| The Chinese alligator Alligator sinensis belonging to Alligatoridae,Crocodilia,Reptilia,is a rare crocodile species endemic to China.It has been listed as a national first-level protected animal because its wild populations are very few.The leather of Chinese alligator is widely used,and its meat is delicious and has medicinal value,what's more,it can be also used as scenic animal,so the artificial breeding can bring high economic benefits.However,the Chinese alligator is a small crocodile with slow growth speed,and the body length is only about a third of the largest crocodile species.It takes too long time and costs too much for artificial breeding of Chinese alligator,so the crocodile farmers always pay attention to the rapid growth breeding technology of Chinese alligator.The genes of the somatotropic axis play critical roles in regulating the growth and development of vertebrates.However,up to date,very little was known about the endocrine regulation of the growth of Chinese alligator.In this study,the molecular structural characteristics of four key genes(GHR,IGF-1R,IGF-2 and IGF-2R)of somatotropic axis in Chinese alligator were analyzed for the first time,and their spatiotemporal expressions during the active and hibernating periods were investigated by the fluorescence quantitative PCR.The complete GHR cDNA of Chinese alligator is composed of 2409-bp nucleotides,including the 5'-untranslated region(UTR)of 326 bp nucleotides,3'-UTR of 325 bp nucleotides and 1848 bp of the open reading frame(ORF).The ORF encodes 615 amino acids including a signal peptide of 18 amino acids and a mature protein of 597 amino acids.The mature protein contains the extracellular domain,transmembrane domain and intracellular domain.The homology comparison analysis showed that the Chinese alligator GHR shares highest identities with American alligator(99%)and higher with turtle(~82%)and birds(71%-81%).Phylogenetic tree analysis indicated that alligators have close genetic relationship with birds.Q-PCR analysis indicated that GHR mRNA is expressed in all the examinated tissues.The highest expression level of GHR is in intestine,higher in liver and muscle while lower in other tissues.During hibernation,the expression level of GHR increasedsignificantly in heart,spleen,lung,kidney,stomach,ovary and muscle(P<0.01),while decreased significantly in intestine(P<0.01),and did not significantly change in other tissues.The various expression of GHR mRNA among different tissues during the active and hibernating periods indicated that the GHR may play an important role in regulating the digestion and growth of Chinese alligator.The complete IGF-1R cDNA of Chinese alligator was obtained for the first time,it is composed of 4681 bp nucleotides,including 69 bp of the 5'-UTR,523 bp of 3'-UTR,and4089 bp of the open reading frame.It encodes a 1363-amino acid precursor molecule of IGF-1R with two subunits-? and ?.The ? subunit contains a signal peptide composed of 30 amino acids,a conservative cystine-rich domain and 11 potential N-glycosylation sites.The ? subunit contains a transmembrane domain,a tyrosine kinase domain and carboxyl terminal.The homology comparison analysis showed that the Chinese alligator IGF-1R shares highest identities with American alligator(99%)and birds(95%-96%).Phylogenetic tree analysis showed that alligators have close genetic relationship with birds.Q-PCR analysis indicated that IGF-1R mRNA is expressed in all the examinated tissues.The highest expression level of IGF-1R is in muscle,higher in heart,kidney,ovary and oviduct while lower in intestine,spleen,lung,stomach,pancreas and liver.During hibernation,the expression level of IGF-1R increased significantly in heart and muscle(P<0.01),and increased in kidney(P<0.05),while did not significantly change in other tissues.The spatiotemporal expressions of IGF-1R mRNA among different tissues during the active and hibernating periods indicated that the IGF-1R may play an important role in regulating the growth and the metabolism of multiple organs of Chinese alligator.The mature IGF-2 of Chinese alligator is composed of 67 amino acids with four domains(B-C-A-D).The IGF-2R consists of 2469 amino acids,which can be divided into four parts,including signal peptide,extracellular region,intracellular region and transmembrane region.Both the deduced amino acid sequence of IGF-2 and IGF-2R share high identities with American alligator and birds.Phylogenetic tree analysis of the IGF-2 and IGF-2R indicated that alligators have close genetic relationship with birds.Q-PCR analysis indicated that IGF-2and IGF-2R mRNA are expressed in all the examinated tissues.The highest expression level of IGF-2 is in spleen and liver,higher in heart,spleen,pancreas,ovarian and muscle while lower in lung,stomach,intestine and oviduct.During hibernation,the expression level of IGF-2 decreased significantly in lung,kidney,stomach and pancreas(P<0.01),and decreased in liver and muscle(P<0.05),while did not significantly change in other tissues.The highestexpression level of IGF-2R is in spleen and stomach,followed by kidney,pancreas and muscle,lower in other tissues.During hibernation,the expression level of IGF-2R decreased significantly in spleen,lung,kidney,stomach,intestine and pancreas(P < 0.01),but increased significantly in heart and muscle(P < 0.05),and did not significantly change in other tissues.The widely expression of IGF-2 and IGF-2R among different tissues during the active and hibernating periods indicated that the IGF-2 and IGF-2R may play an important role in regulating the growth and the metabolism of multiple organs of Chinese alligator.This study not only improved our understanding on the molecular evolution of the key genes(GHR,IGF-1R,IGF-2 and IGF-2R)in the somatotropic axis,but also enriched the basic theory of the endocrine regulation mechanism on the growth of Chinese alligator.It could provide the baseline data for developing the rapid growth breeding technology of Chinese alligator. |
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