Study On Prokaryotic Expression And Interaction Between Determinant Factors Of Self-Incompatibility(SI) And Exploration On SI Related Gene In Brassica Oleracea L.var.capitata L.

Self-incompatibility(SI) is widely distributed genetic mechanism of angiosperm.Plant evolved SI mechanism for cross pollination that allows plants with perfect flowers to avoid inbreeding and keep the diversity and independent,so survive and development could be guaranteed.In the self-incompatibility(SI) response of Brassica oleracea L.var.capitata.L.,the recognition of self-related pollen by the stigma epidermis is affected by two tightly linked and highly polymorphic genes which located in the S locus:â‘ The S locus cysteine-rich protein gene SCR[also designated SP-11]encodes a small hydrophilic and positively charged peptide that is localized to the pollen coat.A series of studies such as mutation analysis,sequence alignment,transgenic pollen acquisition and pollination bioassay finally proved that SCR is the male determinant of SI.â‘¡The S locus receptor kinase gene SRK encodes a single-pass transmembrane serine/threonine kinase,which displayed with its glycosylated N-terminal S domain external to the cell and its C-terminal kinase domain within the plasma membrane of the stigma epidermis.As a female determinant of SI,it shows the specificity recognition of stigma and transfer SI signal to downstream element.An allele-specific interaction between the SRK and SCR is the first and determined step to start SI signal transduction.When SI Brassica oleracea L.pollinated with"self'pollen,the SCR signal molecules within the pollen coat were carried onto the surface of stigma epidermis,a further diffusion through the cell wall makes it interact with the SRK receptor on the plasma membrane and activates the SRK kinase. In turn,this activation presumably triggers a signaling cascade that inhibits pollen hydration and germination.Many signal factors involved in sophisticated SI signal transduction,but only few factors which are SRK,SCR,THE1,THL2,ARC₁ and MLPK were found.Isolation of an unknown phosphoprotein from Brassica oleracea L.,which is assumed as a SI related protein,by D.Wu in 2003 might gives a clue to further studies.In this paper,the highly self-incompatible cabbage(Brassica oleracea L.var.capitata.L)'ZQ' was taken as plant materials.Female and male determinates of SI were cloned and expressed in vitro respectively and an interaction detection system between the two determinates was established.By the research we went to provide the theoretical and technical way for further study on mechanism and reagent control of the interaction between SRK and SCR.On the other hand,RT-PCR and 3'-RACE were used to clone the SI related gene in cabbage'E₁' for new information and clues.The main results were as below:1.Cloning and expressing of the coding sequence of S locus cysteine-rich protein/S locus protein 11(SCR/SP-11)The cDNA of SCR was cloned from pollen total RNA of the cabbage.'ZQ' by nested RT-PCR. Sequence analysis shows that ZQ belongs to the class-â… S haplotypes S₂₈ of Brassica oleracea L., This S class haplotypes exhibit strong SI phonotype gives another proof besides the low ISC(index of self-compatibility).Analysis of the coding sequence of SCR indicated that it encodes a 7.8kD protein covered the predicted three amino acid residue VEA in the C terminate of signal peptide and the whole mature peptide of SCR_fZQ amino acid sequence.Besides the eight conserved cysteine residues and one conserved glycin residue,two nonconservative cysteine residues were found in the SCR_ZQ encoding amino acid sequence.The predicted disulfide bonds in SCR_ZQ showed that:Except for Cys57 could not form the disulfide bonds with any other cysteine residues in the protein,the other conserved cysteine residues formed the disulfide bonds to stabilize SCR protein.The 3D model indicated that SCR_ZQ formed a crimple coil,including anα-helix and three-stranded antiparallelβ-sheets.After SCR_ZQ was transformed into DH5αpET43.1a and the pMD18-T-SCR_ZQ were double digestion and recovering,later ligation by T4 DNA ligase constructed expression plasmid pET43.1a-SCR_ZQ.The recombinant strain BL21/Pet43.1a-SCR_ZQ were induced by IPTG to express Nus·A-SCR_ZQ fusion protein.The expression products were analyzed by SDS-PAGE,and result showed that SCR_ZQ fusion protein was expressed at expected molecular weight 74kD. Orthogonal design was used for analyzing the expression adjusted volume.The result showed that inducing conditions have little influence on expression of SCR_ZQ fusion protein.When expression process was finished,SCR_ZQ was purified by MagneHis^TM Protein Purification System and analyzed by SDS-PAGE.2.Cloning and expressing of the extracellular domain of S locus receptor kinase (mSRK)2.1 Cloning and sequence analysis of the extracellular domain of S locus receptor kinaseThe 1319 bp cDNA of mSRK was amplified from the total RNA of stigma.of'ZQ'.Nucleotide sequence analysis of mSRK reached the same conclusion of SCR_ZQ sequence