ARC1-Exo70A1.In addition,comparing the ?-galactosidase activity level,it was not difficult to find that the interaction strength of SRK-ARC1 was less than that of ARC1-Exo70A1.(2)Based on the RNA-seq,three novel PUB proteins were screened out that interacted with S-locus receptor kinaseThrough comparing the transcriptional level of self-pollination 0 min and 30 min stigma,we identified five differentially expression genes,named as Bo PUB3,Bo PUB9,Bo PUB34,Bo PUB43 and Bo PUB44.Among them,Bo PUB3 and Bo PUB9 showed a significantly downregulated expression after self-pollination 30 min.Bo PUB34,Bo PUB43 and Bo PUB44 displayed an upregulated transcriptional level after self-pollination 30 min.The cloning and sequence analysis of candidate genes found that all of them belonged to plant U-box family.In addition to Bo PUB34 belonged to the Ser/Thr kinase subfamily,the other was the PUB-ARM subfamily.Phylogenetic tree analysis revealed Bo PUB9 was the most closely related to ARC1,and Bo PUB34 was the most distantly related to ARC1.Moreover,the yeast two hybrid screening and GST-pull down assay found that ARM domain of BoPUB3,BoPUB9,BoPUB43 and ARC1 could interact with SRK kinase domain,and their interaction strength was Bo PUB9>Bo PUB3?Bo PUB43>ARC1,but no interaction was detected between SRK with Bo PUB44 and Bo PUB34.Expression analysis showed Bo PUB3 is mainly expressed in stigma,and showed an obvious continuous downregulated transcriptional tendency within 15 min,30 min and 60 min after self-pollination.Bo PUB9 and Bo PUB43 were widely expressed in petal,sepal,anther,stigma and leaf.We speculated that Bo PUB3 might participate in the self-incompatibility response.(3)Cloning and expression analysis of MLPK and its orthologs gene MLPKn1 in Brassica oleraceaThe cDNA of Brassica oleracea "A4" stigma was used as templates.Bo MLPKf1/2 and Bo MLPKn1 was amplified usingDNA polymerase.Sequencing results showed that the CDS of Bo MLPKf1 was 1,215 bp in length,encoding 404 amino acids.The full-length CDS of Bo MLPKf2 was 1,233 bp in length,encoding 410 amino acids.Bo MLPKn1 was a new transcript,and had a length of 1,257 bp,encoding 418 amino acids.Bo MLPKn1 and Bo MLPKf1 had a similar gene structure,both their deduced amino acid sequences contained a typical plant myristoylation consensus sequence at their N terminus,while Bo MLPKf2 had a hydrophobic region in N-terminal.Compared to the Bo MLPKf1/2,Bo MLPKn1 contained two fragment insertions: one was a 12 bp insertion located between 1,102 to 1,104 bp,another was a 30 bp insertion located between its 1,152 to 1,182 bp.The three different MLPK transcripts contained a conservative Ser/Thr protein kinase domain.Semi-quantitative RT-PCR analysis showed Bo MLPKn1 were widely expressed in petal,sepal,anther,stigma and leaf,similarly to Bo MLPKf1.Bo MLPKf2 was predominantly expressed in the stigma.QRT-PCR analysis displayed Bo MLPKf2 had sharply increased expression within the initial 15 min that sharply declined 30 min after self-pollination.The expression levels of Bo MLPKf1 and Bo MLPKn1 were slightly increased after self-pollination,but the change was not significant.Both Bo MLPKf1 and Bo MLPKn1 localized to the plasma membrane by transiently expressed green fluorescent protein fusions two MLPKs in protoplasts.Bi FC assay revealed Bo MLPKn1 could interact with SRK and ARC1 in the plasma membrane.Synteny analysis revealed that Bo MLPKn1 was the orthologous genes of At APK1 B,not the formerly reported Bo MLPKf1/2.Brassica MLPKf1/2 might have emerged after speciation of Brassica and A.thailiana,and that it was recruited to the SRK triggered SI signaling cascade in Brassica.MLPKn1 was highly conserved in evolution of mustard family and did't be involved in SI response.