Cloning And Expression Analysis Of 5-HT7 And DA2 In Several Physiological Processes Of Eriocheir Sinensis

Biogenic amines are widely distributed in the nervous system of crustaceans,among which 5-HT and DA involved in regulating animal organism of many important physiological functions,including reproduction,feeding and aggressive behavior,and the role of DA and 5-HT were not studied at present on Eriocheir sinensis.To explore the role of 5-HT and DA on Eriocheir sinensis reproduction,light adaptation and feeding,5-HT7 receptor of Eriocheir sinensis and DA2 receptor have cloned on the preliminary exploration,and analyzed the expression levles in different tissues,determined the tissues with the highest expression level,analyzed the expression levels of the two genes on the above physiological processes,at the same time,we studied the different concentrations of tryptophan feed for Eriocheir sinensis 5-HT receptor,molt related receptors and the effect of digestive enzymes.This paper adopts the Reverse transcription PCR(RT-PCR)and Rapid amplication of cDNA ends(RACE)for full-length of 5-HT7 Eriocheir sinensis(GenBank accession number: GM601745)and DA2(GenBank accession number: MF683118)receptor gene sequence.Among them,the 5-HT7 receptor gene contains 2328 nucleotides,and the open reading frame(ORF)contains 1773 nucleotides(including the terminator codon),encoding 590 amino acids.DA2 sequence is 2369 bp,and the open reading frame is 1770bp(including the termination codon),encoding 589 amino acids.Both belong to the G-protein coupled receptor superfamily,with seven transmembrane domains,highly conservative DRY residues and multiple conservative areas.By Quantitative real-time PCR(qPCR)technology research the two genes expression levels in different tissues and different stages of ovarian development,different photoperiods,and different feeding stages in Eriocheir sinensis,the results show that these two genes in hepatopancreas,thoracic ganglia,cranial ganglion,eye stalks,intestine,muscle,gill,haemolymph and heart are expressed in the nine tissues,and have a high level in thoracic ganglia and cranial ganglion;the expression of DA2 mRNA in the intestine was not significantly different from thoracic ganglia and cranial ganglion(P>0.05),which was significantly different from other tissues(P<0.05).With ovarian growth,5-HT7 receptor expression levels in stage Ⅲ and Ⅳ are significantly higher than stage Ⅰ(P<0.05),while DA2 expression level in stage Ⅰ-Ⅴ showed a trend of decline,stage Ⅳ significantly lower than stage Ⅰ and Ⅱ(P<0.05).In thoracic ganglia,the expression level of 5-HT7 mRNA was significantly increased in constant light.In the cranial ganglion,constant darkness significantly increased the expression level of DA2.After feeding,the expression levels of 5-HT7 and DA2 receptors in hepatopancreas and intestine increased significantly.These results indicated that the 5-HT7 receptor has a certainly promoting effect on ovarian maturation,while the DA2 receptor has an inhibitory effect on ovarian maturation;and the 5-HT7 receptor and the DA2 receptor were involved in the light adaptation and ingestion process of Eriocheir sinensis.By feeding different concentrations(0.36%(group A),0.47%(group B),0.73%(group C)and 1.05%(group D))of tryptophan feed,this paper studied the effects of tryptophan on 5-HT receptor gene(5-HT1 B,5-HT2 B,5-HT7),the molting related genes(Chi,EcR,MIH)expression levels in the tissues including hepatopancreas,thoracic ganglia,cranial ganglion,and intestine,and digestive enzymes of the hepatopancreas of Eriocheir sinensis.The results showed that,with the increase of tryptophan concentration in feed,the level of 5-HT in haemolymph gradually increased.The expression levels of 5-HT1 B receptor mRNA in the cranial ganglia and thoracic ganglia gradually increased.The expression level of 5-HT2 B receptor mRNA in the intestine gradually decreased.The expression level of 5-HT7 mRNA in cranial ganglion and intestine also gradually increased.In addition,the expression level of chitinase(Chi)gene mRNA in thoracic ganglia increased with the increase of tryptophan concentration.The expression level of ecdysone receptor(EcR)in hepatopancreas was decreased.The expression levels of the molt-inhibiting hormone(MIH)gene in the intestine of group B and in the thoracic ganglion of group C were significantly higher than other three groups.In group C,the activity of hepatopancreas protease was significantly higher than that in group A and B,and there was no significant difference between group C and group D.The activity of amylase and lipase activity decreased with the increase of tryptophan concentration.These results suggested that as the precursor of the 5-HT,the tryptophan played a significant role on the expression levels of the 5-HT receptors,and participated in the regulation of molting,but had certainly inhibition effect on digestive enzymes activity in the Chinese mitten crab.Research has shown that 5-HT7 and DA2 receptor gene express in all tissues of Eriocheir sinensis,and participate in several physiological processes such as reproduction,light adaptation and digestive,play the important role in the growth and development in Eriocheir sinensis.In addition,as a precursor of the 5-HT,tryptophan can adjust Eriocheir sinensis molting and digestion.