alignment:'ZQ' shared the same sequence with Brassica oleracea L.S₂₈,and was closely related to Brassica rapa S₅₄. Analysis of the encoding amino acid sequence of mSRK showed that,the encoding protein contains 439 deduced amino acids with the molecule weight 49.3 kD.The first 16 amino acids encoded hydrophobic signal peptide and the final 10 amino acids were deduced transmembrane domain sequence,12 conserved cysteine residues as the sign of the S-family protein and a number of active sites including six N-glycosylation sites were scattered in the amino acid sequence of mSRK.mSRK protein structural analysis results showed that:mSRK contains B-Lectin domain,SLG-domain and PAN_APPLE domain.The three polar amino acid sites P(350),T(352),R(369) in PAN_APPLE domain were putative protein or sugar-binding sites.Two 3D models of mSRK were acquired from Bio-information websites.Model 1 corresponds to 14-264 amino acid residues in mSRK protein, which contains B-Lectin domain and part of SLG domain,forming a hippocampus-shaped configuration It consists of two 12β-stranded -prismâ…¡fold and a linker located in the abdomen of the hippocampus.Model 2 covers the 285-391 amino acid residues of mSRK protein,which encodes partial SLG domain and PAN_APPLE domain,and shows one a-helix and fiveβ-sheets,containing the twelve conserved cysteine residues and three putative protein or sugar-binding sites.Further analysis of functional sites prediction shows that functional amino acids sites are highly conserved on cysteine residues in mSRK protein,so these conserved cysteine residues might play an important role in mSRK biological functions.Predictions also indicated that 10 of the 12 conserved cysteine residues would form five pairs of disulfide bonds within the protein,while the remaining two cysteine residues Cys366 and Cys370 might remain free,or forms disulfide bonds with other proteins. 2.2 Prokaryotic expression of the extracellular domain of S locus receptor kinase(mSRK)mSRK was constructed into the prokaryotic expression vector in two forms,pET43.1a-Nus·A-mSRK expression vector is conducive to further analysis of its interaction with the SCR protein. Because the Nus·A fusion peptide could significantly increase the mSRK solubility and activity when expressed in E.coli.While mSRK expression vector without Nus·A fusion peptide was constructed to exclude the possibility of that the His tag in Nus·A fusion peptide might get false positive result in the later interaction detection,as an important supplement to verify the Nus·A -mSRK and SCR interactions.Both Nus·A-mSRK and mSRK were expressed in E.coli BL21 with their expected molecule weight about 112.0kD and 50.0kD respectively.Analysis of the expressing of mSRK protein under different induction conditions,the result shows that temperature was significantly effect the expression of Nus·A-mSRK,so best-induced conditions were suggested as follows:temperature:25℃;concentration of IPTG:0.1 mmol·L-1;time:1 hour.While there were insignificant differences on expression of mSRK in different inducing conditions.The BL21 Bacterial cells which expressed Nus·A-mSRK protein were first sonicated and then resuspend in the cell lysis reagent.Aftet incubated with Ni-Particles and recovered by Magnetic stand,purified Nus·A-mSRK fusion protein was finally obtainded.3 In vitro assay of the interaction between SCR and SRKmSRK and Nus·A-mSRK prokaryotic expression proteins were incubated with SCR_ZQ fusion protein respectively for 2 hours in suitable interaction reagents.Because SCR_ZQ fusion protein contained a 6×His Tag,which could chelate with Ni⁺,the complex of SCR_ZQ and SRK interaction product could purified by the affinity between Ni⁺ and Magnetic stand,all products were analyzed by SDS-PAGE.The result showed that SCR_ZQ could combine with mSRK in vitro and possibly form a stable complex.4 Probe into SI related gene induced by pollination of Brassica oleracea L.According to the N-terminal amino acid sequence of phosphoprotein found by Dr.Wu, degenerate primers were designed for 3'-race,and then analysis the encoding amino acid of the obtained nucleotide sequences,we screened out a candidate gene and e-splicing it with EST sequences,expect to find out the full-length sequence encoding phosphoprotein.However,by analyzing the seven amplified sequences,we finally failed to get a sequence encoding the same amino acid with the N-terminal phosphoprotein.But we found out a new sequence,and designed as NG,no sequences could be found homologous to it in other species,only a few ESTs could align in the Brassica EST database.The deduced amino acid sequence of NG had the highest homology with the heat shock protein-like factor of Arabidopsis thaliana.RT-PCR analysis showed that the expression of NG gene is higher in stigma,lower in leaf but have no expressions in other tissues. The putative opening reading frame of NG were cloned and ligated to pET43.1a vector to express the NG in vitro,finally a molecular weight of 91.2kD of NG fusion protein was expressed and purified for further study.