Multiple Spliced Transcripts And Differential Expression Pattern Of The Glycogen Synthase Kinase-3β(GSK3β) Gene In Goat(Capra Hircus)

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that is mainly regulated by phosphorylation of its target substrates or itself. GSK-3 shown to be a key component in the regulation of over hundred diverse proteins and has been continuously identified in connection with multiple pathways such as Wnt, Hedgehog and IGF-I, etc. It plays a crucial role in the regulation of cell fate, regulating processes such as embryonic development, cell proliferation and apoptosis. In mammals, GSK-3 is primarily generated from two known genes: GSK-3a and GSK-3β, and several alternative spliced transcripts of GSK-3β have been isolated from human, mouse and pig tissues. Alternative splicings of GSK-3β has been identified in many animal models but limitted in domestic animals. In this study, we cloned and characterized the GSK-3β gene and identified six GSK-3β spliced transcripts. RT-PCR, molecular cloning, qPCR and western blotting were used to determine the mRNA and protein expression pattern in heart, liver, spleen, kidney, brain, longissimus dorsi muscle and uterus, that all tissues were collected from three female Nanjiang Brown goats at 120 days after birth. The results are as follows:1. Six GSK-3βtranscripts were identified in different tissues and designated as GSK-3β1l, 2,3,4,5 and 6. RT-PCR was used to further determine whether the six GSK-3β transcripts existed in different goat tissues. Bioinformatics analysis revealed novel features in the genomic structure that were detected in GSK-3β2, GSK-3β4 and GSK-3β6. Coding nucleotide and protein sequences alignment showed that GSK-3 β was highly conserved in mammals. The cDNA sequences of the six goat GSK-3β transcripts were deposited in GenBank as KJ649149-KJ649154.2. qPCR analyses revealed that all the GSK-3β transcripts were expressed at the highest level in the heart and brain (P<0.01), whereas their expression levels in the liver, spleen, kidney, longissimus dorsi muscle and uterus were different.3. In a western blot analysis, two bands were observed corresponding to two major predicted sizes. Western blotting revealed that abundant GSK-3β proteins were observed in the higher molecular weight band obtained from the heart, brain and longissimus dorsi muscle. In the lower molecular weight band, GSK-3β can be detected in all tissues but liver.4. Moreover, we constructed the RK-Flag-Goat GSK-3βAS recombinant plasmids that can use for further research on the function of GSK-3β isoforms.