Study On The Application And Mechanism Of Sox-HMG Molecular Markers

Heredity and variation are key subjects of biological study. Genetic marker is one of the essential techniques to acquire the biological information of genetic variation and to understand the inherent laws. The improvement of genetic markers is the embodiment of bioscience development, which embraces not only the progress in technology or the harvest of theory but also the deepened and systematized cognition of the essence of life. As a new generation of genetic markers, DNA barcoding provide fast and low-cost methods, with which even non-taxonomist can easily get biosystematic information. It is suggested that to develop new molecular markers some guidances could be followed:the nucleus gene regions are good choice to avoid the partiality of phylogenetic analysis when unisexual genetic mitochondrial genes are used; gene family members with conserved region could be considered to achieve multi-gene cross-validation; the target gene region should be highly conserved to be amplified in various kinds of animals of distant phylogenetic relationships.Sox genes are characterized by a conserved DNA sequence encoding a high mobility group (HMG) domain of80amino acids, which is responsible for specific DNA sequence binding. With the pair of special degenerate primers that was designed against the conserved sequences of Sox gene HMG-box, the novel Sox-HMG markers were used to amplify multiple DNA fragments from genomic DNA samples of different animals. The amplified DNA fragments of each animal species possessed certain amount and length and formed specific Sox-HMG DNA band patterns; by cloning and sequencing, specific DNA sequences of these DNA fragments could be obtained; specific Sox-HMG genetic markers were achieved with multi-gene cross-validation at double levels. With consideration on gene family members as subject that was investigated to develop innovative molecular markers, this study provides reference to study Sox genes and molecular markers. The major results in this paper were presented as follows:1. Genomic DNA samples were collected from animal species of various kinds. The special pair of degenerate primers was design against the conserved sequence in HMG region of Sox genes to amplify genomic DNA samples by PCR. The PCR products were separated through agarose-gel electrophoresis to obtain specific Sox-HMG DNA band patterns of each animal species. The separated PCR products were further purified, cloned and sequenced to obtain specific Sox-HMG DNA sequences.2. Comparative analysis of the amplified Sox-HMG DNA sequences was carried out. By combining with SSCP technology for further separation of Sox-HMG DNA bands in agarose gel, the principle of Sox-HMG DNA band pattern diversity was studied and the mechanism of Sox-HMG molecular markers was uncovered. By comparing to the popular DNA barcoding method, the advantages of Sox-HMG markers in biosystemic study, genetic breeding project and practical application were discussed.3. The coding sequences of Sox gene HMG region, which had been obtained by using Sox-HMG markers, were employed for phylogenetic study. The DNA sequences of Sox9gene were amplified from many animal species by Sox-HMG markers. Structure analysis was carried out to study the HMG region of Sox9genes. Phylogeny of intron in Sox9HMG region was discussed.4. The5'end regulation region of Sox4gene was amplified from allotetraploid crucian carp by using Sox-HMG molecular markers. After PCR amplification and restricted enzyme digestion, the promoter of Sox4gene was obtained to construct recombinant plasmid. With green fluorescent protein as a reportor gene, the transcription pattern of Sox4promoter in embryonic development of allotetraploid crucian carp was studied and the potential function of Sox4gene was discussed.In this study, it is proved that the Sox-HMG molecular markers were novel DNA markers based on the HMG conserved region of Sox genes. By revealing the basic principle and mechanism of action, the novel markers were proved to possess extensive applicability for biosystemic study on various kinds of animal species. The novel Sox-HMG markers were competent for distant crossing breeding project to distinguish and identify different strains with complicated genetic background. In this study, the gene family with highly conserved region served as subject investigated for creative study on molecular markers. The idea of this study provides a novel way for Sox gene study and molecular marker development. Phylogenetic study and intron evolution analysis were carried out on the basis of the obtained Sox-HMG relative sequences, the results of which proved a wealth of genetic information that had been provided by the novel Sox-HMG markers. The transcription pattern of Sox4gene in allotetraploid developing embryo was studied and the gene function was discussed, which further proved the potential value of Sox-HMG molecular markers in the field of gene function research.