CLONING OF RED-SPOTTED GROUPER TSHβ PROMOTER AND ITS LOCALIZATION EXPRESSION IN PRIMITIVE GONAD AND PITUITARY OF ZEBRAFISH EMBRYOS

Abstract

Thyroid-stimulating hormone(TSH) regulates the growth and function of thyroid for formation of thyroid hormones,which are involved in regulation of growth,development,metabolism and reproduction.TSHβ and their thyroidal receptors have been investigated extensively in mammals,but comparative studies were considerably less in lower vertebrates.Our lab had cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone β-subunit(TSHβ) from orange-spotted grouper(Epinephelus coioides),and first reported TSHβ expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary.Further analysis found that TSHβ also expressed in the gonads of the red-spotted grouper(Epinephelus akaara).To elucidate the mechanisms regulating TSHβ expression,we isolated a 5.1kb fragment of the 5'-flanking region of TSHβ promoter from red-spotted grouper by Genome walking.1.8 kb of this upstream region was used to produce GFP reporter construct(pBK-TOL2) for analysis of tissue-specific expression in zebrafish embryos.During 12—30hpf stage,GFP expressed in the PGC;36hpf—7dpf stage,GFP expressed not only in the PGC but also in pituitary.Western blot analysis showed that GFP protein was detected early in the 10hpf stage embryos.