| Self-incompatibility (SI) is controlled by S-locus in Brassica. SRK gene is the key factor for SI, and was cloned from Brassica oleracea and Brassica napus. The mutation and expression of SRK gene would affect the self-incompatibility. S-domain is polymorphism and often occurs in the coding region. Compared to the S-domain, kinase domain is more conservative. Kinase domain of SRK gene was cloned and analyzed first time, in order to investigate the kinase domain for the role of SI from an evolutionary of view. MLPK was also proved an essential factor in SI and was the role of downstream component of SRK. In the paper, we located the SRK and MLPK genes associated with SI of Brassica oleracea on different extend chromosomes by dual-color FISH. The result showed that:1. By PCR and RT-PCR, The gDNA and cDNA fragments of the gene encoding S-locus receptor kinase(SRK) kinase-domain was amplified from Brassica oleracea 263, Brassica oleracea AY627 and Brassica napus. And we constructed a phylogenetic tree of SRK. The lengths of gDNA an d cDNA were 1694 bp and 1307 bp,1705 bp and 1229 bp,1606 bp and 1214 bp, respectively. Sequence analysis indicated that the genomic DNA of Brassica oleracea 263 contained five exons and four introns, encoding 433 amino acids, and both the genomic DNA of Brassica oleracea AY627 and B.napus contained six exons and five introns, encoding 407 and 402 amino acids, respectively. Based both three nucleotide sequences cloned in this study and SRK sequences of twenty-first species released by NCBI, we constructed a phylogenetic tree of SRK. The three sequences were in the same group with classâ… , and classâ… and classâ…¡was divided into two groups. Arabidopsis thaliana and the classâ…¡were in the same group, but Raphanus sativus was't formed a single group. By comparing of the three cDNA fragments, we found that there is a 70 bp of DNA insertion in Brassica oleracea 263, which may be one of the cause leading to the phenotype of the SC. We also found there were not any insertion and deletion in Brassica oleracea AY627 and Brassica napus, indicating that the SC of Brassica napus may not be caused by the kinase domain. The analysis of phylogenetic tree indicate that differentiation between Brassica and Raphanus sativus was later than the differentiation between classâ… and classâ…¡of SI in Brassica.2. In the FISH process, we changed some conditions between prometaphase chromosome and DNA fibers. In the prometaphase chromosome. Our research have acquired the most suitable time of cold temperature treatment, which was 20 hours. We use enzyme mixture to treat the prometaphase chromosome for 90min, And we obtained the best cytological target slide without background and with better stretched state. We also changed some conditions to obtain the DNA fibers. Through improved method, such as the different treatment with TritonX-100, we obtained Nuclei with symmetrical. Through these changes,we obtained extended DNA fibers.3. Two color hybridization signals were detected on a homologous chromosome, respectively. But the detection rate was low. One hand, the reason may be that the quality of homologous chromosome was not so well. Another hand, the single-copy and small fragments of probe affected the FISH. And SRK and MLPK gene was only located on Chromosome 1 and Chromosome 3, respectly. We couldn't detect the signal on DNA fiber. And the reason may be that the physical and chemical characteristics was undermined.
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