Study On Cloning And Expression Of T/Brchyury Gene During Early Embryos Development In Short-tail Sheep

Hulunbuir sheep is one of the sheep economic varieties developed vigorously in Inner Mongolia Autonomous Region.According to the tail characters,it can be divided into two main strains:Baler tiger sheep(semi-oval tail)and Hulunbuir short-tail sheep(taillless and small peach tail).According to the resequencing of Hulunbuir sheep,the mutation at 334 and 333 of the second exon of T/Brachyury gene may be related to the short-tail trait of Hulunbuir short-tailed sheep.Mesodermal inducers such as TGF-βfamily,fibroblast growth factor and activin can up-regulate the expression of T/Brachyury gene in early embryonic development,which plays an important role in embryo mesodermal development,and then its expression is shut down.Based on the above experimental background,this experiment is divided into three parts:1.The average tail lengths of 25 Barhu sheep and 25 Hulunbeier short-tailed sheep were counted as 13.4 cm and 23.4 cm,respectively.After DNA sequencing,it was found that there were a small number of heterozygotes in the T/Brachyury gene in 25 Hulunbuir short-tail sheep populations.The asymmetric high-resolution melting curve method was successfully established and the genotype identification of 50 Hulunbeier sheep was carried out.After comparing with the DNA sequencing results,it was found that the asymmetric high-resolution melting curve could accurately distinguish the two Hulunbeier short-tailed sheep T/Brachyury genes.The mutation sites c.G333C and c.G334T are homozygous and heterozygous.2.Use SMART RACE method to clone the full-length c DNA of T/Brachyury gene from 16 day short-tail sheep embryos.The length of the c DNA is 2426 bp,of which 3’UTR,3’ploy(A)tail and5’UTR are 763 bp and 328 bp,respectively The CDS(protein coding region)is 1335 bp,and the protein physicochemical properties of the amino acid sequence after the translation of the CDS sequence are analyzed,and it is found that the Random coil structure in the BRACHYURY protein accounts for the largest proportion.3.The complete CDS fragment of the sequenced T/Brachyury gene was successfully connected to the p EASY~?-Blunt E1 expression vector for prokaryotic expression of BRACHYURY protein,and the protein expression conditions were explored,the target protein was purified,and the polyclonal rabbit anti-sheep BRACHYURY serum antibody was successfully prepared.After ELISA The experiment identified that the maximum dilution ratio of serum antibody was 1:1500.At the same time,the 16 day,20 day,25 day,and 30 day embryos of Hulunbeier short-tail sheep were used to identify the T/Brachyury gene expression level.The results showed that the 16 day embryonic T/Brachyury m RNA expression level was the highest.