Molecular cloning of growth hormone-encoding cDNA of an Indian major carp, Labeo rohita, and its expression in Escherichia coli and zebrafish.
The cDNA clones encoding for growth hormone (GH) of an Indian major carp rohu Labeo rohita were isolated from a cDNA library constructed from the poly(A)(+) RNA extracted from the pituitary glands of rohu. Partial GH cDNA of the rohu (3'-end) was amplified by RT-PCR and used as probe to screen the cDNA library. Full-length GH-specific cDNA clones (1180 bp) were isolated and sequenced. The sequence contains 48-bp 5'-noncoding region followed by an ORF of 621 bp and a 3'-noncoding region of 521 bp. The peptide shares about 90% identity with the GH of Cyprinus carpio (Linn.) and >84% identity with GH sequences of other cyprinids. The GH-encoding cDNA of rohu has been cloned into expression vectors and GH protein has been over expressed in Escherichia coli and purified as a soluble protein. The GH cDNA was cloned into a bicistronic vector with EGFP; injection of in vitro transcribed GH-EGFP mRNA into zebrafish embryo has resulted in EGFP expression confirming the cloned GH cDNA is functional in fish and the IRES element could be effectively used in fish for bicistronic expression of foreign genes.
Thayanithy
V
V
Department of Genetics, Madurai Kamaraj University, Madurai, 625021, India.
Venugopal
T
T
Anathy
V
V
Pandian
T J
TJ
Gong
G Z
GZ
Mathavan
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002347:Carps / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection; D015027:Zebrafish / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013006:Growth Hormone; D004262:DNA Restriction Enzymes