Study On Clong, Expression In Vitro And Interaction Of ARC1 And SRK In Self-Incompatible Signal Transduction Pathway Of Brassica Oleracea L.

Many bisexual flowering plants possess a reproductive strategy called self-incompatibility(SI) that enables the female tissue(the pistil) to reject self but accept non-self pollen for fertilization.SI response represents cell signal transduction process that allows the pistil to distinguish between self and non-self pollens and prevented self pollens germination.Pollination and fertilization in flowering plants involve a series of complex events with tightly regulated cell-cell interactions and signaling between the pollen and the pistil.More than half flowering plants possess a SI and involve 70 family,250 genus.Brassica oleracea L.is a classical Brassicaceae plant with the sporophytic self-incompatibility that was controlled by a single multi-allelic S locus genetically.In recent years,much progress has been made in determining the male and female S determinant in Brassica species.To date,in the signal transduction pathway of self-incompatible Brassica,three highly polymorphic genes with linked S locus have been identified.One is the S locus glycoprotein(SLG) gene that encoded a secreted glycoprotein abundantly present in the papillar cell of the stigma surface,one is S-locus receptor kinase(SRK)gene,the other is S-locus cystein-rich(SCR) gene.One after the other,three gene(SLG,SRK,SCR) isolated and characterized implies that upstream events of the SRK-mediated signal transduction pathway are more perspicuous.In the signal transduction pathway,ARC1 is a most interesting target that can interact with SRK specifically and transfer self-incompatible signal. Therefore,ARC1 band to SRK or not and stability of the formed compound is the key to regulate SI responses.In order to answer the former questions,we test the interaction between ARC1 and SRK, further analyze interaction mechanism,regulate separation ang aggregation of ARC1-SRK,and establish a screening system for chemical regulation of the interaction.Sequentially,these studies provide condition for further screening downstream targets of ARC1.In this paper,the highly self-incompatible Brassica oleracea E1 was taken as plant material. ARC1 and SRK were cloned,expressed respectively and in vitro confirmed interaction between interaction ARC1 and SRK.A detection system between the two determinates was established.By the research we provide the theoretical and technical foundation for further selecting controlled reagent and artificially regulating the aggregation and dissociation of SRK-ARC1 complex.The main results were as below:1 Cloning of coding sequence of ARC1 from B.oleracea E1 and expression in E.coli BL211.1 Cloning and sequence analysis of ARC1 geneThe DNA and cDNA fragments of ARC1 were amplified from genomic DNA and stigma total RNA of Brassica oleracea E1 by PCR and RT-PCR methods.Sequencing assay demonstrated that no intron existed in DNA sequences,and the cDNA of ARCl was 1,992 bp in length,encoding 663 amino acids,molecular weight was 73.5 kD.The nucleotide sequence of B.oleracea E1 ARC1 respectively showed 97%,94%,93%,95%,70%and 70%sequence identity to B.oleracea kale ARC1(Accession number in NCBI is EU344909),B.napus ARC1(Accession number in NCBI is AF024625),B.oleracea 20010197ARC1,A.thaliana PUB17(Accession number in NCBI is NM102674),and A.thaliana ARC1(Accession number in NCBI is AY150512).Little difference was showed in the bases of five ARC1 alleles(B.oleracea E1 ARC1,B.oleracea kale ARC1,B. oleracea 20010197ARC1,B.oleracea ARC1 and B.napus ARC1),but more difference in N-terminal of B.oleracea E1 ARC1 and A.thaliana ARC1.This explained that the C-terminal of ARC1 had more conservative than N-terminal.In addition to there were miss bases in the nucleotide sequence of B.oleracea and B.napus,and this miss bases with 'codon' didn't bring code mutation.The alignment amino acid sequence of B.oleracea E1 ARC1 respectively showed 97.9%,97.6%,93.2% and 53.2%sequence identity to B.oleracea kale ARC1,B.oleracea ARC1 20010197,B.napus ARC1 and A.thaliana ARC1.Phylogenetic tree of ARC1 proteins showed that B.oleracea E1 ARC1 was the closest to B.oleracea kale ARC1,B.oleracea ARC1 and B.oleracea ARC1 20010197,but the furthest to A.thaliana PUB17,A.thaliana cress ARC1.Database searches with both the DNA and amino acid sequences revealed that there were five arm repeat domains of C-terminal encoded amino acid 42 and a U-box domain that was located between amino acid 282^th and 347^th in B. oleracea E1 ARC1.In contrast to B.oleracea E1 ARC1,five arm repeat domains and a U-box domain that was located between amino acid 283^th and 347^th existed in B.napus ARC1,only an arm repeat in A.thaliana cress ARC1 and A.thaliana PUB17.The significant difference potentially determined the function discrepancy of ARC1in B.oleracea and A.thaliana.Prediction of active site of ARC1 showed that B.oleracea E1 ARC1contained active site of armadillo and the active site motif were poorly conservative in B.oleracea.In addition to ARC1 also contained Protein kinase C phosphorylation site,cAMP-and cGMP-dependent protein kinase phosphorylation site,Casein kinaseâ…¡phosphorylation site,Leucine zipper site,N-glycosylation site,N-myristoylation site, Amidation site,Tyrosine kinase phosphorylation site.They probably took part in the signal transduction of SI.In ARC1 protein,the three dimension models covered the 264-663 amino acid residues which wereα-helix and formed a hippocampus-shaped configuration and a wide groove and a narrow groove,which may be the binding