| Rubber is a kind of important strategic material in China. Hevea brasiliensis has become the only commercial source of natural rubber around the world for its high yield, excellent quality, ease to collect and long economic life. At present, researchers had cloned more than420kinds of important genes or cDNA from Hevea brasiliensis. However, these genes were obtained from the rubber tree genomic or cDNA library, not only need heavy work but also unable to confirm their location and linkage on chromosomes. The saturation is not enough about these reported genetic maps of rubber tree which constructed by a variety of molecular markers, it is also difficult to be used for rubber tree breeding research. These problems could be resolved effectively if the single chromosomal DNA library of Hevea brasiliensis was constructed. Related researches about the rubber tree libraries were from the whole genome or cDNA libraries. However, the research on Hevea brasiliensis single chromosome library construction has not been reported. In this thesis, construction of the single chromosome library of rubber tree, by the means of single chromosome microdissection and microcloning technology, will provide a new perspective for the study of rubber tree genome and a molecular assisting way for breeding of rubber tree. The results of the study are as follows:(1) In this study, the chromosome samples were prepared with the enzyme and low osmotic method using tender leaves of Hevea brasiliensis RY-7-33-97as material. The preparation of chromosome was optimized, so that it can better be used for single chromosome microdissection of rubber tree.(2) The process about making glass needle and the method of chromosome microdissection were explored. An efficient technique of Hevea brasiliensis chromosome microdissection was initially established. And57chromosomes were successfully isolated from different metaphase cells of Reyan7-33-97by using the method of slender glass needles. There were18chromosomes from one metaphase cell of Reyan7-33-97.(3)Three chromosomes were amplified by linker adaptormediated PCR(LA-PCR) for two rounds. The products were verified by Southern blot hybridization. The LA-PCR products were homogeneous with the Hevea brasiliensis genomic DNA, indicating that DNAs from the chromosome have been successfully amplified. The three chromosomes were verified by fluorescence in situ hybridization (FISH) and Karyogram. They were the chromosome1, the chromosome5and the chromosome9in Hevea brasiliensis. A complete verified system was constructed.(4)Libraries of the three chromosomes were constructed by their LA-PCR products. The library of chromosome1contains9.6xlO4clones, the main size range of inserts from300bp to1200bp, the inserted fragments averaged600bp, and the library coverage reached1.6times of the chromosome length; The library of chromosome5contains of6.3x104clones, the main size range of inserts from250bp to1100bp, the inserted fragments averaged600bp, and the library coverage reached1.2times of the chromosome length. The library of chromosome9contains of4.9×104clones, the main size range of inserts from300bp to1150bp, the inserted fragments averaged650bp, and the library coverage reached1.0time of the chromosome length.(5) One hundred random positive clones from the library of chromosome5were sequenced. A total of78sequences were obtained and there were66sequences which the size range from196bp to1030bp left after removing duplicates. These sequences were performed by BLAST online. Results showed that there are15sequences were more than90%homogeneous with the sequences of Hevea brasiliensis WGS (RRIM600) database, and3sequences L-5Chr-16, L-5Chr-88and L-5Chr-57have high similarity more than90%with some EST sequences from NCBI. It will facilitate specific probe screening, gene cloning and locating, genetic mapping of this chromosome. |
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