MOLECULAR CLONING AND CONSTRUCTI ON OF EXPRESSI ON VECTOR OF THE 5′ FLANKING REGI ON OF BLUNT SNOUT BREAM (M EGALOBRAMA AMBLYCEPHALA) GONADOTROPIN I β2SUBUNIT GENE

In teleosts, as in other vertebrates, the pituitary gonadotropic hormones, GtH I and GtH II, play an important rolein regulating gametogenesis. Nowadays, the mechanism of gonadotropin I β2subunit gene transcriptional regulation has notbeen thoroughly understood yet. The objective of the current studywas to get basic information of the possible cisacting elements that involved in transcriptional regulation of the GtH I β gene expression of blunt snout bream (Megalobrama am2blycephala), and provide preconditions for further research on the molecularmechanism of these cis2acting elements in theGtH I β gene transcriptional regulation.According to cDNA sequence information of blunt snout bream gonadotropin I β2subunit (bGtH Iβ) gene, the 5′ regionof bGtH I βwas cloned by a simple method for cloning genomic DNA segments outside the boundaries of known sequences.In the first step of the method, a library of single-stranded flanking sequences is generated by linear amplification with oneprimer in the known region. Then a homooligomeric cytosine tail is added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments are amplified by PCR with a nested primer in the known regionand a poly-guanine primer complementary to the cytosine tail in the unknown region. Finally, the different fragments areseparated by cloning and characterized by sequencing.Sequence analysis reveals that the length of the 5′ flanking region of bGtH I β gene is 479 bp, and the region containssome potential transcription factors which may have important functions for the transcriptional regulation of the gene, suchas TATA box,ARE, PRE, ERE, SF2 1 and Ptx1, etc. Based on the above information, three partial deletion fragments aswellas the full length of the 5′ flanking region were cloned from the genome by PCR and linked to a luciferase reporter gene.These partial fragments contained 365bp (-345 to+20), 230 bp (-210 to+20), and 150 bp (-130 to+20), respec2tively. All these luciferase plasmid expression vectors will be used for the coming research.