Cabbage SI determinant of the prokaryotic expression and interaction studies, and SI-related genes

Title: Cabbage SI determinant of the prokaryotic expression and interaction studies, and SI-related genes ofAuthor: Yang YangDegree-granting units: Southwestern UniversityKeywords: self-incompatibility;; SRK;; SCR/SP11;; interaction;; S locus-related factors;; cabbageSummary:Self-incompatibility (self-incompatibility, SI) is a widely present in ancient genetic trait in angiosperms. Plants evolved through a long cross-pollination in favor of the SI genetic mechanism for flowering plants to avoid inbreeding depression and self-pay, is conducive to maintaining species diversity and independence, for the species survival and development of a strong of protection.In cabbage, the stigma of the pollen S locus Neodymium Magnets  recognition depends on the simultaneous presentation of two closely linked highly polymorphic genes: ① S locus cysteine-rich gene (S locus cysteine-rich protein, SCR), and called the S locus protein 11 gene (S-locus protein11, SP11), the gene encoding a pollen shell at polar hydrophilic small molecule protein. By mutant analysis, sequence alignment, identification of genetically modified and pollination range of evidence that it is the male-determining factor. ② S locus receptor kinase Keegan (S locus receptor kinase, SRK) encodes a transmembrane serine / threonine kinase receptors, the kinase in the stigma papilla cell membrane, rich in glycosylation sites of the N-terminal S prominent in the region outside the cell membrane, C-terminal serine / threonine kinase integrated in the cell membrane. As self-incompatibility and female determining factor signal transduction, the decision stigma specific S genotype and delivered self-incompatibility and signals. S genotype of the same allele SRK and SCR interaction is induced by self-incompatibility and the first step in signal transduction pathway, activated the self-incompatibility and the signal transduction pathway.Self-incompatibility of cabbage in the self-pollination, when pollen adhesion to the stigma, pollen can be carried by the signal molecule diffusion SCR was transported to the stigma surface, and by the further proliferation in the papilla cell membrane of protoplasm SRK signal receptor interaction. SRK is activated after receiving the signal, causing a cascade of http://www.everbeenmagnet.com/en/products/110-sintered-neodymium-magnets intracellular signaling, and ultimately inhibited pollen germination. SI in the complex signaling network, has been fixed in self-incompatibility factors and signal transduction only SRK, SCR, THL1, THL2, ARC_1 and MLPK, there are many signal factor was not found. In 2003, Wu table by ~ (32) P labeled autoradiography from the self-incompatibility and plant for a specific unknown protein phosphorylation, suggesting that the pollination-induced self-incompatibility and related factors, and may be used as self-incompatibility and downstream signal transduction factors, for the self-incompatibility of the molecular mechanism of the study provide new content.In this study, a high degree of self-incompatibility of cabbage ZQ materials, cloned, and self-incompatibility signaling in male and female determining factor, and for in vitro expression analysis, established for in vitro interaction between SRK and SCR detection system for further screening of chemical control agents to achieve the SRK-SCR complex aggregation and dissociation of human control and provide a theoretical and technical basis. While the gene encoding the site-directed mutagenesis and the use of large-scale protein interaction research methods such as yeast two-hybrid, mass spectrometry, protein chips, and bioinformatics-based analysis, in-depth study of the mechanism of interaction between SRK and SCR provides an interaction system technology platform. Also use a high degree of self-incompatibility of cabbage material E_1 design degenerate primers with 3'-RACE and for self-incompatibility gene screening and analysis, with a view to self-incompatibility and the unknown signal separation and identification of downstream provide new information and clues. Main work and the results are summarized as follows:1.S locus cysteine-rich protein / S locus protein 11 (SCR/SP11) Cloning and expression of the coding regionThe use of nested RT-PCR, with kale ZQ anther cDNA as a template, the SCR sequence was amplified ZQ. SCR_ (fZQ) Sequence analysis showed that the S-genotypes of ZQ materials are cabbage S_ (28) haplotypes. According to the known classification of S haplotype analysis, ZQ is the first class Ⅰ S haplotypes, with a high degree of self-incompatibility, which is field testing the self-affinity index was 0.004 the same conclusion. Further coding region clones SCR_ (ZQ) sequence analysis showed that: SCR_ (ZQ) encoding SCR_ (ZQ) protein C-terminal signal peptide and mature peptide of three amino acids VEA to predict protein-coding size 7.8kD. Encoding 64 amino acids in SCR proteins containing eight conserved cysteine ​​and a characteristic amino acid glycine site, also contains two non-conserved cysteine. Of these 10 cysteine ​​disulfide bond formation analysis showed that, in addition to conservative Cys57 can not form disulfide bonds within proteins, and the remaining conserved cysteine ​​within the protein disulfide bonds are formed to stabilize the SCR_ (ZQ) of protein conformation. Three-dimensional structure analysis shows that SCR_ (ZQ) protein was wrinkled spiral, contains an α-helix and three antiparallel β-sheet. SCR_ (ZQ) is converted into digestion and connected to build a fusion expression vector pET43.1a-SCR_ (ZQ), and received a size of approximately 74kD induced the Nus · A-SCR fusion protein. By orthogonal design, quantitative analysis of the expressed protein derived from the relative intensity of the correction value, SCR_ (ZQ) in