Cloning Of MOD Gene In Brassica Oleracea Associated With Self-incompatibility And Localization Of MOD And ARC1 In Brassica Oleracea By FISH

SI (self-incompatibility, SI) is a prezygotic breeding barrier that allows a stima to distinguish between self- and cross-pollen. ARC1 and MOD are two very important downstream proteins among members of the signaling process. SI signal components have been further studied by genetically modified and gene knock-out technology to various studied these years. However, genome copy numbers and location of signal elenments studies little in brassica plants. MOD genes have been cloned and sequence analysis in Brassica oleracea, peculiarly preserved in our school in order to make clear the structure and its genetic MOD water way. In addition, MOD gene and ARC1 gene were located in the medium-term, medium-term and broccoli period of thick thread chromosome by by fluorescence in situ hybridization technology, so we can understand both the Physical location and copy Numbers in Brassica oleracea genome. The main results obtained as follows:1. The gDNA and cDNA fragments of MOD gene were amplified in B.oleracea and B. napus by PCR and RT-PCR. We initially obtained three gDNA fragments with length of 1350bp,1449bp and1477bp respectively and one cDNA fragment with length of 867bp contained the whole ORF frame of MOD. Through homologous and phylogenetic analysis of amino acid sequences, we speculated MOD coding protein of B.oleracea belongs to the PIP aquaporins and one member of MIP family. Compairison between gDNA and cDNA sequences indicated:MOD gDNA of B.oleracea and B.napus were all contained 3 introns and they all followed the splicing rule of "GT-AG". Variations exists in introns were higher than those in exons by 14.4%-17.3%. There were 53 differences in coding sequence which only cause only 3 amino-acid variations between BoMOD and BnMOD, indicating that the low-frequency variations of coding regions were mainly nonsense mutations. There were 4 possible phosphorylation sites in amino acid sequence of MOD, being in agreement with the phosphorylation as a final step to regulate water transportation in the molecular process of Brassica SSI. 2. Preparation of prometaphase and pachytene chromosomes by flame drying the FISH DNA vector, and explored the key factors of moviemaking production procedures, making of the prometaphase and pachytene chromosomes moviemaking technique has been further optimization. For the prometaphase chromosome, through of the optimization to obtain more prometaphase splitting collection after by recovery culture to 1.4-17cm long apical; the most appropriate time of cold treatment should be controlled between the 16h-20h; optimal pretreatment time within 50-60min at best 0.002mol/L 8-hydroxyquinoline; the best deal 40min with 0.075mol/L of potassium chloride solution for before and after the hypotension; he best baking sheet timing 5s.The best results for early pachytene Brassica chromosome, use of mobile baking sheet 10s-15s and with Lateral flame.3. Based on the former, we improved some parameters of the FISH technique to obtain a FISH system that applied to different extended cytological target in Brassica oleracea. We found, disgeste prometaphase chromosome 40min and early pachytene chromosome 30min respectively with lug/ml pepsin may increase the chance of success in hybridization. During FISH, plus 7.5% dextran sulfate can improve probe concentration obviously, so we only selected 1.0nguL-1 probe and got high noise ratio hybrid result and fewer false signals. The elution conditions controlled as follows:washing at 37℃in 2×SSC,4×SSCT (0.2% Tween20) and 1 x PBS all for 5 minutes.4. We located SI releted gene MOD and ARC1 in Brassica oleacea on prometaphase and early pachytene by FISH. Results show, ARC1 labed with digoxin and MOD labed with biotin only detected one pair signal at most in early pachytene, which indicate that the both gene may be single copy Brassica oleacea genome. According to karyotype analysis we found the karyotype of Brasscica oleacea was 2A. Through repeat hybrid twice on one film, we got one red signal point of MOD on prometaphase, but the chromosome morophology was too vague to do physical location of it. We didn't get hybride signal of ARC1 on prometaphase. The results indicated it is possible that MOD and ARC1 are both single or low copy gene in Brassica oleracea genome.