| In China,researchers studied them very early,the emphasis of domestic study of Onchidiidae have changed from morphology to evolution.We selected the dorsal skin from two species of Onchidiidae as the objects of study.Onchidium struma were mainly terrestrial living and Paraoncidium reevesii were predominantly aquatic living.The two species were seleted to further explored the differences in protein level of dorsal skin adapt to the environment.Based on the biopsy staining observation and proteomic sequencing analysis of dorsal skin from two species,we got the differences in protein level,and screen out important protein related to biological phenomenon from two species:(1)the amphibian skin played an irreplaceable role of survival and adaption in habitat conditions change.From the observation of two species of Onchidiidae dorsal skin biopsy TUNEL staining,we could saw that apoptosis signal of dorsal skin from O.struma more stronger than that from P.reevesii,and apoptotic cells closed to the skin parts were more concentrated distribution,the papillae region had apoptosis signal obviously.We suspected dorsal skin cells from two species of Onchidiidae continuously updated to adapt to the environment.The P.reevesii had obvious molting and this phenomenon requires constant new cells,O.struma dorsal skin cell also had phenomenon of cell differentiation to adapt to the terrestrial life and kept skin wet without obvious molting,thus the body to maintained balance,induction of apoptosis signal.(2)Using the Label-free non-tag quantitative research methods to identified proteomic and analysed dorsal skin in O.struma and P.reevesii.The collagen protein family was identified for many times in the dorsal skin from two species of Onchidiidae,explained that onchidiids have high nutritional value and potential economic value.Difference analysis showed that purine metabolic pathway had most homologous sequences.P.reevesii dorsal skin had more vitamin C and heme albumin protein than O.struma in differences proteins.Those proteins played an important role in respiration and osmotic adjustment in P.reevesii.Quantitative analysis showed that there had many kinds of differential homologous proteins in two species of Onchidiidae dorsal skin,differential homologous proteins were closely linked with different biological character,at the same time to participate in a variety of pathway regulation.There were also more differential homologous sequence in Glycolysis / Gluconeogenesis pathway,suggested that two species of Onchidiidae dorsal skin tissue had differences in energy metabolism process.Data analysis showed that the key enzyme hexokinase of Glycolysis / Gluconeogenesis pathway is high expression in P.reevesii dorsal skin,to ensured adequate energy supply,promoted cell proliferation,thus the level of P.reevesii dorsal skin glycolysis ability was stronger,related to the phenomenon of molting(skin cutin membrane change frequently),needed more energy and rapid cell proliferation to support.Corneous layer in the surface of the skin,was the important barrier to defense physical and chemical damage from external environment.The keratinization degree of dorsal skin from O.struma is higher,and the main components ceramide of the lipid matrix were express high in dorsal skin of O.struma.The ceramide was the important second messenger in a variety of cytokines and growth factors signaling pathways,played an important role in the skin osmotic pressure regulating pathway,made a significant contribution to the skin moisturizing,was an embodiment of O.struma to adapted to the terrestrial life.Found in apoptosis enrichment,proteins related in promote cell proliferation and inhibit apoptosis P.reevesii dorsal skin high expression,associated with the phenomenon apoptosis signal was weak in P.reevesii dorsal skin through TUNEL staining observation.There were many different homologous sequences exist in purine metabolic pathway of dorsal skin from two species of Onchidiidae.studies suggested in the process of animals from aquatic to terrestrial evolution,urease gene in land animals disappear completely,or lose activity,transcriptome and proteome variance analysis of and P.reevesii showed that urease gene and protein in O.struma dorsal skin were in high expressed,and preliminary experiments showed that urea level in the secretion mucus of P.reevesii were much higher than O.struma,suggested that urea of O.struma produced by purine metabolic pathway was mostly decomposed by urea enzyme,O.struma preferred to aquatic biology characteristics,belonged to the special species.We presumed that when O.struma received stimulation,they were given priority to secreted mucus and sent out an irritant gas.The excitant gas contained ammonia,but P.reevesii were given priority to jet mucus in response to external stimuli.(3)Using RACE method,we got full sequences of Os-PAK2 and Os-14-3-3 in O.struma,and also got Pr-14-3-3 cDNA in P.reevesii.The 29-64 sites amino acid position in Os-PAK2 were typical PBD(P21 binding sites),222-473 was a serine/threonine structure,had self phosphorylation regulation effect,was main parts to regulate the activity of PAK.The mRNA and protein expression level of PAK2 in O.struma dorsal skin were both higher than P.reevesii.Combined with TUNEL staining results,surmise in O.struma dorsal skin tissue Os-PAK2 promote apoptosis effect more apparent.The fluorescence quantitative expression of Os-14-3-3 in intestinal tract and nerve tissue of O.struma showed that the expression were much higher than that in P.reevesii.As a result,we surmised that O.struma to adapt to the terrestrial life lead to the intestinal tract and nerve tissue activity decreased and update rate of tissue cell was slower.Pr-14-3-3 expression levels in dorsal and foot from P.reevesii were higher than those from O.struma,surmised P.reevesii dorsal skin cell proliferation differentiation,and apoptosis signal is restrained,ensure that have added new cells in place while the cutin membranes often change.The dorsal skin of O.struma and P.reevesii formed their unique biological adaptation for the environment in long-term evolution.This study gave a further understanding on the differential proteins and amphibious adaptability of dorsal skin from two species of Onchidiidae,provided the foundation and some candidate proteins of proteomics for comprehensive analysis environmental adaptability of two proximal species,provided direction for further study. |
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