The Primary Study Of Related Genes Of Ca²⁺ Signal Pathway Sensing Tidal Rhythm In Onchidium Reevesii

In order to adapt to environmental changes,marine invertebrates that inhabit intertidal zones have exhibited a variety of biological rhythms,such as circadian rhythms,tidal rhythms and lunar rhythms,which are coincided with the plasticity of the nervous system.Compared with higher vertebrates,with a simple structure of the nervous system,invertebrates are best materials for studying the related functions of the nervous system.Onchidium reevesii inhabiting the intertidal zone is widely distributed in the coastal areas of southeastern China.Due to the simple structure of the nervous system,it is a good material for studying the plasticity regulation of the nervous system and can be developed into an excellent marine mollusk model.Four kinds of Onchidium in coastal areas of China have been investigated in the early stage of this laboratory,among which the structure of the O.reevesii is highly simple,providing convenience for studying effect system plasticity on tidal rhythm also supplying a good material for studying the biological rhythm mechanism of intertidal marine animals.In this experiment,the expression of the related gene in Ca²⁺signal pathway were detected by q RT-PCR,the positioning analysis of the Or Ca MKII protein was detected by Western blot and the low frequency acoustic wave frequency simulates the tide to explore its learning and memory function.1.Cloning and tissue expression analysis of Or Ca M,Or Ca MKII and Or Ca MKIVgeneThe Ca²⁺signal pathway is one of the most important pathways of rhythmic regulation,has the function of neuroplasticity regulation.During the stimulation of external factors such as environmental stress,virus invasion and mechanical damage,the Ca²⁺influx in organism,combined with Ca M to form the Ca²⁺/Ca M complex which activated the Ca MKII of downstream signaling pathway,followed by the phosphorylation of Ca MKII that induce the expression of c AMP-responsive element-binding protein（CREB）,causing corresponding physiological behavioral changes.To explore the role of Ca²⁺signaling pathways in the evolution of O.reevesii from ocean to land,furthering understood the physiological functions of this signal pathway in the neuromodulation of O.reevesii.In this study,the c DNA sequence of Ca M,Ca MKII and Ca MKIV gene were cloned by RACE-PCR using nervous tissue of O.reevesii,and bioinformatics analysis and QRT-PCR experiment were performed,respectively.The results suggested that the full-length c DNA of Ca M gene consists of 2 123 base pair（bp）,with 465bp open reading frame（ORF）which encode a total of 155amino acids,has four Eh-hand domains.The full-length c DNA of Ca MKII gene consists of1656 base pair（bp）,113 bp 5’non-coding region（UTR）,196 bp 3’non-coding region（UTR）and 1347 bp open reading frame（ORF）which encode a total of 349amino acids.The atom of the polypeptide chain encoded by this gene was predicted.The Ca MKII protein was predicted to contain 7 178 atoms,the molecular weight is about 50.8 KDa,the theoretical isoelectric point is 7.24,the molecular formula is C₂₂₇₇H₃₅₉₃N₆₂₉O₆₆₆S₁₃,and the N-terminal signal peptide is composed of 1-29 amino acids.The results indicated that the full-length c DNA of Ca MKIV gene consists of 1603 base pair（bp）,315 bp 5’non-coding region（UTR）,256 bp 3’non-coding region（UTR）and 1032 bp open reading frame（ORF）which encode a total of 343 amino acids.The atom of the polypeptide chain encoded by this gene was predicted.The Ca MKIV protein was predicted to contain 5 490atoms,the molecular weight is about 3.873 7KDa,the theoretical isoelectric point is 6.12,the molecular formula is C₁₇₄₁H₂₇₆₈N₄₅₂O₅₁₄S₁₅,and the N-terminal signal peptide is composed of 1-29 amino acids.The amino acid sequence alignment and phylogenetic tree results suggest that the Or Ca MKII and Or Ca MKIV gene were respectively closest to the Ca MKII and Ca MKIV gene of Aplysia californica and the Biomphalaria glabrata.Their phylogenetic analysis was consistent with the traditional morphological classification.The results of real-time PCR indicated that the Ca MKII gene was expressed in all tissues,and the relative expression in ganglion was the highest,followed by the intestine and hepatopancreas,and the expression level in the plopod,dorsal skin,mouthpart and albumen gland were low.The results of real-time PCR displayed that the Ca MKIV gene was expressed in all tissues,and the relative expression in the foot was the highest,followed by the dorsal skin and mouth,and the expression level in the ganglion,albumen gland,intestine,hepatopancreas and other organs were low.It was preliminarily speculated that the Ca MKII and Ca MKIV gene may play an important role in synaptic plasticity of the ganglion in O.reevesii.2.Effect of tidal and diel orchestration on related gene expression of Ca²⁺signal pathway in an intertidal Onchidium reevesiiMarine animals inhabiting the intertidal zone have long been exposed to high tide and low tide environments,and their behavior and physiological state are largely affected by the tidal cycle caused by the sun and the moon.O.reevesii was exposed to tidal movements.According to tide table on May 15 to 20,2019,the lowest tide samples were selected.The rising tide samples and ebb tide samples of tidal height from 300 cm to 350 cm were selected,respectively.The O.reevesii was submersed by seawater when it was at highest tide.The seawater will be touched the sample when the tidal height between 300 cm and 350 cm.Next,the daily sampling time was delayed by about 60 minutes and the samples for 6consecutive days were selected.The m RNA expressions of Or Ca M,Or Ca MKII and Or CREB1 at lowest tide of night were higher than in the other groups after exposure to tidal cycle and day-night cycle.We hypothesized that these genes expression may be in connected with the regulation