Cloning And Expression Of Sox9 Gene Related To Sex Determination In Tetraploid Hybrids Of Common Carp (Cyprinus Carpio L.) With Red Crucian Carp (Carassius Auratus Red Var.)

In the present study, a series of molecular biological techniques including subcloning of polymerase chain reaction(PCR) product, RT-PCR, Northern blot, Southern blot, were used to analyze Sox9 genes related to sex determination in allotetraploid fish. The main results were summarized as follows:(1) Using degenerate primers within the HMG-box (high mobility group-box) , the Sox gene of the male and female allotetraploid fish and their original parents (red crucian carp and common carp) was amplified by PCR. Four DNA bands with the length 200bp, 600bp, 900bp and 1900bp were observed in the PCR products of allotetraploid fish whereas only three bands existed in their original parents with the length 200bp, 600bp, 900bp and 200bp, 500bp, 900bp, respectively, indicating that there were homologous genes of SRY in allotetraploid fish and their original parents, but no sex-specific band was found in male and female individuals. Allotetraploid fish not only possessed sex-linking bands which almost covered all the bands their parents had, but also had some variation, providing further evidence that tetraploid fish was allotetraploid fish instead of autotetraploid fish. Since F3, the tetraploidy was maintained from one generation to another and to date FH allotetraploid hybrids were produced(F:!-FM)and a stable and new allotetraploid fish population was formed.(2) The excised 200bp, 600bp and 900bp fragments were extracted, subcloned and sequenced. The sequencing showed that an HMG-box with the length 200bp and two Sox9 genes (Sox9a and Sox9b) yielding a putative 71 amino acids protein separately were obtained. Both of them had an intron within the HMG-box, whose intron junctions were in accordance with the "gt-ag" rule by analyses of computer and RT-PCR.(3) Using the 900bp fragment labeled by DIG as probe, dot blotting analysis demonstrated Sox9 was obviously present in the allotetraploid fish genome. To futher the study on tissue-specific expression, Northern blot was performed with total RNA extracted from different tissues like testis, ovary, lung, brain and heart, indicating Sox9 was expressed in testis, brain and heart, but not in ovary. Morever, strong hybridization signals were observed in testis , which suggested Sox9 likely played an important role during spermatogenesis and sex determination and differentiation. In addition, it was confirmed by RT-PCR and Southern blot.(4) Two transcripts, approximately 1. 7kb and 2.1kb were found in Northern blot, suggesting two Sox9 genes existed in allotetraploid fish but that whether they were Sox9a and Sox9b respectively, relied on further study.