Expression Characteristics Of Dopamine Beta Hydroxylase And Its Effect On The Growth And Metamorphosis Development Of Sinonovacula Constricta

Sinonovacula constricta is one of the four economically farmed shellfish in China,and it needs to go through an important larval metamorphosis period during its development.This period is also an important stage that affects the yield of seedlings and the breeding efficiency.Neurotransmitters are one of the chemical substances required in the process of biological growth and developments,including dopamine,norepinephrine have important regulatory effects on the growth of shellfish and metamorphosis of larvae.Dopamine beta hydroxylase is an important synthetase in the synthesis of catecholamines and catalyzes the conversion of dopamine to norepinephrine.Its role is very important.At present,there are few reports on the effect of shellfish dopamine beta hydroxylase,and there are few reports on the use of RNAi to study shellfish larvae.In this paper,the sequences of two dopamine ?-hydroxylase of S.constricta were identified and their expression characteristics and their roles in the development of S.constricta and metamorphosis of larvae were studied,in order to provide a certain way for further elucidating the mechanism of metamorphosis of shellfish larvae and improving the breeding efficiency of seedlings reference.1.Aligned the two D?H gene fragment sequences from the S.constricta transcriptome database,verified them and cloned the full-length cDNA,named ScD?H and ScD?H-?,respectively.The ScD?H gene cDNA sequence is 1959 bp in length,including a 15 bp untranslated region(UTR)at the 5 'end,a 222 bp 3'-UTR,and an 1722 bp open reading frame(ORF),which encodes 573 amino acid residues.The predicted molecular weight of the gene is 64.50 k Da and the theoretical isoelectric point is 5.40.The cDNA sequence of the ScD?H-? gene is 2079 bp in length,including a 67 'bp 5' terminal untranslated region(UTR),a 272 bp 3'-UTR,and a 1740 bp ORF,which encodes 579 amino acid residues.The predicted molecular weight of the gene is 64.97 k Da and the theoretical isoelectric point is 5.35.Phylogenetic tree results show that the D?H gene is divided into two branches,the S.constricta D?H gene and one of the invertebrate D?H genes come together,and ScD?H and ScD?H-? belong to the same subtype.2.Real-time PCR fluorescence quantitative detection of ScD?H and ScD?H-? genes in adult tissues of S.constricta(including liver,gill,foot,hemolymph,mantle,gonad and siphon)and larval development(including embryos,trochophores,veliger larvae,umbo larvae,creeping larvae,and juveniles).The results showed that ScD?H and ScD?H-? were widely expressed in various tissues.The expression of ScD?H gene was highest in liver.And the relative expression levels in hemolymph and gill tissues also increased significantly..Similarly,the relative expression of ScD?H-? gene was highest in liver and lowest in siphon tissue,while the expression level in other tissues was not significantly different.The expression levels of ScD?H and ScD?H-? gene in the early stage of larval development were not high.The expression levels gradually increased from umbo larvae and creeping larvae,respectively,and the gene expression of juveniles was the highest.3.Screening of suitable inhibitors of D?H and their concentrations.Screening was performed from three commonly used inhibitors including Nepicastat HCl(Nep),Fusaric acid(Fus),Disulfiram(Dis).The results showed that Nep was the most suitable inhibitor,and its optimum concentration was 1 mg / L.A Nep inhibitor was used to immerse S.constricta larvae during metamorphic development.The results showed that the inhibitors could effectively inhibit the development of S.constricta larvae and had a lethal effect.At the same time,inhibition experiments in S.constricta juveniles showed that juveniles' body weight and shell length were significantly consistent.It was found that in the Nep inhibitor group,the body weight and shell length of juvenile shellfish were significantly suppressed.4.Using RNAi interference technology to detect the role of D?H gene in S.constricta growth and larval metamorphosis.First,a suitable siRNA chain was designed and selected,and a suitable siRNA concentration was 1.1 ?g / ml.Secondly,RNA interference experiments were performed on S.constricta larvae using the immersion technique.The results showed that the metamorphosis rate of the siRNA group was significantly suppressed,and the survival rate of the high-concentration siRNA group was also significantly reduced.RNA interference experiments were performed on S.constricta juveniles using siRNA injection technology,and their shell length and body weight were significantly suppressed.Further detection of related growth factors downstream of the D?H pathway indicated that when the D?H gene partial silenced,the expression of related growth factor genes is suppressed to varying degrees.The results revealed that the D?H gene has an important effect on the growth and development of S.constricta larva metamorphosis.