of tidal rhythm and circadian rhythm.These findings will strengthen the notion that the molecular mechanism of circadian clock genes in intertidal organisms,and it will provide the reference for understanding the behavior of O.reevesii affected by circatidal rhythm and circadian rhythm.3.Expression of Ca M-like and Ca MKII genes in Onchidium reevesii under low frequency sound wave stimulation.In this experiment,we stimulated O.reevesii in the laboratory by simulating the low-frequency sounds of the tides.The c DNA sequence of Ca M-like gene was cloned by RACE-PCR,bioinformatics analysis and q RT-PCR experiments were performed.The results showed that the full-length c DNA of Ca M-like gene was 2321 bp,366 bp 5’non-coding region（UTR）,1 337 bp 3’non-coding region（UTR）,618 bp open reading frame（ORF）which encoded a total 206 amino acids.The gene encodes a polypeptide chain was predicted with an atomic number of 3 165,the molecular weight is about 23 029.64 KD,the theoretical isoelectric point is4.64,the molecular formula is C₁₀₁₈H₁₅₄₄N₂₇₄O₃₂₀S₉,and the N-terminal signal peptide is composed of 29 amino acids.The phylogenetic tree was constructed by amino acid sequence alignment.The results suggested that the Ca M-like gene of O.reevesii was closely related to the Ca M-like gene of Lymnaea stagnalis and Biomphalaria glabrata,which was consistent with traditional morphological classification.The distribution of Ca M-like gene in different tissues was detected by q RT-PCR.The results indicated that Ca M-like m RNA was distributed in different tissues of O.reevesii,but the expression of ganglion was significantly higher than dorsal skin,pleopod,intestines,liver,mouthparts and albumen gland（P<0.05）.We speculated that it may be involved in the plasticity regulation of ganglion system.Fluorescence quantitative results indicated that the Ca M-like and Ca MKII genes were highly expressed underlying the stimulation of low frequency sound wave frequency of 12.4 h at 25 Hz and 50 Hz,respectively,which was presumed that it could sense the 25-50 Hz sound frequency.This may be related to the tidal memory formed by long-term sense 12.4 h half-day tidal cycle rhythm in the intertidal zone.This study will lay a foundation for further understanding of the molecular mechanism of O.reevesii sensing tidal rhythm,and also provide a basis for exploring the environmental adaptation mechanism of marine animals from ocean to land evolution.In this experiment,we investigated the functions of related genes of the Ca²⁺signaling pathway that involved in the plasticity regulation of neural signal transduction in O.reevesii.And the basic functions of these genes were preliminarily predicted by software.Real-time PCR was used to detect the expression of those gene m RNA in different tissues.The results showed that the Or Ca M,Or Ca M-like and Or Ca MKII gene were mainly expressed in the ganglion compared with other tissues,while the Or Ca MKIV gene was mainly expressed in the gastropod compared with other tissues.Through field experiments,the expression of Or Ca M,Or Ca MKII and CREB1 genes were higher in lowested tide of night than that of the control group.Western blotting detected the levels of the endogenous Or Ca MKII protein in response to tidal changes.Finally,using low frequency sound waves to simulate the tidal frequency,it is found that the expression of Ca M-like and Ca MKII is higher when the 25 HZ-50HZ acoustic frequency is stimulated for 12.4 h,and the expression trend is consistent.Therefore,we speculated that the O.reevesii can sense the tidal rhythm to adjust its behavior in accorandant with environmental changes,which may be related to the low-frequency sound produced by the tide.4.Effect of pathogenic bacteria on a novel C-type lectin,hemocyte and superoxide dismutase/alkaline phosphatase activity in Onchidium reevesiiBacterial infection in the marine environment is a serious problem to maintain the stability of marine ecosystems.Nevertheless,there is little report so far for the biological effects of pathogenic bacteria in coastal ecosystems.Hence,we investigated the responses of shell-less Onchidium reevesii to resist against pathogenic bacterial infection.Analysis of data here could be used as fundamental information for assessment of innate immune response of O.reevesii.The full-length Or CTL c DNA was cloned and consists of 1849 base pair（bp）encoding protein of 192 amino acids.Constructing multiple alignments suggested that Or CTL has conserved carbohydrate recognition domain（CRD）of CTLs,containing an EPS（Glu-Pro-Ser）motif that may imply the function of recognition of carbohydrates like others invertebrate.Or CTL m RNAs were mainly detected in ganglion and hepatopancreas,and expression was highly up-regulated from 2 h after Vibrio harveyi challenge,rapidly decreased at 4 h,and significantly increased at 12 h.In addition,after challenge with Vibrio parahaemolytics,Or CTL gene expression was slightly up-regulated from 2 h,peaked at 12 h.Enzyme activity（in the hepatopancreas）and cell immune（in the hemolymph）were investigated along with Superoxide dismutase（SOD）activity,alkaline phosphatase（ALP）activity and cell cycle.SOD activities were significantly higher after V.harveyi and V.parahaemolytics challenge than that in the control group,respectively.By contrast,ALP activities were significantly inhibited after challenged with bacteria than that in the control group,respectively.Enzyme activities in the hepatopancreas obviously fluctuated,and ALP activity was more sensitive to bacteria.Cell responses illustrated that there were a significant higher percentage of cells in the S and G2/M phase in hemolymph after challenged with bacteria.Our results suggested that the immune response of O.reevesii could be activated by pathogenic bacteria,and the data will provide referent for the disease prevention of systematic investigation in aquatic animal.