Molecular Cloning And Expression Analysis Of α Type Calcitonin Gene Related Peptide (α-CGRP) In Gekko Japonicus

Objective: To clone the full length ofαtype calcitonin gene related peptide (α‐CGRP) in Gekko japonicus, and investigate the expression changes ofα‐ CGRP in the spinal cord regeneration after tail amputation. Also, to detect the distribution ofα‐CGRP mRNA in spinal cord by ISH and to locateα‐CGRP peptide in gecko Gsn1 cells by transient transfection.Methods: Using the technique of Rapid Amplification of cDNA Ends (RACE) to obtain the full length ofα‐CGRP cDNA from gecko brain and spinal cord; and the size of its transcript and expression in different tissues were analyzed by Northern Blot and RT-PCR, respectively. Then the expression and distribution ofα‐CGRP in the proximal spinal cord was compared at different time point after tail amputation , by both in situ hybridization (ISH) and RT-PCR. The cellular localization ofα‐CGRP in Gsn1 cells was observed through eukaryotic transfection.Results:(1)α‐CGRP cDNA spans 1,002 bp, codes 124 amino acids, and mature peptide contains 37 amino acids. Alignment ofα‐CGRP mature peptide sequence with those of other species, we found that geckoα‐CGRP shares 86.5% amino acid identity with that of the zebrafish , around 80% with those of mammalian animals, and about 94% with that of chicken.(2) Northern blot and RT-PCR analysis revealed that the size ofα‐CGRP transcript was about 1.0 kb.α‐CGRP mRNA was expressed in all tissues examed, including brain, spinal cord,, muscle, liver, lung, ovary , kidney and testis, and the highest expression in the spinal cord.(3) By in situ hybridization, theα‐CGRP was expressed mainly in the gray matter of spinal cord, scarcely in white matter, and with more location in anterior horn than posterior horn. After tail amputation, in the proximal spinal cord of injury, expression ofα‐CGRP was significantly increased at 1d and the increased expression was concentrated in the anterior ,posterior horn and central canal. When at 2w, the expression was reduced proximally to the level of control.(4) RT-PCR results showed thatα‐CGRP was remarkably up-regulated in the spinal cord proximal to the amputation at 1d after tail amputation, and reached the peak level. And the expression ofα‐CGRP was slightly decreased at 3d, but the level was still higher than control. At 1w and 2w, the expression was continually decreased.(5) In transfected cells, CGRP peptide was located in the whole cell body.Conclusion:(1) This research obtained the full length ofα‐CGRP cDNA. It spans 1,002 bp, codes 124 amino acids, and mature peptide contains 37 amino acids.(2) The size ofα‐CGRP transcript in gecko was about 1.0 kb.(3) Theα‐CGRP was extensively distributed in all tissues examed, and the expression in the spinal cord was the highest.(4) Cell transfection results indicated that,α‐CGRP peptide was located in the whole cell.(5)α‐CGRP was remarkably up-regulated in the spinal cord proximal to the amputation at 1d after tail amputation. CGRP synthesized and released from neural system maybe participate in regulating immunoreaction or wound healing.