| Sinonovacula constricta is our country’s important economic shellfish,with good prospects for development.As an important neurotransmitter receptor in the nervous system,dopamine receptors are concerned by scholars both at home and abroad.The growth of dopamine receptor in sensation,movement,immune,feeding,growth and other biological processes are important regulators and participants,dopamine,epinephrine and norepinephrine and their derivatives constitute the system of the various catecholamines,catecholamine neurotransmitters and receptors in close cooperation,launched a variety of signaling pathways,forms of biological information transfer complex network.1Cloning,selective of dopamine receptor of Sinonova culaconstricta and discover of polyadenylation variantTwo dopamine receptors ScDopR1 and ScDopR2 gene fragments from sinonovaculaconstricta transcriptome,after race cloned the full-length cDNA of two genes,the full-length 2216bp ScDopR2-1 gene,171bp 5 UTR region,ORF region1814bp,231bp 3 UTR region,encoding 597 amino acid residues;ScDopR2-2 gene found two variable polyadenylation variants contain the same ORF region and 5 UTR region,ORF region is 1440bp,encoding 479 amino acid residues,5’UTR region is34bp,the difference between the two variants is 3’UTR region length of the ScDopR2-2b gene in polyA before the end of the ScDopR2-2a gene than a 960bp,ScDopR2-2a variant cDNA the full-length 1824bp ScDopR2-2b variant of cDNA was2758bp.The results of prediction analysis and functional domain sequences showed that two receptors have the typical domains of G protein coupled receptors,the third have long outer ring and C-end is short,the preliminary judgment of two dopamine receptors are D2 dopamine receptor.2 Temporal and spatial expression of Sinonova culaconstricta dopamine receptorThe fluorescence quantitative detection of adult tissues showed that the highest expression level of ScDopR2-1 gene was water pipe and labial flap,and the expression level in axon foot,gill and mantle was the second,and the expression level in liver and gonad was the lowest.The ScDopR2-2 gene expression pattern of the homologous segment and ScDopR2-2b gene is basically the same,the highest expression of the tissue is water,expression in water,gill,axe foot was significantly higher than in other tissues,ScDopR2-2 gene homology compared with ScDopRb,the expression level is different,the expression of ScDopR2-2 gene homologous segments in the gills than to axe foot high,while the expression of ScDopR2-2b gene in the gills and axe foot in roughly the same.During the development stage,the expression level of ScDopR2-1gene was highest in creeping stage,followed by D stage.In these two periods,the peak of two expression levels increased,then decreased,then increased and then decreased.The expression level of ScDopR2-2 gene was highest at the top stage,and increased gradually from the cleavage stage,the wheel stage to the D stage.The expression level reached the peak at the top of the shell stage,and the creeping period began to slightly decrease,and the expression of creeping stage and juvenile stage was almost the same.3 Selecting of dopamine receptor antagonists and verification of dopamine receptor typesIn the antagonist selecting experiment,three antagonists,Domperidone（Domp）,sulpiride（Sulp）and Chlorprothixene hydrochloride（Chlo）,were tested as subjects,and the respective solvents were used as the control group.The concentration of cAMP and the changes of the expression of dopamine receptor gene were measured by Q-PCR in each dentisy(10-2M,10-3M,10-5M)at 2 and 6 hour.The selection of the type and concentration of dopamine receptors in Sinonova culaconstricta was carried out.The experimental results showed that the expression of gene expression at two time points in Chlo group was significantly lower than that of the control group at the concentration of2 hours and 6 hours at 6 hours,and cAMP was also in line with the increase of the expected experimental results compared with the control group.The suitable inhibitor of dopamine receptor was screened for Chlo,and the suitable concentration of 10-2M was screened out.According to the known pharmacological characteristics,the inhibitory ligand stimulates the cAMP level of the D2 receptor to verify the dopamine receptor type as D2 dopamine receptor.4 Water damage experiment of Sinonovacula constricta and Vibrio anguillarum infection experimentThe water pipe damage experiment,water pipe damage treatment and the expression of dopamine receptors were detected at different time points.The results showed that after the injure of the inlet pipe,the expression level was significantly up-regulated at 12 and 48h,while the 8,24 and 72h decreased significantly,suggesting that the dopamine receptor gene of the Sinonovacula constricta participates in the reaction process after the water pipe injury.Results the expression pattern of rise,down and up was presumed to be related to the compensatory effect of dopamine.In the infection experiment of Vibrio anguillarum,three experimental groups were set up:seawater injection of 100ul（group H）,injection of seawater 50ul+bacteria 50ul（HJ group）,and injection of inhibitor 50ul+bacteria 50ul（CJ group）,among which the group H was the control group.The results showed that the expression of ScDopR2-1gene in 2 and 4h increased,and 8,12 and 24h decreased.In the three experimental groups,the expression of CJ group was lower than that of the HJ and H groups.The expression of CJ group in 4h,8h,and the expressions decreased significantly.The expression of ScDopR2-2 gene increased in 2 and 4h,and the expression of 8,12 and24h decreased.In the three experimental groups,the HJ group decreased significantly in2 and 4h than that in H group.The CJ group was significantly lower than the H group.CAT detection results showed that the immune response initiated,the inhibitor had an effect on the immune process,and dopamine D2 receptor was involved in regulating the immune response.5 The role of dopamine receptors in Sinonovacula constricta in the developmental stagesThe effect of dopamine receptors in the early development of Sinonovacula constricta with receptor inhibitors was investigated by setting 4 inhibitors(10-2M,10-3M,10-4M,10-5M).The anaphase of shell top larva of Sinonovacula constricta was treated with 24h,and to calculate the rate of foot-growing larva.The results showed that the larval rates of the 4 concentrations of antagonists were 20%,35%,40%,40%respectively.while the control group was 60%,and the D2 type dopamine receptor antagonists played a certain inhibitory effect.It was concluded that the dopamine receptor had a regulatory role in the developmental stage of shell top larva in Sinonovacula constricta. |
PDF Full Text Request