Studies On Cloning And Localization Of Three Genes Encoding Key Self-incompatible Elements In Stigma Of Brassica Oleracea And Corresponding Protein Interaction

Brassica have sporophytic self-incompatible (SSI) characteristics which prevent self-fertilization by rejection of'self pollen'by the stigma. Initial SSI response results from interactional binding between SRK and SCR encoded by two genes at S locus. Then the response of recognition between stigma and self pollen is transmitted from outer cells to internal cells. At the same time, ARC1is activated by the kinase domain of SRK. That activated of ARC1can interact with EXO70A1possibly triggers subsequent cascade reactions which results in self-incompatibility. After acquiring the genes of SRK, ARC1and EXO70A1, bioinformatics analysis was done in the thesis, and orientation of three genes at the chromosomes were confirmed by means of fluorescence in situ hybridization and Blast. At the same tine, the interaction of corresponding three proteins was analyzed by use of yeast two-hybrid system.1In this study, CDS and the corresponding gDNA of EXO70A1were cloned by PCR from Brassica oleracea, Brassica rapa and Brassica napus. Sequence analysis was carried out by means of bioinformatics. RT-PCR was used to analyze expression characteristics of EXO70A1in Brassica oleracea. And BoEXO70A1was transformed into yeast Y187in order to its expression in yeast. The results showed:The gene lengths of BnEXO70A1, BrEX070A1and BoEXO70A1were3797bp,3752bp and3770bp respectively, and all consisted of12exons and11introns; their identity positions reached91%. Sequence conservativation was higher in exons than that in introns (excluding4th,5th,6th and8th intron). CDS of three EXO70A1genes consisted of1917base pairs and sequence similarity was97.1%. Three proteins of EXO70A1conduced all consisted of638amino acids; consensus positions and identity positions of three EXO70A1were99.8%and98.1%respectively. BnEXO70A1, BrEXO70A1and BoEXO70A1had similar secondary structures and three-dimensional structures. All introns start from the sequence GU and end with the sequence AG (in the5'to3' direction). They are referred to as the splice donor and splice acceptor site, respectively. Another important sequence "CU(A/G)A(C/U)" in all introns of EXO70A1genes was located20～50bases upstream of the acceptor site. The corresponding sequence lengths of all12exons of EXO70A1were identical among three Brassica species and Arabidopsis thaliana; and similarity of4coding sequences was90.1%. Consensus positions and identity positions of four EXO70A1proteins reached99.8%and93.7% respectively. EXO70A1was a subunit of EXO70family which showed high conservative in Magnoliophyta. It showed low expression of BoEXO70A1in Y187. EXO70A1was a constutive gene which could be expressed in stamens, young stems, petals, pistils, young roots and leaves. However, its expression quantity is different in different tissues, with highest expression quantity in pistils and lowest one in stamens.2. CDS and the corresponding gDNA of ARCl were cloned by PCR from Brassica oleracea, Brassica rapa and Brassica napus. Sequence analysis was carried out by means of bioinformatics. The results showed:There existed no introns in ARC1from three kinds of plants. CDS of BoARC1gene consisted of1992base pairs (bp). However, the counterparts of BnARC1and BrARCl both consisted of1986bp. Identity positions of three ARC1's, CDS reached93.8%and there existed differences of144base pairs among them. The consensus positions and identity positions of three ARC1proteins were99.4%and92.2%respectively, there were differences of51amino acids among them. Three ARCl had identical domains and motifs, namely N-terminal UND (N-terminal domain), U-box (U-box domain) and C-terminal ARM (armadillo repeat). In addition, three ARCl had highly similar secondary structures and three-dimensional structures.3. CDS of BoSRK6was cloned, and31sequences of SRK were obtained from Genbank. Sequence analysis was carried out by means of bioinformatics. The results showed:SRK consisted of seven exons and six introns. It encoded proteins consisting of816to860amino acids. Signal peptides and S domain of SRK were encode by the first exon. Though different SRK had similar protein sequences, SRK showed high polymorphism due to three highly variable regions in its extracellular domain which played an important role in the interaction between SRK and SCR/SP11. The extracellular domain of SRK was highly similar to SLG in structure and sequence. That possibly originated from the gene duplication in the S locus.4. Young roots from Brassica oleracea were used to prepare for cell-wall free nuclei. The method of receding interface was used for subsequent preparation of chromatin fibers, and fluorescence in situ hybridization (FISH) with5S rRNA-targeted was selected to test the quality of chromatin fibers. The results showed that:Cell-wall free nuclei from young roots could be used to prepare for chromatin fibers free of pigments from chloroplast, which was favorable for subsequent FISH. The compactness of plant metaphase chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to FISH for single-copy or low-copy DNA sequences whose lengths were2-4kb or so. Only about5%slides were detected with FISH signal. If prolonging the time of chromosomes treated with pepsin, more FISH signals could be detected because of chromosome deaggregation. More signals could be detected when FISH on prometaphase chromosomes and pachytene chromosomes. FISH onto the chromatin fibers could get the optimal results.5. Localization of SRK:FISH onto metaphase chromosomes, pachytene chromosomes and chromatin fibers for gene localization on chromosomes with SRK gene as probes. At the same time, the location of SRK was verified by searching with BLAST at BRAD Brassica genome database (http://brassicadb.org/brad/blastPage.php). The result of FISH onto mitotic metaphase chromosomes is identical to that onto pachytene counterparts. Only one pair of green signal was detected on a pair of homologous chromosomes, which indicated that only one-copy SRK gene possibly existed in Brassica oleracea's genome. However, it was hard to define correctly the localization of SRK in the genome of