Establishment Of Technique For Preparing Extended DNA Fibers And Studies Of Localization Of Brassica Oleracea S Genes

Studies of effects of S genes on self-incompatibility in phanerogam have been the focus since the first S gene (S-locus glycoprotein, SLG) was identified. There are three genes in the S-locus, namely SLG, SCR (S-locus cystein rich protein) and SRK (S-receptor kinase). They are closely linked during the course of inheritance. As a type of brassica species, chromosomes in brassica oleracea exhibits shortness and compactness. Localization of S-locus on brassica oleracea genome didn't put in practice because of possible single- or low-copy S-locus, so that location of S locus in brassica oleracea hasn't been identified up to now. However, it became possible until the technique of fluorescence in situ hybridisation was set up. That S genes existed in brassica oleracea as the possible form of single copy made it difficult for us to make clear the accurate location of S locus. In consideration of the problems we met across, we decide to attain high-resolution physical mapping of S genes in brassica oleracea genome by FISH to extended DNA fibres. However, classic methods for preparation of DNA fibres weren't suitable to brassica oleracea with short chromosomes. So we attempted to set up a new kind of technique for preparing for EDFs. Then we localized brassica oleracea S locus using SRK and SCR/SP11 as probes by fluorescence in situ hybridisation to extended DNA fibres.Main contents in this thesis are as follows. We try several methods of preparing for EDFs, and contrast experiment results of protein lysis and stretching DNA. In the same time, we set up a new efficient technique to extending and straightening DNA. Then use two kinds of S genes as probes hybridized to brassica oleracea pachytene chromosomes and extended DNA fibers. We analyzed the copy number of S locus and resolution of EDFs in accordance to the experiment results.This study primarily groped a new technique to stretching DNA. In order to acquire perfect EDFs, interphase nuclei chromatin and metaphase chromosomes were all straightened by means of conventional experiment methods. In comparison with EDFs prepared using the technique newly founded, we educed optimal technique. Fluorescence in situ hybridization onto EDFs was done for verifying exercisable character of the newly founded technique. Results also tell us that S locus in the genome of brassica oleracea is single-copy except feasibility of DNA fibers we prepared used for hybridization. In addition, superiority of EDF-FISH over other forms of ISH was distinctly exhibited in contrast with the result of FISH to pachytene chromosomes. The brief experimental manipulation and results are as follows:1. Groping for technique of preparing for EDFsIn order to find a simple but effective method to preparing for EDFs, we set about our scheme in two aspects as follows: one was selection of the experimental materials, the other one was to grope for the optimal stretching buffer solution.According to different type of tissues and the different phase in the cell division, experimental materials selected primarily include three types: (1) mitosis metaphase meristematic tissue of root tips; (2) mitosis interphase young leaves; (D pollen mother cells at the prophase I of meiosis. The stretching solutions selected are pepsin and proteinase K, The buffer component of the former one is 0.1-0.2 mol/L hydrochloric acid, and those of latter one are STE buffer. Through many repetitive experiments, we drew the conclusion that meiotic prophase I PMC is the optimal materials and result of stretching DNA using proteinase K is obviously superior to that by pepsin. In comparison, among the four STE buffer solutions STE4 is the optimal selection. Except proteinase K, components of the STE4 stretching solutions mainly contain 0.1% SDS, 10mmol/L Tris-HCl pH7.8, and 0.1mmol/L EDTA. After several repetitive trials, the conclusion was drawn as follows:For materials in meiotic prophase I, the optimal concentration of proteinase K and the most effective treatment time are respectively 45ug/ml and 7.5 minutes. For mitosis metaphase brassica oleracea material, optimal concentration proteinase K and time of treatment are respectively 60ug/ml and 7.5 minutes. And for interphase brassica oleracea chromatin, optimal concentration proteinase K and time of treatment are respectively 40ug/ml and 7 minutes.