Cloning Of KAPP Gene Associated With Self-incompatibility In Brassica Oleracea And Localization Of KAPP And THL1 In Brassica Oleracea By Dual-color FISH

Self-incompatibility (SI) is a very important genetic mechanism to avoid close relative heredity and increase mutation for many flowering plants. Actually, it is a cell signal transduction pathway after the recognition between the ligand in pollen and the receptor in pistol. In Brassica, SI recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus. However, the physical location relationship between S-locus and downstream relative genes in SI signal transduction has not been reported, and the linkage relationships of S-locus and the SI relative genes have also not been identified up to now. Additionally, Brassica oleracea is one of the three basic species in Brassica. In the process of its evolution and produce new types, there had occurred lots of recombination and genetic mutation, which obviously influence transfer and genetic variation of SI genes among Brassica. Furthermore the research of KAPP gene which encodes the general negative regulator in RLK (Receptor-like kinases) signaling is not so deep. In this paper, we have not only cloned and analysed the KAPP gene but also located the KAPP and THL1 genes associated with self-incompatibility of Brassica oleracea on different extended chromosomes by dual-color FISH. Our results showed that:1. The gDNA and cDNA fragments of KAPP gene were amplified from genomic DNA, bud RNA and leaf RNA in Brassica oleracea by PCR and RT-PCR and other molecular biology methods. We initially obtained a fragment of KAPP gDNA with length of 3247 bp, a fragment of KAPP cDNA with length of 1699 bp, as well as a bud cDNA with length of 1578 bp and a leaf cDNA with length of 1581 bp which were called KAPP2 cDNA of bud and KAPP2 cDNA of leaf respectively. Compared gDNA with cDNA of KAPP, we found that there were 11 introns in KAPP gene, and these introns all comply with typical GU-AG rule. Between KAPP cDNA which we cloned and KAPP cDNA released there are 6 nucleotide differences, but they encode a same amino acid sequence. Sequences analysis of the KAPP1 cDNA which we cloned from bud cDNA and leaf cDNA in Brassica oleracea showed that they share 85.2% and 85.0% identity with the released KAPP cDNA respectively. We also found that the nonsense mutation in 590 bp of KAPP2 of bud cDNA and 593 bp of KAPP2 of leaf cDNA led to the early appearance of termination codon whose blast indicated that both of them shared more identity in Arabidopsis thaliana than in Brassica oleracea. Based on KAPP cDNA sequence of eight species released by NCBI and two sequences of KAPP2 cDNA we cloned, a phylogenetic tree of KAPP gene were constructed, which showed that the two KAPP2 sequences were in the same group with released KAPP cDNA in Brassica oleracea. Based on all the above analysis, as well as some view of the comparative mapping research, we speculated that KAPP gene may have more than one copy in the genome of Brassica oleracea, the KAPP2 sequence we cloned may be the other copies of KAPP gene. Moreover, they are likely to be the pseudogene which was inactive by mutation during evolution process. It has an important significance for the deep research between KAPP and Self-incompatibility. The results will provide some new insights into the research of molecular mechanism in SI and molecular evolution in Brassica oleracea.2. Through regulating the relative technique system, we acquired preferable technique system of FISH targets preparation in Brassica oleracea. Zygotene chromosomes, pachytene chromosomes, prometaphase chromosomes and other two types of chromosomes were successfully prepared by using this technique system, which were applied to fluorescence in situ hybridization. Our research have acquired the most suitable time of cold temperature treatment, which was 12 hours. Except this, we found that the best pretreatment time of 8-hydroxyquinoline was 50-65 minutes. We use enzyme mixture of 2.5% cellulose and 2.5% pectase to treat the zygotene chromosomes, early pachytene chromosomes and pachytene chromosomes for 1 hour, and then we obtained the best cytological target slide without background and with better stretched state through roasted and pressed the slide. 3. We obtained dual-color FISH system be applied to different extended cytological targets through improving the reported FISH system. The treatment time of enzyme mixture of 2.5% cellulose and 2.5% pectase for prometaphase chromosomes and metaphase chromosomes was 40 minutes, but the treatment time of enzyme mixture for Zygotene chromosomes, early pachytene chromosomes and pachytene chromosomes was 30 minutes. During FISH, the dextran sulfate was removed in the hybridization mixture in which the proportion of the two probes with different tag was 1:1. The condition of washing at 37℃for single or low copy probe was adjusted to washing in 4xSSCT(0.2%Tween20),2xSSC and 1xPBS respectively for 4 and a half minutes. We can get a better FISH results with low backgrounds and high rate of detected signal through that way.4. Studies of KAPP and THL1 genes for self-incompatibility of Brassica have been gradually widespread since they were identified. However, the position and copy number of KAPP and THLl genes in Brassica oleracea genome are still unclear. In this paper, localization of KAPP and THL1 genes for self-incompatibility of Brassica oleracea on zygotene chromosomes, pachytene chromosomes, prometaphase chromosomes and other two types of chromosomes was conducted successfully by dual-color fluorescence in situ hybridization. The results indicated that the signals of KAPP probe which labeled by biotin were detected on two pairs of homologous chromosomes and the signals of THL1 probe which labeled by digoxingenin were detected on one pair of homologous chromosome. According to karyotype standard of Armstrong, the karyotype analysis of prometaphase chromosome shows that KAPP gene was located on the chromosome 4 and 9, and their percent distances from centromere to the signal point were respectively about 60.46±5.14 and 79.5±3.35, THLl gene was located on the chromosome 5, its percent distance from centromere to the signal point was 65.42±2.82. Integrating hybridization signals from five kinds of cytological targets shows that KAPP gene might have two copies and located at two different loci in Brassica oleracea genome, but THL1 might be a single copy gene in Brassica oleracea genome. Additionally, we discussed the collinearity relationship of KAPP as well as THL1 between Brassica and Arabidopsis thaliana on the basis of comparative genomics. Combined the existing results of the research in S-locus, we can primarily concluded that neither KAPP nor THL1 is linked to S-locus. The research on the distribution characteristics of KAPP, THL1 and S-locus in Brassica oleracea genome will have an important significance for the exploration to the time and space specificity of the regulation of SI related gene expression.