Brassica oleracea because of the compactness of Brassica oleracea's chromosomes. SRK gene lay in the region of8732037-8675105bp of the B. oleracea' chromosome7by searching BRAD Brassica genome database. The corresponding region in Brassica rapa's genome was the region of8732037-8675105bp in the chromosome7.6. Localization of ARC1:The searching result by BLAST showed that:ARC1was also a single-copy gene in both Brassica oleracea'genome and Brassica rapa' genome. It lay in the region of35662808-35664799bp of B. oleracea's chromosome4. It located at the region of17204000-17300000bp in Brassica rapa's chromosome4.7. Localization of EXO70A1:The result of FISHing onto metaphase chromosomes and pachytene chromosomes for EXO70A1localization on chromosomes with EXO70A1gene as probe showed that:Only one hybridization signal was detected on mitotic metaphase chromosomes of Brassica oleracea, but sometimes two or three hybridization signals were detected on Brassica oleracea's pachytene chromosomes. After searching searching at BRAD Brassica genome database with BLAST, only one copy EXO70A1gene was detected and it lay in the region (39669464-39673197bp) of Brassica oleracea's chromosome9. In contrast, it lay in the region (17076191-17079967) of Brassica rapa's chromosome10. The result was different from that of FISH on pachytene chromosomes. That was possibly because there were one or two highly homologous genes of EXO70A1lying in Brassica oleracea's genome. 8. S locus receptor kinase (SRK) and ARM repeat containing (ARC1) are two key SI elements in Brassica. Interaction of SRK-ARC1possibly plays essential role in downstream SI signal transmission. In this study, in order to identify the interaction of SRKJ-ARC1during the course of SI, the segmental and full-length CDS of SRKJ and ARC1were amplified from B. oleracea var apitata and Brassica oleracea L var. acephala and then ligated to the plasmids of pGADT7and PGBKT7respectively. Then two recombinant plasmids were transformed to yeasts Y187and Y2HGold. At last the interaction of SRKJ-ARC1was tested by yeast two-hybrid system. The results showed that:The bait was confirmed not toxic to yeast and not to activate the expression of reporter genes by the test for autoactivation and toxicity. There existed five ARM repeats in ARC1, which had98%similarity at amino acid level between B. oleracea var acephala and B. oleracea var apitata. That three experimental groups Y2HGold[pGBKT7-ARC1]×Y187[pGADT7-SRKJ],Y2HGold[pGBKT7-ARC1T3]×Y187[pGADT7-SRKJ] and Y2HGold[pGBKT7-ARC1T4]×Y187[pGADT7-SRKj] could grow on QDO/x/A nutritional media with transcription activation of the reporter genes AUR1-C, MEL1, HIS3and ADE2indicated that there existed interaction between ARC1from B. oleracea var acephala and SRK from B. oleracea var capitata and the interaction domain was located at the domain of ARM repeats, and the difference at amino acid level with the ARC1of B. oleracea var capitata was not enough to change the conformation in the interaction region. All above mentioned provides some insights into the molecular mechanism of self-incompatibility in Brassica.9. EXO70A1is possibly an important SI element in Brassica. EXO70A1-ARC1possibly plays essential role in downstream SI signal transmission. In order to identify the interaction of EXO70A1-ARC1during the course of SI, the segmental and full-length CDS of ARC1and EXO70A1were amplified from B. oleracea and Brassica napus and ligated to the plasmids of pGADT7and PGBKT7respectively. Then two recombinant plasmids were transformed to yeasts Y187and Y2HGold. At last the interaction of EXO70A1-ARC1was tested by yeast two-hybrid system. The results showed that:Sequence analysis showed that ARC1consisted of663amino acids in Brassica oleracea and661amino acids in Brassica napus, and there existed45amino acids difference between them. Sequence alignment showed that similarity positions and identity positions reached95.9%and93.9%between BoARC1and BnARCl, whereas there existed only six-amino-acid difference between BoEXO70A1and BnEXO70A1. The Similarity positions and identity positions reached99.4%and98.9%between BoEXO70A1and BnEXO70A1respectively. The homology of EXO70A1was higher than that of ARC1. Yeast two-hybrid results indicated that the strong interaction existed between ARC1and EXO70A1, and it could activate the expression of four reporter genes (ADE2, HIS3,AUR1-C, and MEL1) in diploid yeasts. However, low interaction existed between EXO70A1and ARC1N with316amino acids deleted from C-terminal, and it only activated the expression of three reporter genes (ADE2, AUR1-C, and MEL1). This provides an insight that the interface of interaction between ARC1and EXO70A1may not consist of the domains of arm repeats in ARCl. N-terminal domains of ARC1play an essential role in the interaction of ARC1-EXO70A1. The influences of the differences in amino-acid composition between B1ARCl and BnARCl on the interaction of ARC1-EXO70A1couldn't be detected by means of yeast two-hybrid system, which probably resulted from that the binding interface between ARC1and EXO70A1was not altered by sequence difference of two proteins in two Brassica species.Thus conclusions can be drawn that:1. ARC1and EXO70A1, in Brassica species, are both high conservative; EXO70A1perhaps plays very important roles in plant cells because of its different expression characteristics in different plant tissues. The conservation of SRK was relatively slightly lower than that of ARC1and EXO70A1in Brassica. High polymorphism of resulted from the three high variable regions in SRK, and it was an important reason of molecular evolution of S locus genes leading to self-incompatibility of Brassica.2. Young roots can be prepared for cell-wall free nuclei. Gene localization using FISH and searching genome databases can obtain the same or similar results. However, FISH seems not to be suitable for the orientation of DNA sequences with which there are one or more highly similar sequences. It indicated that FISH has some limitations.3. ARC1can interact with SRK and EXO70A1. The interface of interaction between ARC1and kinase domains of SRK consists of the domains of arm repeats in ARC1. N-terminal domains of ARC1, containing U-box, interact directly with EXO70A1.