| Body size is the most intuitive phenotypic difference between animals.In order to survive and reproduce better during the long-term evolution of animals,their body shape has changed significantly.Approximately 53 million years ago,cetacean ancestors transformed from a fully terrestrial quadruped to an obligate aquatic habitat,and fully adapted to the ocean,resulting in a series of adaptive changes in their morphology and physiology,the most obvious of which is the body size.For example,the largest blue whale(Balaenoptera musculus)and the smallest vaquita(Phocoena sinus)among the existing whales have a weight difference of 3000 times.The regulation mechanism of animal body size is complicated.Through the model animals,such as mouse and drosophilid,have found some body-related signaling pathways and genes,but the role of them in the regulation mechanism of cetacean body shape is unclear.Based on early bioinformatics analysis,we selected three genes(Grb10,Galns,p27)that have undergone adaptive evolution in cetaceans and related to body size regulation for the explore in vitro.We used mouse cell lines to perform a series of experiments,including q RT-PCR,Western blot,flow cytometry and EDU(5-Ethynyl-2'-deoxyuridine)cell proliferation,to analyze the functional differences at the cellular level of these 3 genes in different body types,such as sperm whale(large cetacean),bottlenose dolphin(small cetacean),cow(cetacean-related terrestrial mammal),and mouse(small mammal),and to explore the regulatory mechanism of mammalian body type differences.First,we explored the influences of the Grb10 gene in mouse,cattle,sperm whale,and bottlenose dolphin on the growth of myoblasts.Overexpression the Grb10 gene of the 4 species in myoblasts respectively,the results of EDU cell proliferation experiment showed that the Grb10 of mouse inhibited myoblast proliferation,but the Grb10 of cattle,sperm whale and bottlenose dolphin didn't inhibit the myoblast proliferation significantly.The results of q RT-PCR showed that compared with the control group,the expression of cyclin A2 m RNA in the group of mouse was significantly reduced,and the expression of P27 m RNA was significantly increased;while the expression of P27 m RNA of the sperm whale group and bottlenose dolphin group was reduced significantly.Further Western blot experiments showed that compared with the control group,the expression of cyclin A2 in the mouse group was significantly reduced,and the expression of cyclin-dependent kinase inhibitor P27 in the sperm whale group and bottlenose dolphin group was reduced significantly.The results of flow cytometry experiments showed that compared with the control group,overexpressing the Grb10 gene of 4 species in the cells respectively,they did not significantly affect the cycle operation of the myoblasts.The above results suggest that the Grb10 gene of mouse inhibits the growth and development of myoblasts,and the Grb10 gene of cattle,sperm whale and bottlenose dolphin weakens the growth of myoblasts,and there is no significant difference between the two cetaceans.Second,we explored the influences of the P27 gene in mouse,cattle,sperm whale,and bottlenose dolphin on the growth of chondrocytes.We overexpressed the P27 gene of 4 species in ATDC5 cells respectively and grouped them,the results of EDU cell proliferation experiment showed that the P27 of mouse inhibited myoblast proliferation,but the P27 of cattle,sperm whale and bottlenose dolphin didn't inhibit the myoblast proliferation significantly.Western blot showed that compared with the control group,the expression of cyclin A2 and cyclin D1 in the mouse group were significantly reduced,but there was no significant difference of the two cyclins in the group of cattle,sperm whale and bottlenose dolphin.The results of flow cytometry experiments showed that compared with the control group,the cells in the mouse group were retained in the G1 phase,resulting in a significant reduction in the number of the cells in S phase and G2 phase;and the cycle of myoblasts in the cattle group,sperm whale group,and bottlenose dolphin group didn't change significantly.The above results indicate that the P27 gene of mouse blocks the G1/S phase transition of cells and inhibits the growth and development of chondrocytes;while the P27 gene of cattle,sperm whale and bottlenose dolphin weakened the inhibition of chondrocytes growth,and there was no statistical difference between sperm whale and bottlenose dolphin.Finally,we explored the effect of the Galns gene in mouse and sperm whale on the growth and development of chondrocytes.Early analysis found that the 265 amino acid of Galns gene in large animals is arginine(R),and that in small animals is lysine(K),in addition,mutations at certain positions of the gene will affect the animal's body shape.In order to observe the effect of Galns gene in large size animals and small size animals on the growth of chondrocytes,and whether the mutation of the265 amino acid position of the Galns gene affects the function of this gene,we transferred the Galns gene of mouse,sperm whale and the gene with amino acid mutation at position 265 into ATDC5 cells,and divided them into 5 groups(group of control,group of mouse,group of sperm whale,group of mouse mutation,and group of sperm whale mutation).The EDU experiment showed that the Galns gene of mouse and sperm whale promoted the proliferation of chondrocytes;compared with the group of mouse and sperm whale,the cell proliferation of the group of mouse mutantion and sperm whale mutation didn't change.The results of q RT-PCR showed that compared with the control,the expression of P27 and caspase3 m RNA in mouse and sperm whale were significantly reduced,and the expression of Bax m RNA in the group of sperm whale was significantly reduced.Western blot showed that the Galns gene of mouse and sperm whale decreased the expression of cyclin-dependent kinase inhibitor P27,and the Galns gene had no effect on the expression of cyclin A2 and pro-apoptotic protein Bax.ELISA experiments showed that compared with the control group,the Galns gene of mouse and sperm whale increased the GALNS enzyme activity in cells,and the mutation of amino acid at the position of 265 increased the enzyme activity.The above results indicate that the Galns gene promotes the proliferation of chondrocytes,and the K265 R mutation in mouse and R265 K mutation in sperm whale increase the GALNS enzyme activity.In summary,the above results suggest that the Grb10 and P27 of cetaceans have weakened functions of inhibiting growth and development,and there is no significant difference of these two genes in large and small cetaceans,suggesting that Grb10 and P27 maybe not the key genes that caused huge differences in cetaceans.In addition,the Galns gene of cetacean promotes the growth of chondrocytes,prompting that this gene may be involved in the regulation of sperm whale body enlargement,the research laid an experimental foundation for us to further explore the regulation mechanism of cetacean body size. |
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