Studies On Sox Genes Of Allotetraploid Fish And Steroidogenic Enzymes Of Rice Field Eel

The Sox family of transcription factors were found throughout the animal kingdom. They were characterized by the presence of a HMG domain, involved in the regulation of such diverse developmental processes of early embryogenesis as sex determination, chondrogenesis, haemopoiesis, neural development and lens development. The formation of tetraploid stock of red crucian carps (Carassius auratus red var., (?), 2n=100) and common carps (Cyprinus carpio L., (?), 2n=100) provided a very important experimental material for the studies on the mechanism of sex determination and differentiation of polyploidy fishes. In the present study, Sox genes of allotetraploid fish were addressed in order to provide new materials and views for the molecular mechanism of sex determination and differentiation in fish, the origin and evolution of Sox genes, and so on. Meanwhile, investigations on the expression patterns of steroidogenic enzymes (P₄₅₀arom, P₄₅₀11β, P₄₅₀SCC and P₄₅₀C₁₇) and some genes potentially involved in male gonadal development of natural sex-reversal rice field eel, Monopterus albus, were carried out in this study. It will not only contribute to clarification of the mechanism of sex determination and sex conversion of rice field eel, but also play a significant role in artificial sex-control of animals with economic interest as well as in enrichment of the theory of sex determination and differentiation in fish. The major results in this paper were presented as follows:1. We isolated a Sox9 homologue, termed Sox9b from allotetraploid testis and analyzed its expression pattern in different tissues. The results demonstrated that Sox9b was highly expressed in the testis but at lower levels in the heart and brain, while no expression of Sox9b mRNA was discovered in the ovary of adult allotetraploids. Moreover, two transcripts of Sox9 (2.1kb and 1.7kb) with overlapping expression were observed for the first time in teleosts. Based on the above results, it indicated that allotetraploid Sox9b might be involved in gonadal development of male allotetraploids. Additionally, the diverse tissue-specific expression profiles of Sox9b provided significant molecular evidence for the lineage-specific subfunction partitioning of some ancient genes in teleosts like Sox9 gene.2. Analysis of intron sequence variability of the conservative HMG-box of Sox9 genes in allotetraploids and their original parents were conducted in this paper. The results showed that introns of the HMG-domain of Sox9s in allotetraploids and their original parents have not only the length polymorphism but also intron variability. Furthermore, the research obtained had profound effects on properly understanding the information of position, function, evolution and origin of introns.3. The partial Sox4b gene and its activity of 5' flanking regulatory region with the promoter structure in the allotetraploid fish were cloned and analyzed. The results indicated that 600bp promoter of allotetraploid Sox4 gene was able to conduct the expression of exogenous enhanced green fluorescent protein (EGFP), which played a significant role in further studying the regulatory mechanism of Sox4 gene of allotetraploids during the transcription and translation, as well as regulatory factors involved in this process. 4. Investigations on expression profiles of four steroidogenic enzymes, i.e., P₄₅₀arom, P₄₅₀11β, P₄₅₀SCC and P₄₅₀C₁₇, and three genes potentially participated in gonadal development of male individuals were performed during three different gonadal developmental stages of female, intersex and male rice field eel. The present study will provide vital molecular evidence for revealing the mechanism of sex reversal in rice field eel.5. We investigated in this study the expression analysis of cytochrome P450 aromatase (P₄₅₀arom) responsible for estrogens production , and estrogen receptorα(ERα) during oogenesis of rice field eel. Moreover, the relationship of P₄₅₀arom and ERαwere explored, which will provide significant experimental data for further shedding light on the function of estrogens during oogenesis of rice field eel.