Construction Of Gene Expression Profile Of Gonads And Screening And Preliminary Validation Of The Gonadal Development Gene In Apostichopus Japonicus

Sea cucumber (Apostichopus japonicus) is an invertebrate which has high nutrition value and health care function and the highest economic output within mariculture species in China. Nowadays, the cultivation of sea cucumber has become one of the pillar industries of China marine aquaculture. But gender identification of sea cucumber is still a major problem that needs to be solved in the breeding process. The mechanism of gonadal development is the basis of breeding regulation, and it also provides a theoretical basis for sex determination mechanism. Sea cucumber is an ideal model for gonadal growth for its special evolutionary status and its periodical gonadal development. In this study, gene expression profiles of gonad from male and female sea cucumber are constructed using a high‐throughput transcriptome sequencing technology. Using comparative transcriptome analysis, candidate major regulative genes or related transcriptional regulatory factors for gonad development are selected. Then temporal and spatial expressions of the selected candidate genes are detected using real‐time quantitative PCR and conventional PCR.The main results are listed as follows:Gonads gene expression profiles of male and female sea cucumber are constructed using PTS‐454high throughput sequencing techniques from sea cucumber gonads which are in the growing phase. Using454sequencing run,909332reads are produced and then assembled into13544contigs. A total of23506unique protein‐coding genes were identified from a variety of developmental stages and adult tissues based on sequence similarities with known proteins. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes recovered diverse biological functions and processes. Transcripts putatively involved in binding，catalytic activity，protein binding，nucleic acid binding，hydrolase activity，transferase activity， oxidoreductase activity， kinase activity were identified. Using comparative transcriptome analysis,3318significiant differentially expressed genes are selected which involves1135up‐regulated genes and2734down‐regulated genes.15266single nucleotide polymorphisms (SNPs) and1117simple sequence repeats (SSRs) were also detected.Twenty‐five genes are identified using bioinformative analysis according to gene annotation information and gene expression abundance. Of which,15genes named A1‐A15are seleted from male gene expression profile and10genes named B1‐B10are selected from female gene expression profile. Then16pairs of real‐time PCR primers (11from male and5from female) are designed accroding to the25gene sequences and15pair of primers can product good amplification curve. Real‐time PCR were conducted to dectect differential expression in male and female gonads which in growth phase of the15candidate genes. The result shows that all of the selected genes have significiant different expression in male and female gonads. Expression levels of11genes selected from male gene expression profile in male gonads is significiant higher than that in female gonads, the expression levels of A1, A6, A10, A14in male gonads is123~6000000times to the female gonads; the expression level of A3, A4, A5, A7, A8, A9, A13in the male gonads is4.38~650000times to the female gonads. Expression levels dectection are also conducted in4genes selected from female gene expression profile. The expression levels of B2, B8in female gonad is significiant higher than the male gonads, the expression level in the female gonads is115~524,417times to the female gonad. However, the expression level of B6, B7in the male gonads is significiant higher than the female gonads, the expression level in the male gonads is3.52~936times to the female gonads. The concordance rate of the454sequencing results and quantitative results is81%.Gene expression model of the15gene in different gonad development periods of male and female sea cucumber are detected using real‐time period. The results showed that the gene A4, A7, A9only express in growth Phase II and mature Phase of the sea cucumber female gonads, but express in the male gonads of proliferative phase, growth phase I, growth Phase II, mature phase; A8express in growth phase II and mature phase of female gonads, the male gonads of proliferation phase and growth phase I, and didn’t express in the remaining phases of the male and female gonads. The others genes express in the sea cucumber gonad development period, the expression levels of A1, A3, A5, A6, A10, A13, A14, B6, B7in the male gonads was higher than the female gonads, the expression levels of B6, B7in the female gonad was higher than the male gonads. In addition, the spatial expression model of the16genes were conducted using conventional PCR in detected gene expression in the body cavity fluid, body wall, longitudinal muscle, tube feet, respiratory tree and intestinal of sea cucumber. The results show that all the genes did not express in the coelomic fluid, longitudinal muscle, the body wall and tube feet. A7express in intestinal of sea cucumber. A4, A5, A7, A8, A9, and B4express in the respiratory tree.In this study, we construct the male and female gonad gene expression profiles, through bioinformatics analyzing, differentially expressed genes were screened, and completed the study of temporal and spatial expression of related genes, provide the basic data to analyze molecular mechanism of sea cucumber sex differentiation and gonad development from functional genomics, as well as provide a theoretical basis to genetic mechanism and evolution mechanism of gonad development in marine vertebrate, and sea cucumber breeding and reproduction regulation.