(4)The functional analysis of MLPKn1's orthologs gene At APK1 b in ArabidopsisBo MLPKn1 was the orthologs gene of At APK1 B.According to the gene structure of At APK1 b,two sg RNAs,sg R1 and sg R2,were designed to contain guide sequences in the first exon regions and the third exon regions.Sg RNA expression constructs were synthesized using overlapping PCR with p UC119-At U6::g RNA plasmid DNA,and then inserted into the multiple cloning sites of expression vector Cas9-p G626.The constructed vector was transformed into A.thaliana successfully by Agrobacteriummediated transformation.0.03% glufosinate-ammonium(Basta)was used as the selective agent to select transgenic lines.After T0 Basta-resistant screening and PCR sequencing,we got 13 T1 generation At APK1 b transgenic mutations.Among them,there were deletion in the length of 21-509 bp,and there was also the phenomenon of additional insertion and mutation.The preliminary phenotypic observation found that transgenic plants was small and weak,but they could normally blossom and bear fruits.Further phenotypic observations were required when the homozygous mutations were obtained.(5)The coding sequence analysis of S-locus receptor kinase related factor in the self-compatibility and self-incompatibility materialsThe cDNA of self-compatible Brassica rapa "Yellow sarson" and self-incompatible Brassica oleracea "A4" stigma were used as template to amplify SRK,MLPK,ARC1 and Exo70A1 gene encoding sequence.SCR gene encoding sequence was amplified from the cDNA of their anther.Sequence alignment of SCR and SRK revealed that Brassica oleracea "A4" was consistent with the S28 haplotype,and the "Yellow sarson" material was consistent with the f2 haplotype.Amino acid sequence comparison found that Bo SRKA4 with Br SRKB shared sequence identity as high as 99%,and with Br SRKB was 97%.The sequence identity of Bo SCRA4 and Br SCRB/Q was 92%,and there were five amino acid differences.The sequence identity of Bo ARC1 and Br ARC1B/Q was 93%.Bo Exo70A1 shared 99% identity with Br Exo70A1B/Q,respectively,and there was only one amino acid difference.These results indicated that SCR,SRK,ARC1 and Exo70A1 genes were highly conserved in "Yellow sarson" and Brassica oleracea "A4".Comparison Bo MLPK and Br MLPK amino acid sequence,we found there were six amino acid differences,and they were K116-R,L183-I,G194-R,S288-N,T350-N and A365-E in Bo MLPK.According to the former reports,the G194 R form of MLPK exhibited no autophosphorylation activity.Therefore,there was no direct correlation between self-incompatible and gene sequence of SCR,SRK,ARC1 and Exo70A1,but it was not unknown whether MLPK mutation caused the self-compatible of "Yellow sarson" material.(6)The effect of MLPKf2 overexpression on “Yellow sarson” phenotypeMLPK occured mutation in "Yellow sarson" material,and Bo MLPKf2 was specificly expressed in the stigma.In order to explore the MLPK function in “Yellow sarson”,we designed the primers with restriction sites according to the full-length sequence of Bo MLPKf2 and multiple cloning sites of plant expression vector.The MLPKf2 was inserted into plant binary vector p CAMBIA1300,resulting in the generation of overexpression vector p CAMBIA1300-Bo MLPKf2,contained a stigma-specific SLR promoter.The expression vector was transformed into hypocotyls and cotyledons of "Yellow sarson" seedling using Agrobacterium mediated method.After pre-culture,infection,dark culture,callus differentiation,adventitious buds,rooting and transplanting process,the transgenic plants were obtained.Then gDNA identification of transgenic plants by PCR showed that Bo MLPKf2 were intergated to the genomicDNA of "Yellow sarson" plants successfully.Besides,the real-time fluorescence quantitative PCR analysis displayed Bo MLPKf2 expression levels in transgenic plants were much higher than those of wild-type plants.The phenotype showed T0 transgenic plants have reduced seed production in comparison to wild-type plants that produces full seedpods,suggesting that the overexpression of MLPKf2 could partially restore the self-incompatibility of "Yellow sarson".