| Exopalaemon carinicauda is mainly distributed in shallow and low salt waters of the Yellow Sea and Bohai Sea.It is widely salted and has the advantages of fast growth and strong adaptability.In recent years,the aquaculture in coastal saline-alkali waters has achieved initial success.As a potential model organism to study the reproductive development of crustaceans,Exopalaemon carinicauda can be reproduced multiple times during the breeding season.At present,it is not possible to achieve full-year reproduction by artificial control technology.Therefore,it is very necessary to understand the ovarian development mechanism for the selection and breeding of fine varieties and improve the economic benefits of pond culture of Exopalaemon carinicauda.Studies have shown that estrogen-related receptors(ERR)may be involved in regulating ovary development in crustaceans,but the regulatory mechanism remains unclear.Therefore,this study cloned the estrogen related receptor gene of Exopalaemon carinicauda,interfered the expression of ERR gene by RNAi technology,and analyzed the comparative transcriptome sequencing to preliminarily explore its mechanism in ovarian development,so as to provide basic information for further study on the regulation mechanism of estrogen related receptor in crustaceans.Studies have shown that carbonate alkalinity stress can affect the ovarian development of Exopalaemon carinicauda.However,its molecular regulatory mechanism is still unclear.In this study,a comparative transcriptome analysis was conducted on the ovarian tissue of Exopalaemon carinicauda under carbonate alkalinity stress to preliminarily study its response mechanism under carbonate alkalinity stress.The main results are as follows:1、Cloning and sequence analysis of estrogen-related receptor gene in Exopalaemon carinicaudaIn this study,the full-length ERR cDNA of Exopalaemon carinicauda,named EcERR,was cloned by RACE method.The total length was 2025 bp,and the 5 ’noncoding region and 3’ non-coding region were 241 bp and 410 bp,respectively.The open reading frame of EcERR was 1374 bp,and the protein encoded by this gene was composed of 457 amino acids,and the predicted molecular weight and isoelectric point were 50.83 kD and 7.84,respectively.EcERR contains two conserved functional domains,including one nuclear receptor C4 zinc finger structure(ZnF＿C4),one ligand binding domain(Holi domain)and one low complexity domain.The average value of predicted hydrophilicity of EcERR amino acids is-0.331.In the ligand-bound region,nine hydrophobic regions can be seen.The amino acid sequence encoded by ERR gene of Exopalaemon carinicauda was compared with the amino acid sequence encoded by ERR gene of other species.The results showed that the amino acid sequence of EcERR and Macrobrachium rosenbergii had the highest homology of 98.47%.Phylogenetic analysis of EcERR amino acid sequence was conducted using MEGA6.0 software.The results showed that EcERR amino acid was closely related to Macrobrachium Rosenbergii,and it clustered with crustaceans to form a branch,and then with insects to form a branch.2、Preliminary study on the function of EcERR gene in Exopalaemon carinicaudaThe tissue distribution of EcERR gene and its expression in various stages of ovarian development were discussed.It turns out that EcERR was expressed in all tissues,and the relative expression was the highest in ovary.The expression level of EcERR was high in the early stage of ovarian development,and decreased with the gradual maturation of the ovary.The expression level of EcERR was the lowest in the stage III of ovarian development,and then gradually increased,suggesting that EcERR was involved in the ovarian development of Exopalaemon carinicauda.5-HT is widely distributed in crustaceans,and some studies have found that 5-HT can effectively promote ovarian development.5-HT,as a biological amine,is widely distributed in crustaceans.Studies have found that 5-HT can promote ovarian development.The Vg and EcERR in the ovary of the experimental group were 15.15 and 4.48 times higher than those of the control group,respectively.These results indicated that5-HT had a certain promotion effect on the ovarian development of Exopalaemon carinicauda,and EcERR was involved in the ovarian development process of Exopalaemon carinicauda,but its regulatory mechanism needs to be further studied.3.Transcriptome sequencing analysis of ovarian tissue after interference ERR geneInterference ERR gene expression,in GFPvs ERR group the difference of gene,eight screening differentially expressed genes,among them,involved in estrogen signaling pathways of cathepsin D(CTSD)and heat shock protein 90(HSP90)lower expression,studies have found that CTSD and HSP90 participated in the genetic regulation of vitellogenin speculation EcERR gene may participate in the regulation of vitellogenin genes,which affect the growth of oocytes and mature.The expressions of adenylate cyclase(AC)and calmodulin(CaM)involved in cAMP signaling pathway were significantly up-regulated.The upstream gene AC of c AMP can promote the synthesis of c AMP,make the intracellular cAMP level in high concentration,and inhibit the meiosis of oocytes.It is speculated that EcERR is involved in the regulation of ovary development,possibly through the regulation of oocyte meiosis and vitellogenin,and then affect ovary development of Exopalaemon carinicauda.4、Sequencing analysis of transcriptome of Exopalaemon carinicauda under alkalinity stressAfter sequencing the transcriptome,a total of 41,162,692-64,591,170 Raw Reads were produced in 6 libraries,and a total of 41,024,540-64,383,978 Clean Reads were obtained.In all libraries,Q20 is greater than 98,Q30 is greater than 93,and the size of clean reads is about 18.5GB.The Clean Reads were assembled by Trinity software,and123,732 unigenes were obtained.The N50 and average length of unigenes are 1337 bp and 748.31 bp,respectively.According to the length distribution statistics of assembled unigenes,there are 80,890 pieces distributed between 200 bp and 500 bp,accounting for 65% of the total,with the largest number;There are only 9633 pieces with a bp greater than 2000,accounting for only 8% of the total.In the KB vs SC＿2 group,8differential genes were screened for verification,and the expression trend was consistent with that of RNA-seq,which indicated that the sequencing results were reliable.The expression levels of differential genes Mth1 and VMO-1 were downregulated,which suggested that the formation of yolk outer membrane would be affected by carbonate alkalinity stress,and then the quality of ovum might be affected. |
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