Dissertation > Excellent graduate degree dissertation topics show

Cloning of KAPP Gene Associated with Self-incompatibility in Brassica Oleracea and Localization of KAPP and THL1 in Brassica Oleracea by Dual-color FISH

Author: WuWeiZuo
Tutor: ZhuLiQuan
School: Southwestern University
Course: Biochemistry and Molecular Biology
Keywords: Cabbage Self-incompatibility KAPP THL1 Fluorescence in situ hybridization
CLC: S635.1
Type: Master's thesis
Year: 2010
Downloads: 54
Quote: 0
Read: Download Dissertation

Abstract

Self-incompatibility is an important mechanism to prevent close genetic variation increased flowering plants, and its essence is a mutual recognition of ligands and receptors in the pollen and stigma caused by cellular signal transduction process . Including cabbage Brassica self-incompatibility by a polymorphism S gene locus with multiple alleles. However, the S-locus with SI downstream signal transduction factor encoding gene in the chromosome of the physical positional relationship also been reported between them, whether or not in the the Brassica genome of the distribution chain and downstream signal transduction factor The location is not yet known. In addition, cabbage as three basic species of the genus Brassica, in its evolution and new species of the synthesis process, a lot of restructuring and genetic variation events. This greatly affects the Brassica self-incompatibility and gene transfer between genetic variation in Brassica. Secondly, KAPP this widely from the downward effect on the protein factor receptor protein kinase-mediated cell signal transduction pathway gene encoding not very well studied. Therefore, this study not only the cabbage KAPP gene cloning and sequence analysis by two-color FISH technique KAPP with THLl two SI signal transduction pathway related genes on different target DNA vector to locate. The main results are as follows: 1. PCR.RT-PCR and other molecular biology methods, cabbage genomic DNA, RNA bud and leaf RNA as a template, the cabbage KAPP gDNA and KAPP cDNA amplification length of the 3247bp KAPPgDNA fragment length 1699bp Kapp cDNA fragment of 1578 bp bud KAPP2 cDNA fragment and 1581bp leaves KAPP2 of cDNA fragments. Cloned the cabbage KAPPgDNA and cDNA (combined reported KAPP cDNA) than Brassica KAPP gene contains 11 introns, are in line with the GU-AG splicing rule, and the cloned the Kapp cDNA sequence reported KAPP cDNA sequence has a single nucleotide at the difference, but the difference between the amino acid sequences were identical. However buds KAPP2 cDNA fragment reported leaves KAPP2 cDNA KAPPcDNA sequence similarity of 85.2% and 85.0%, respectively. These two sequences were 590bp and 593bp at the earlier onset of a nonsense mutation caused by a stop codon. Blast analysis showed that the two genes encoding the amino acid sequence of Arabidopsis KAPP highest similarity of the amino acid sequence, followed by the Brassica KAPP amino acid sequence. The reported eight kinds of plant KAPP CDS sequences and the experimental cloning two KAPP2 sequence constructed phylogenetic tree showed that the sequence of Brassica KAPP sequence clade. Comparative mapping and molecular phylogenetic tree, suggesting that KAPP gene has two copies in the Brassica genome, and the author cloned into the KAPP2 cDNA sequence is another copy, KAPP evolution of the proposed gene mutation inactivated. FISH target DNA preparation technology through the exploration of the program so that the cabbage has been further optimized. The use of these optimized preparation technology and thus suitable for FISH cabbage before the mid-term and the metaphase chromosomes producer, and zygotene to pachytene chromosomes of. In this study, optimization of the cold treatment time synchronization has been the most appropriate cold treatment time of 12h. In addition, optimization of 8 - hydroxyquinoline pretreatment time more suitable for FISH chromosome pretreatment time was 50-65min. Zygotene to pachytene carrier preparation with 2.5% (w / v) cellulase and pectinase 2.5% (w / v) mixture of 37 ℃ enzymatic treatment of about 1h, and then baked the state of the cleanest stretch of the the tablet press chromosomes of background. Improve existing FISH technique, the proper conduct of the trial and error for different target DNA vector-color FISH technology system. Former medium-term and medium-term pre-treatment of the chromosomes of the hydrolysis time was 40min, zygotene to pachytene chromosomes of processing time are 30 min. Removed in the process of hybridization probe mixture ratio of dextran sulfate, the two different labeled probes mixing ratio of 1:1, no changes of the concentration of other components. After hybridization elution conditions found by screening to maintain 37 ° C constant temperature in 4 × SSCT (0.2% Tween 20), 2xSSC and 1 × PBS each elution 4min 30 sec lighter background of the hybridization results, signal detection the highest rate. 4-color fluorescence in situ hybridization, cabbage self-incompatibility gene KAPP to meiosis zygotene to pachytene THL1, mitotic metaphase and before the mid-five different target DNA vector to locate . The results indicate that the the biotin the KAPP labeled probe signal is detected to two pairs of homologous signal, and the digoxigenin-labeled THL1 is detected only a pair of homologous signal. Armstrong karyotype analysis standard karyotype analysis of mitotic metaphase chromosomes found, Kapp probe signals are located on chromosome 4 short arm of chromosome 9 short arm ends, the percentage of distance from the centromere were 60.46 ± 5.14 and 79.5 ± 3.35; THLl probe signal is located a short arm of chromosome 5, the percentage of distance from the centromere of 65.42 ± 2.82. The integrated carrier FISH results show that KAPP cabbage genome may have two homologous sequences Block the two copies THL1 cabbage genome single-copy gene is only one homologous sequences Block. In addition, from the perspective of comparative genomics hybridization results, combined with the existing research results to determine the initial S-locus linkage relationship. Kapp, the THL1 and S-locus in the Brassica genome study on the characters to the SI signal transduction pathways related to the exploration of gene expression and regulation of the temporal characteristics of great significance.

Related Dissertations

CLC: > Agricultural Sciences > Gardening > Vegetable gardening > Cabbage > Cabbage
© 2012 www.DissertationTopic.Net Mobile