the induction of expression under the conditions of both high-volume, in which the expression of factors influence the expression of the fusion protein did not reach significant levels. SCR_ (ZQ) expression by protein purification kit bacilli after treatment, can be significantly mixed with the purified protein-free.2.S locus receptor kinase extracellular domain coding region (mSRK) Cloning and expression of2.1 S locus receptor kinase extracellular domain coding region (mSRK) Cloning and sequence analysisZQ in cabbage successfully cloned mSRK stigma length 1319bp cDNA sequence, nucleotide sequence analysis and comparison SCR_ (fZQ) sequence alignment analysis results are consistent: ZQ materials, the S genotype and cabbage S_ (28) gene exactly the same type of sequence, followed with the rape of S_ (54) close genetic relationship. mSRK amino acid sequence analysis of deduced amino acid 439, encoding proteins the size of 49.3 kD. The first 16 amino acid hydrophobic signal peptide sequence, the last 10 amino acid sequence for the transmembrane domain, the middle with a sign S multigene family of 12 conserved cysteine. It also contains six N-glycosylation sites and a number of active sites. mSRK encoded protein analysis showed that high-level structure: mSRK contains B-Lectin domain, SLG domain and PAN_APPLE domain. Which PAN_APPLE domain of three polar amino acid sites P (350), T (352), R (369) speculated that the protein or sugar binding sites. Division of mSRK protein modeling, to obtain two mSRK 3D models. Model 1 corresponds to the mSRK protein first 14-264 amino acids, including the B-Lectin domain and part of the SLG domain, forming a configuration such as the hippocampus, contains two 12-strand β-prism-like fold and hippocampus abdomen short Ⅱ connection area. Model 2 corresponds to the mSRK protein first 285-391 amino acids, covering some of the SLG and the PAN APPLE domain domain. Mainly by an α-helix and five β-sheet consisting of, containing 12 conserved cysteine ​​and the putative 3 protein or sugar binding sites. Further functional site prediction, conservative amino acid high concentration of functional active site cysteine ​​in mSRK protein locus, inferred mSRK cysteine ​​protein has an important biological functions. 12 conserved cysteine ​​10-cysteine ​​is predicted to form five pairs of disulfide bonds within the protein, while the other two cysteines, Cys366 and Cys370, or preserving the free status, or is other proteins in the paired cysteine ​​residues form disulfide bonds.2.2 S locus receptor kinase extracellular domain coding region (mSRK) prokaryotic expressionIn this study, two forms of mSRK constructed prokaryotic expression vector. pET43.1a-Nus · A-mSRK expression vector containing Nus · A fusion peptide, used to increase mSRK soluble in E. coli expression and expression of activity, help to further analyze the interaction of proteins with SCR. Away except Nus · A fusion peptide pET43.1a-mSRK expression vector, mainly to exclude Nus · A fusion peptide containing the His tag detection system in the interaction may result in false-positive effects, as a Nus · A- mSRK protein SCR_ (ZQ) validate protein interactions important supplement. Will be constructed pET43.1a-Nus · A-mSRK with pET43.1a-mSRK expression vector successfully expressed in E. coli BL21, respectively, express the size of about 112.0kD and 50.0kD of the target protein. In Nus · A-mSRK expression in prokaryotic expression temperature effects on protein expression significantly, induced the best conditions: temperature 25 ℃; IPTG concentration of 0.1 mmol · L ~ (-1); time 1h. mSRK by the expression conditions was not significant. Lysis by sonication expression bacilli, adding Ni ~ + bead recycling target protein, to obtain a purified Nus · A-mSRK protein.3.SCR_ (ZQ) and mSRK protein interactions in vitro testingUse SCR_ (ZQ) fusion protein on the histidine-tagged beads with Ni ~ + chelation, the SCR_ (ZQ) fusion protein, respectively, and the construction of the two prokaryotic expression system pET43.1a-mSRK/pET43.1a -Nus · A-mSRK induced expression of protein and Ni ~ + beads in a suitable mSRK and SCR_ (ZQ) interacting protein extract in the incubation 2h, washing purification of the complexes were purified by SDS-PAGE analysis, we verified mSRK protein and SCR_ (ZQ) protein in vitro is the ability to combine with each other to form a stable binding complex. Also shows mSRK protein and SCR_ (ZQ) protein interaction in vitro does not arise from a combination of the moment.4 pollination-induced genes of Brassica SIAccording to Wu tables phosphorylated proteins found in the N-terminal amino acid sequences of degenerate primers for 3'-race PCR, and nucleic acid sequences coding for amino acids derived from the analysis, we screened for candidate genes and EST sequences of electronic splicing. Analysis of the six amplified sequence encoding the phosphorylation do not meet the N-terminal protein sequence. However, we found a new sequence named NG, currently not found in other species with homologous sequences, but in the EST database to find the cabbage can be spliced ​​expressed sequence tags. NG according to analysis of the open reading frame encoding the amino acid sequence with the Arabidopsis heat shock protein-associated factor with high homology. Preliminary RT-PCR analysis showed that NG have high levels of gene expression in the stigma, micro expression in leaves, anthers, stems and roots no expression. Then we will speculate NG sequence coding frame in accordance with primers designed to amplify the coding region after the connection to the pET43.1a expression vector was constructed pET43.1a-NG expression vector. Successfully induced in E. coli BL21 expression and purification of the fusion protein of about 91.2kD purpose, in order to facilitate a new protein-coding sequence NG function of speculation.Degree Year: 2009