Cloning Of Chicken Stra8Gene Promoter And Its Related Inducers Screening

Sperms are derived from primordial germ cells through complex process of spermatogenesis, the differentiation of male germ cells go through several stages:Firstly, the spermatogonial stem cells produce many spermatogonial cells that are ready for sperm by proliferation. And then the primary spermatocytes enter meiosis. The first meiotic division can form two secondary spermatocytes. Secondly, after the first meiotic division, the secondary spermatocytes can form four round sperm cells. And then the sperm stem cells can eventually form Sperm cells by a complex sperm process. RA plays a decisive role in start of meiosis, and Stra8plays a important role in the process of meiosis,With the purpose of studying the mechanism of regulation of Stra8, in this experiment, the promoter of Stra8gene was cloned, biology information analysis was employed, and series of promoter missing mutants were directly subcloned into pGL3-Basic vector, promoter luciferase activity was detected after transient transfection. As there are biting sites of RARs and RXRs in the region of Stra8promoter, dependent on epigenetic principle of gene regulation, we selected Am80and TSA to check the regulation of Stra8promoter activity. This study achieved the following results:1Biology information analysis of Stra8gene promoter region。The study has cloned DNA fragment of5’promoter region with the length of2947bp, using online software Matlnspector and AliBaba2.1to predict and analysis regulated sequence and potential biting sites of transcription elements. The predictions show there is not eukaryotic typical TATA box existing in Stra8gene regulation region, but transcription elements RARα。RXRa and RARβ are founded in the region -1003bp～-664bp. Meanwhile using online software MethPrimer and CpG Island searcher to predict and analysis the CpG island, and find required CpG island region.2Deletion expression vector construction and relative luciferase activity of Stra8gene promoter. Using bioinformatics methods to find chicken Stra8gene5’ regulatory sequences and predict the location of promoter as well as transcription factor biting sites. Application of different lengths of promoters fragments by PCR, which connected to the luciferase reporter gene vector pG13-Basic. Seven recombination expression vectors, such as PGL3-P1、PGL3-P2、 PGL3-P3、PGL3-P4、PGL3-P5、PGL3-P6and PGL3-P7were successfully constructed identified by restriction enzymes digestion and sequencing. Deletion expression vectors and Renilla luciferase reporter vector as25:1were co-transfected DF-1and GC-1cells, by using dual-luciferase reporter assay system to check luciferase activity, and analyzing activity of deleted promoter, screen that PGL3-P2exhibits the highest activity, PGL3-P4follows behind.3The effects of Am80and TSA on the Stra8gene promoter activity. We use pGL-P4as main study subject, check the effects of promoter activity when Am80and TSA were used singly or in combination. When used singly, TSA has the biggest effects on Stra8gene promoter activity at the concentration of, combination of Am80and TSA promotes promoter activity best. Replace the CMV promoter of vector EGFP-N1with the promoter fragment of recombinant pGL3-P4, and construct the vector Stra8-EGFP; during the period of culturing P19cell transfected by vector Stra8-EGFP, add different inducer into medium and observe the expression of green fluorescent protein. Induced by ATRA, Am80, TSA and combination of Am80and TSA, results suggest that induction group of combination of Am80and TSA expressed green fluorescent protein strongest, and the intensity of fluorescence expression of each group corresponded to their promoter activity.4The impacts on Stra8gene expression by Am80and TSA. Dissociate the left ovaries from chicken embryos with hatching to12.5days, and culture them in tissue culture medium for48h, in the process add different inducers into the experiment group. Extract RNA from ovaries, detect the expression of meiosis maker gene Stra8and SCP3by using qRT-PCR. Results show that combination of Am80and TSA made the expression of Stra8and SCP3significantly higher than ATRA induction group, Am80and TSA replace ATRA as inducers for Stra8gene.