The Expression Of Goat Tet Protein Family Genes In The Main Tissues, Tet3 Gene CDS Cloning And Bioinformatics Analysis Of It

DNA demethylation is a essential process during early embryonic development and somatic cell reprogramming process,and Ten-eleven translocation(Tet) protein family plays an important role in the process of DNA demethylation. Researches have shown that Tet3 protein plays a key role in activing demethylation of paternal pronucleus in mousezygote, which also is essential to activate the expression of pluripotency genes for maintaining the embryonic development. However, the DNA demethylation patterns are different between different species, for example, there are differences between goat and mouse in the mechanism of DNA demethylation in early embryos development, and the mechanism of DNA demethylation in goat is still unclear. Similarly it is also uncertain that whether the Tet3 protein can improve the cloned embryos DNA methylation reprogramming efficiency, or even reduce the probability of abnormal methylation. In addition, there is much incomplete DNA demethylation prevalently taking place in the cloned embryos, which may be one of the reasons for the inefficiency of animal cloing. In this study, we used Real-Time PCR to detect the relative expression of goat Tet protein family genes in different tissues, and we also used Touchdown PCR to amplify Tet3 CDS, which was divided into three segments from liver that showed a high expression level of Tet3. Then we cloned the the full-length sequence of Tet3 CDS by In-Fusion Cloning, and we used Tet3 CDS to construct the expression vector p EGFP-N1-Tet3. After that, We used a lot of bioinformatics analysis tools such as Ex PAsy, MEGA, Net Pho, Net NGlyc, SOPMA, SWISS-MODEL, STRING and so on, to analysis and forecast the goat Tet3 gene’s bioinformatics infotmations for a better knowledge of it theoretically and technically, in order to research the roles of Tet3 protein in the process of DNA methylation reprogramming of goat early embryos. The main contents and results are as follows:1. Detection of the relative expression of goat Tet protein family genes in different tissues by Real-Time PCRWe took 12 kinds of tissue from three goats respectively, then used Trizol to extract organizations Total RNA and obtained c DNA by RT-PCR. By Homologous sequence alignment through NCBI we gained the goat Tet conserved sequence and designed the primers of Tet protein family for Real-Time PCR, then we used Real-Time PCR to detect the relative espression of goat Tet protein family genes in different tissues. By using SPSS software for statistical analysis of the experiment results that we used 2-ΔΔCт methods and chosed heart tissue as a reference sample(mark as 1), we found that the goat Tet1 relatively highly expressed in skin(20.05), spleen(11.10), and ovary(7.70); goat Tet2 relatively highly expressed in the spleen(13.89); goat Tet3 relatively highly expressed in the spleen(59.32), pancreas(53.95), lung(47.22) and ovary(39.58).2. Amplifing fragments of goat Tet3 CDS by Touchdown PCR and full-length sequence by In-Fusion CloningBy Homologous sequence alignment through NCBI we gained the goat Tet3 m RNA predicted sequence,then we used RT-PCR to gain c DNA from liver which showed a high expression level of Tet3. We divided Tet3 partial m RNA which contains Tet3 CDS into three segments then amplified them with liver c DNA by Touchdown PCR with additional DMSO(Dimethyl sulfoxide). Then we cloned the the full-length sequence of Tet3 CDS(4902 bp) by In-Fusion Cloning. The enzyme digestion results showed the cloning was correct.3. Construction of vector p EGFP-N1-Tet3Digesting the p EGFP-N1 vector and the In-Fusion Cloning plasmid, which contains the Tet3 CDS, and purifing them through gel extraction. Then we constructed p EGFP-N1-Tet3 by inserting the Tet3 CDS into the p EGFP-N1, The enzyme digestion results showed the vector was correctly constructed.4. Bioinformatics analysis of goat Tet3 geneWe used a variety of bioinformatics analysis techniques to analyze biological information of goat Tet3 gene, and the analyses showed that the homology between goat and pantholops, bos is above 97%, while mus is 85%, and homo is 89%; and the results also showed that Tet3 protein is a hydrophilic NLS protein, with no signal peptide, has a Tet_JBP function domain which has a demethylation function.