Cloning And Expression Of Pituitary Prolactin Gene In Ailuropoda Melanoleuca

Giant panda, Ailuropoda melanoleuca, belong to Ailuropodidae and Camivora in the Taxonomy. They inhabit montane forests and mixed coniferous and broadleaf forests where bamboo stands are present. Present, they are found only in 6 small forest fragments in small isolated areas of the Sichuan Province, Gansu Province, and Shaanxi Province in China. They are an endangered species and indigenous to China. We can preserve Giant Panda in two ways, protecting they and their surroundings in wild and feeding them in the zoo. But in the captive group, both female and male giant panda exhibit reproductive problems, and breeding the giant panda in captivity has proved difficult. They have specialized reproductive pattern with low fecundity. At the same time, there is little known about their basic reproductive biology at molecular level. So there is a serious need to understand the reproductive biology of the giant panda for the development artificial breeding technology that could be attributable to enhance reproduction of captive pandas.Pituitary prolactin (PRL) is a polypeptide hormone that is synthesized in and secreted from e-cells of the anterior pituitary gland. The hormone was given its-name based on the fact that an extract of bovine pituitary gland would cause growth of the crop sac and stimulate the elaboration of crop milk in pigeons or promote lactation in rabbits. However, we now appreciate that prolactin has over 300 separate biological activities including Reproduction and Immunoregulation. In this paper the genes encoding pituitary prolactin of the giant panda were amplified for the first time by RT-PCR from the panda's pituitary total RNA. And the PCR product was subcloned, sequenced, and submitted to GenBank (with accessions No.AY161285). The sequence analysis revealed that the giant panda prolactin cDNA contains a 687-nucleotide open reading frame encoding the prolactin prohormone of 229 amino acids residues. The signal peptide contains 30 amino acids residues, and the mature giant pandaprolactin is composed of 199 amino acids residues. Then the DNA fragment amplified was subcloned into pGEX-4T-l procaryotic expression plasmid and the protein expression was induced by isopropyl-1-thio-beta-D-galactopyranoside in Escherichia coli strain BL21. SDS-PAGE anayysis revealed the prolactin, expressed in E. coli, is infusible. The multiple sequence alignments revealed that the homology of giant panda over 90% to cat, pig, rabbit and possum, 80% to 70% to human, cow, sheep, goat, and chicken, 52.2% to rat and 46.1% to mouse at the amino acid level. It is interesting that the homology of prolactin is over 90% between giant panda, belong to eutherian order, and possum, belong to metathria order, while the homology is only 46.1% between the panda and mouse belonging to eutherian yet. Our results lay a foundation for produce recombinant giant panda prolactin, which may amply with other hormer in artificial breeding technology aiming to increase its captive reproductive efficiency.