Cloning And Verification Of Low-frequency Hearing Gene And Preliminary Exploration Of Tide-sensing Ability In Onchidium Reevesii

Onchidium reevesii is a brackish water amphibian living in tidal flat wetlands.It belongs to Mollusca,Gastropoda,Pulmonata,and Systellommatophora.O.reevesii is a shellfish with high edible and medicinal value.Its living distribution area is common in the high tide zone at the junction of freshwater and seawater in Jiangsu,Shanghai,Zhejiang,Fujian,Guangdong and other places in China.Its temperature adaptability is very strong,its distribution range is wide,and its wild resources are very rich.Observing from the morphological point of view,O.reevesii has not evolved a complete cardiovascular system.There is only a small amount of blood sinuses in the skin tissue,which makes it incapable of obtaining oxygen through skin respiration."Lung sac" is the main organ that the body uses to obtain oxygen.In terms of behavior,O.reevesii also exhibits a long-term and accurate "homecoming behavior".It lives in nests,caves and rock cracks during high tide,and comes out to absorb humus and food such as algae,O.reevesii from different nests rose again at an hour before being able to go home at the same time.Demonstrates the high degree of accuracy and complexity of this process.The balance capsule and otolith of shellfish are mostly considered as the sensory organs for sensing the acceleration of gravity and angular acceleration,but the otolith structure of O.reevesii is not easy to be separated and obtained.In this laboratory,the study of nerve tissue separation of O.reevesii is relatively mature,and its perception of tide and return home cannot be separated from neural induction.Therefore,in order to explore the high accuracy,complexity and influencing factors in O.reevesii of ability to accurately perceive tides in the special behavior of "homing behavior".This study selected O.reevesii nerve tissue as the research object,for gene cloning,fluorescence quantitative and in situ hybridization technique,a preliminary explore O.reevesii of low-frequency hearing related genes,and more interested in gene expression of time and space analysis,the correlation between listening ability and speculated that perception tide,O.reevesii tidal perception and reaction mechanism provides basic theory data in a timely manner.The main research contents are as follows: 1.Cloning and verification of OnWSF1 and OnPMCA2 genes in O.reevesiiThe full-length sequences of OnWFS1 and OnPMCA2 genes in O.reevesii were 3003 bp and 5091 bp,encoding 814 and 1208 amino acids,respectively.According to amino acid component analysis,the OnWFS1 and OnPMCA2 genes encoding proteins are stable proteins.The software predicted that OnWFS1 protein had 9 transmembrane regions,which were encoding endoplasmic reticulum transmembrane glycoproteins.The OnPMCA2 protein has 8 transmembrane regions,which are also transmembrane proteins.OnWFS1 and OnPMCA2 proteins contain most of the helix structure in the secondary structure prediction,which lays a structural foundation for the formation of the active center of the protein and the catalytic center.Through amino acid sequence comparison,it was found that OnWFS1 and OnPMCA2 of in O.reevesii had high sequence homology with california sea rabbits.The phylogenetic tree analysis of the coded amino acid sequence showed that in O.reevesii had a relatively close evolutionary relationship with the California sea hare.The expression of OnWFS1 and OnPMCA2 genes in in O.reevesii protein glands,nerves,hepatopancreas,gonads,intestines,back,gastropods and oral organs were analyzed.The results showed that the OnWFS1 and OnPMCA2 genes were expressed in all tissues of in O.reevesii,and the q RT-PCR results showed that the expression levels of OnWFS1 and OnPMCA2 genes were significantly higher in tissues of in O.reevesii than in other tissues.2.Multi-spatial and temporal quantitative analysis of OnWFS1 and OnPMCA2 genes in different groups of O.reevesiiThe expression of OnWFS1 and OnPMCA2 genes in O.reevesii nerve tissues was investigated by the use of low-frequency sound wave generators with different frequencies(0Hz,10 Hz,40Hz,70 Hz,100 Hz,130 Hz,160 Hz,190 Hz,220 Hz,250 Hz,280 Hz,310 Hz)and field tides to interfere with tumor fossae sulfonyl.According to the quantitative analysis results,OnWFS1 and OnPMCA2 genes have a significant trend of increasing when frequency interference occurs between 130 Hz and 310 Hz,and OnPMCA2 genes are more sensitive to acoustic stimulation perception in the range of 160 Hz to 310 Hz.Field of three groups of O.reevesii nerve tissue OnWFS1,OnPMCA2 genes and quantitative analysis,the results showed the island May 8 local collection and July 08 directly to temporarily raise in the east China sea in O.reevesii,two months after the acquisition of its nerve tissue OnWFS1 and OnPMCA2 gene expression quantity with local trend,the tide when the water is at high tide,two high gene expression,the tide to the low tide period,quantity of gene expression is relatively low.Onthe other hand,22 OnWFS1 and OnPMCA2 temporarily raised in the east China sea for 10 days on May 18 were not consistent with the tidal trend of the same day in the east China sea,and they were not correlated with each other.3.In situ hybridization analysis of OnWFS1 and OnPMCA2 genes of nerve tissues in O.reevesiiIn situ hybridization experiments were conducted to investigate the expression and dynamic distribution of OnWFS1 and OnPMCA2 genes in the nerve tissues of groups in O.reevesii,including blank untreated group,280 Hz low-frequency acoustic disturbance group and east China sea field tidal disturbance group.Generally speaking,the OnWFS1 and OnPMCA2 genes are widely expressed in O.reevesii neurons,but the hybridization signal intensity is different,which is basically expressed as the blank > group of > east sea field tidal disturbance group of 280 Hz low-frequency acoustic wave interference group.The experimental results are consistent with the fluorescence quantitative results in chapter 3 of this paper,and the experimental results are reliable.The dynamic distribution of OnWFS1 and OnPMCA2 genes in each ganglion of nerve tissues in O.reevesii indicates that the induction of O.reevesii pairs of low-frequency sound waves is interrelated among the ganglia,and is not limited to a single ganglion or a single neuron cell.