Molecular Cloning And Expression Analysis Of Genes Associated With Self-incompatibility Of Brassica Oleracea L.and Their Protein Interaction Analysis

Self-incompatibility is an important mechanism to prevent selfing in close relative, promote outcrossing, and maintain genetic diversity of species. And also provide an ideal pattern system to analysis the signaling transduction in plant cells. Utilizing different haplotype self-incompatible line to produce seeds is an important breeding mode in production, but there have defects like multifarious production process and low efficiency. So this research not only provided new knowledge to explain the signaling transduction of self-incompatibility, but also provided theoretical basis of studying and utilizing self-incompatibility.At prevent, it is widely believed that at the florescence of Brassica crops, when the pollen falling on the stigma, the SCR come from pollen can be recognized and combined with the extracellular domain of SRK, this leads to the challenge of SRK conformation, and then release the THL1/2that combine with the kinase domain of SRK, activate the kinase activity of SRK. And then SRK interact with ARC1, and ARC1phosphorylated by SRK, and then go through a series of unknown pathway to transmit the signal from extracellular, finally leads to germinate blocked of the self-pollen. Although recent years there were so many research achievements about signaling transduction of self-incompatibility, but they were mainly about the recognition and interaction analysis of determinant factors of self-incompatibility, they were focus on the relation between expression regulation of SRK and SCR with self-incompatibility, and the interaction analysis of SRK and THL1/THL2. Relative to these aspects, researches of the isolation and identification of proteins associated with signaling transduction pathway of SI were behindhand, after ARC1identified to the downstream signaling protein at1998, the SI core factor Exo70A1could be interact with ARC1was found until2009. But unlike with each of the SCR and SRK mutation or deficiency would lead to the material completely lost SI character, antisense suppression expression of ARC1and overexpression of Exo70Al would reduce the material SI character just a little.This declared that the ARC1and Exo70A1pathway maybe just a branch of the SRK downstream signaling transduction pathway, there exit other unknown proteins combined with ARC1to transmit SI signaling in stigma. But until now, the isolation and identification researches about downstream signaling proteins have no reported.Based on the international research progress, aimed to explore the research pointcut of signaling transduction of SI, analysis the molecular mechanism of downstream signaling transduction better. This thesis based on the former study about the difference expression of proteins response to self-and cross-pollination in SI stigma, the homozygous lines A4and F1of Brassica oleracea L. as the materials, utilized molecular biological technique to screen and clone these signaling factors maybe participate in downstream signaling transduction pathway, and did the expression analysis of these genes, also utilized yeast two-hybird system to analysis the interaction of these downstream factors. The main research results are as following:(1) Based on the former study about the difference expression of proteins response to self-and cross-pollination in SI stigma, we screened eight proteins maybe associated with self-incompatibility:BosiPA1, BPS1, KPDP, MLP423, Tubulin, Annexinl, Annexin2and Actin; Based on others’ study, we screened there proteins associated with self-incompatibility:ARC1, Exo70A1and MLPK. We cloned these genes by homologous amplification, and