sites of other proteins.1.2 Expression of ARC1 in E.coli BL21The plasmid pMD18-T-ARC1 and pET43.1a were extracted,and then they were digested by restriction enzymes EcoRâ… and Kpnâ… for purification and recovering of ARC1 cDNA and pET43.1a fragments.Two fragments were ligated by T4 DNA ligase to construct expression plasmid pET43.1a-ARC1,identified by PCR and two restriction enzymes digestion by EcoRâ… / Kpnâ… ,then pET43.1a-ARC1 was transformed into E.coli BL21.The recombinant strain BL21/ pET43.1a-ARC1 were induced by IPTG to express ARC1 fusion protein.The expression product were analyzed by SDS-PAGE,result showed that ARC1 fusion protein was expressed with molecular mass as expected at 135 kD.The primary analysis of inducing condition showed that inducing temperature has insignificant influence on expression of ARC1 fusion protein,but,IPTG concentration is little.So best-induced conditions were suggested as follows:temperature,32℃; concentration of IPTG,1.0mmol·L^-1;time:4 hour.When expression process was finish,ARC1 was purified by MagneHis^TM Protein Purification System and analyzed by SDS-PAGE.The result showed the only ARC1 band in the gels,although ARClwas inclusion.2 Cloning of coding sequence of SRK kinase domain from B.oleracea E1 and expression in E.coil BL21 2.1 Cloning and sequence analysis of SRK_E1 geneThe coding sequence of SRK kinase domain(termed SRK_E1) was amplified from Brassica oleracea E1 in this paper.Sequence analysis showed that the amplified fragment of DNA and cDNA were 1 711bp and 1 241bp respectively,SRK_E1 gene contained 6 extrons and 5 introns.The two ends of 5 introns complied with GT-AG rule.Alignment of the amino acid sequence of SRK_E1 with SRK₆, SRK₉₁₀,SRK₁₈,SRK₂₉ showed that the identity was 88.2%,85.7%,87.2%and 85.7%respectively. The amino acid sequence of SRK_E1 began from 4^th amid acid of the transmembrane domain, including 16 amid acids of latter transmembrane domain and the whole kinase domain,Cysteine of transmembrane domain and Lysine of active-site have no mutation,showing the amplified cDNA fragment of SRK_E1 could be used to express the kinase domain of SRK.Analyzing conserved domains of SRK_E1 by Blastp showed that the coding region of SRK contained tyrosine kinase domain and protein kinase domain,and protein kinase domain localized between amino acid 78 and 361.The phylogenetic analysis showed that different SRK alleles were classified into two groups, and classâ…¡,on the basis of their nucleotide sequences.Classâ… S haplotypes represent highly self-incompatibility,classâ…¡S haplotypes represent poorly self-incompatibility,and SRK_E1 clustered with SRK₄₅,SRK₃.So,Based on sequence diversity,the high self-incompatibility of E1 was demonstrated on molecular level.2.2 Expression of SRK_E1 in E.cali BL21The plasmid pMD18-T-SRK_E1 and pET43.1a were firstly extracted from each own strains,and then they were digested by restriction enzymes BamHâ… /Hindâ…¢.After the SRK_E1 cDNA and pET43.1a fragments were purified and recovered,they were ligated by T4 DNA ligase to construct expression plasmid pET43.1a-SRK_E1,identified by PCR and two restriction enzymes digestion by BamHâ… /Hindâ…¢,then pET43.1a-SRK_E1 was transformed into E.coli BL21.The recombinant strain BL21/ pET43.1a-SRK_E1 were induced by IPTG to express SRK_E1 fusion protein.The expression product were analyzed by SDS-PAGE,result showed that SRK_E1 fusion protein was expressed with molecular mass as expected at 74 kD,The primary analysis of inducing condition showed that IPTG concentration and inducing temperature only have a little influence on expression of SRK_E1 fusion protein.SRK_E1 fusion protein expressed in E.coli BL21 was mainly insoluble.3 Establishment way of interaction in vitro between ARC1 and SRK_E1Based on the principle of co-immunoprecipitation method,we put forward a new method in this study.The fusion protein of pET43.1a-SRK_E1 contains a 6×His target,which can combine with Ni⁺. The product of pET43.1a-SRK_E1 combined with Ni⁺ was used as 'bait',ARC1 solution as 'target'. The mixture of 'bait' and 'target' is,then,incubated at 4℃.The protein complex was purified from mixture through the absorbability of Ni⁺ with magnetic stand.In this study,via method,we have got perfect result.This suggests that this method is valuable to validate interaction between proteins. Compared with coimmunoprecipitation method,the new method shows advantages of antibody free, simple operation,and less fault result because of its weak specificity of antibody.In addition, constructed detection system is taken as a screening flat of chemical regulation reagents of the interaction between ARC1and SRK,which provides new information and clues for further analyzing mechanism of interaction and isolating and identifying unknown downstream targets in signal transduction pathway of self-incompatibility.4 In vitro assay of the interaction between ARC1 and SRK_E1The pET43.1a-SRK_E1 fusion protein expressed in E.coli BL21 contained a 6×His-Tag,which can combine with Ni⁺.SRK_E1 fusion protein having combined with Ni⁺ was used as 'bait'.The pET43.1a-ARC1 fusion protein expressed in E.coli was solved in buffer suited for interaction between ARC1 and SRK_E1.The SRK_E1 solution as a 'target','bait' and 'target' were incubated at 4℃for 2 h,mixing gently for every 15 min,then the complex of ARC1 and SRK_E1 was purified by the affinity between Ni⁺ and Magnetic stand,all products were analysis by SDS-PAGE.The result showed that ARC1 and SRK_E1 could act with each other to combine and possibly form a stable complex,and the interaction was not bond momentarily.