gone through bioinformatics analysis. There were two significant genes, one was BosiPA1, it was a bHLH transcription factor, the sequence analysis declared that it maybe participate in SI reaction by directly regulate SI core factors SCR, SRK and Exo70A1. The other one was MLPK, we got four transcripts of this gene:MLPKf1, MLPKJ2, MLPKm and MLPKi, MLPKfl/2were specificity for Brassica, they maybe emerged after speciation of Brassica and A.thailiana, they were positive factors for SI reaction. MLPKi was a new gene, and AtAPK1b was the ortholog of the MLPKi not of the MLPKfl/2. The MLPKm was a mosaic gene when MLPKfl and MLPKi coexpress. These results contribute to provide new knowledge to study SI signaling network.(2) Use the RT-PCR technology, the cDNA of petal, sepal, pollen, stigma and leaf on the flowering day as templates, we acquired these genes expression specificity in different organs. BosiPA1, BPS1, KPDP, Tubulin, Annexinl, Annexin2and Actin expressed in all five organs; MLP423expressed in petal, sepal, pollen and stigma but have no expression in leaf; MLPKf2specific expressed in stigma, MLPKfl and MLPKi expressed in all five organs, MLPKm expressed in petal, pollen, stigma and leaf but have no expression in sepal. Use the Real-time quantitative PCR technology to analysis the Time and Space Expression specificity, non-pollination stigma cDNA, self-pollination5min,30min,1h stigma cDNA, and cross-pollination5min,30min,1h stigma cDNA as templates, we acquired these genes expression specificity in different pollination mode and period, BosiPAi, BPS1, KPDP, MLP423, Annexinl1Annexin2and Exo70A1have uprising expression after self-pollination, Tubulin have down expression after self-pollination, these results provide basis to downstream experiments.(3) Builded yeast expression vectors of Actin、Annexin、Tubulin、ARC1、 Exo70A1、 MLPKfl、MLPKm and MLPKi, they were pGADT7-Actin, pGADT7-Annexin, pGADT7-Tubulin, pGADT7-ARC1,PGBKT7-Exo70A1,pGBKT7-Annexin, pGBKT7-Tubulin, pGBKT7-MLPKf1, pGBKT7-MLPKm and pGBKT7-MLPKi. We detected the interaction by yeast two-hybird system, the couples as followings: pGADT7-ARC1×pGBKT7-Exo70A1, pGADT7-ARC1×pGBKT7-MLPKf1, pGADT7-ARC1×pGBKT7-MLPKm, pGADT7-ARC1×pGBKT7-MLPKi, pGADT7-Actin×pGBKT7-Exo70A1,pGADT7-Actin×pGBKT7-Tubulin, pGADT7-Actin×pGBKT7-Annexin, pGADT7-Annexin×pGBKT7-Exo70Al, pGADT7-TubulinxpGBKT7-Exo70A1and pGADT7-TubulinxpGBKT7-Annexin, the results showed that there were no direct interaction in ARC1×Exo70Al, ARC1×MLPKf1, ARC1×MLPKm, ARC1×LPKi, Actin、Exo70A1, ActinxTubulin, Actin×Annexin,Annexin×Exo70A1,Tubulin×Exo70A1and Tubulin×Annexin.(4) The subunits of Exocyst complexus cloned by homologous amplification, they were SEC3, SEC10, SEC15, SEC84N and SEC84C. And then builded yeast expression vectors of these genes, they were pGADT7-SEC3, pGADT7-SEC10, pGADT7-SEC15, pGADT7-SEC84N, pGADT7-SEC84C and pGBKT7-Actin. We detected the interaction of the subunits of Exocyst complexus and the core factors Exo70A1, Actin, Tubulin, Annexin of SI in Brassica oleracea L. by yeast two-hybird system, those couples as followings:pGADT7-SEC3xpGBKT7-Exo70Al, pGADT7-SEC10×pGBKT7-Exo70A1, pGADT7-SEC15×pGBKT7-Exo70A1, pGADT7-SEC84N×pGBKT7-Exo70A1.pGADT7-SEC84CxpGBKT7-Exo70A1, pGADT7-SEC3×pGBKT7-Actin, pGADT7-SEC3×pGBKT7-Annexin, pGADT7-SEC3×pGBKT7-Tubulin,pGADT7-SEC10×pGBKT7-Actin, pGADT7-SEC10×pGBKT7-Annexin,pGADT7-SEC10×pGBKT7-Tubulin, pGADT7-SEC15×pGBKT7-Actin,pGADT7-SEC15×pGBKT7-Annexin, pGADT7-SEC15×pGBKT7-Tubulin,pGADT7-SEC84N×pGBKT7-Actin, pGADT7-SEC84N×pGBKT7-Annexin,pGADT7-SEC84N×pGBKT7-Tubulin, pGADT7-SEC84C×pGBKT7-Actin,pGADT7-SEC84C×pGBKT7-Annexin and pGADT7-SEC84C×pGBKT7-Tubulin, the results showed that there were direct interaction in Exo70A1and SEC3, Exo70A1and SEC84N.