Yilei Wang

Transcriptome analysis of the differences in gene expression between testis and ovary in green mud crab (Scylla paramamosain)

BackgroundThe green mud crab (Scylla paramamosain) is the most prevalent crustacean on the southeast coast of China. The molecular regulatory mechanism of sex determination and gonadal differentiation in this species has received considerable attention in recent years because of the huge differences—both biological and economic—between male and female crabs. In this study, next-generation sequencing technology was used to develop deep-coverage transcriptomic sequencing data for the testis and ovary of S. paramamosain.ResultsA total of 365,116 reads (testis 171,962, ovary 193,154) with an average sequence length of 285 bp were produced from testis and ovary cDNA libraries. After filtering out contaminating reads, the clean reads were assembled, producing a total of 21,791 isotigs and leaving 22,814 reads as singlets. Using the BLASTX program, 3,471 unique sequences (2,275 isotigs and 1,196 singletons) were annotated with known protein sequences from the NCBI non-redundant (Nr) protein sequence database. The Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses allowed the 224 unique sequences that were annotated with enzyme code (EC) numbers to be mapped into 174 KEGG pathways. After comparing the ovary and testis libraries, 4,021 gonad-differentially, 10,522 ovary-specifically, and 19,013 testis-specifically expressed genes were identified. Moreover, 33 ovary-specific, 14 testis-specific, and 34 gonad-differential transcripts were confirmed by semi-quantitative PCR and quantitative real-time PCR. In addition, 8,610 putative simple sequence repeats (SSRs) and 23,879 potential single nucleotide polymorphisms (SNPs) were identified.ConclusionThis is the first large-scale RNA sequencing of S. paramamosain to be reported. We have identified many important functional genes and made a preliminary attempt to construct the regulatory network involved in the gonadal development of crustaceans. The annotated transcriptome data will provide fundamental support for future research into the reproduction biology of S. paramamosain. A large number of candidate SSRs and SNPs were detected, which could be used as genetic markers for population genetics and functional genomics in this species.

Characterization and expression profile of Vitellogenin gene from Scylla paramamosain

The full-length (7816 bp) cDNA of Vitellogenin (Vtg) encoding 2560 aa with an estimated molecular mass of 287.743 kDa was cloned from the green mud crab Scylla paramamosain. Semi-quantitative PCR (sq-PCR) revealed a specific expression pattern of Sp-vtg gene in ovaries and hepatopancreas. With the development of ovaries, the expression level of Sp-vtg gene showed an increasing trend both in ovaries and hepatopancreas, and the expression level of Sp-vtg gene in hepatopancreas and ovary was stable after stage IV. By in situ hybridization, the positive signals of Sp-vtg gene were detected in the cytoplasm of oocytes in stage I, in the follicle cell and the surrounding of the nucleus in stage III, and in the nucleus in stage V. Furthermore, the signals become stronger with the later development stages of ovary. Moreover, in situ hybridization analysis revealed that positive signals of Sp-vtg gene are present in the hepatopancreatic tubule, and the signals increase during the development, becoming the strongest in stage V. Our results indicate that both ovaries and hepatopancreas are sites of Vitellogenin gene synthesis in S. paramamosain.

Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor

Mammal interleukin-1 receptor-associated kinases (IRAKs) have been demonstrated to play important functions in TLRs (Toll-like receptor) signal pathway and T cell proliferation, but there is less knowledge available on mollusc IRAKs. In this study, a molluscan IRAK-4 gene, saIRAK-4, was cloned for the first time from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence was 2062 bp, with a 1548 bp open reading frame encoding a protein of 516 aa. The molecular mass of the deduced protein was approximately 57.8 kDa with an estimated pI of 5.23, and showed highest identity (47%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed saIRAK-4 shares conserved signature motifs with other IRAK-4 proteins, including the death domain (DD), serine/threonine/tyrosine protein kinase domain (STYKc), protein kinases ATP-binding region signature, serine/threonine protein kinases active-site signature and prokaryotic membrane lipoprotein lipid attachment site. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK-4 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK-4 mRNA could be detected in all examined tissues, with the highest expression level in gills, and was up-regulated in hemocytes and gills after bacteria injection. Additionally, saIRAK-4 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK-4 could respond to pathogenic infection and may play an important role in the adult abalone immune system and early innate immunity in the process of abalone larval development.

EST analysis on the gonad development related organs and microarray screen for differentially expressed genes in mature ovary and testis of Scylla paramamosain

A total of 5160 high quality ESTs (expressed sequence tags) averaging 357 bp were collected from normalized cDNA libraries created from testis, ovary and mixed organs of mud crab Scylla paramamosain. Clustering and assembly of these ESTs resulted in a total of 3837 unique sequences with 576 overlapping contigs and 3261 singletons. Comparisons with the GenBank non-redundant (Nr) protein database (BLASTx, e-values <10(-5)) revealed putative functions or matched homologs from other organisms for 847 (22%) of the ESTs. Several gonad development related genes such as cathepsin C, thioredoxin peroxidase, vitellogenin receptor precursor, 50S ribosomal protein L24 and ubiquitin-conjugating enzyme E2 isoform 2 were identified from this EST project and demonstrated as gonad differential expression genes by rqRT-PCR. Sixty five different types of SSRs (simple sequence repeats) were identified from the total 411 EST-SSR motifs. A home-made cDNA microarray containing 5664 spots was developed and the hybridization results indicated that 39 unique transcripts were differentially expressed in testis and ovaries (P<0.05). The expression levels of eleven unique transcripts examined by rqRT-PCR were matched with microarray fairly. These results will provide a useful resource for functional genomic studies on the biology of reproduction of mud crab.

Molecular characterization and expression profiles of cdc2 and cyclin B during oogenesis and spermatogenesis in green mud crab (Scylla paramamosain)

The maturation promoting factor (MPF) is a key regulator of controlling G2/M phase transition in the meiotic maturation of oocyte and spermatocyte in animals, which is a complex of CDC2 (CDK1) and cyclin B. To better understand the molecular mechanism of oocyte and spermatocyte maturation in mud crab (Scylla paramamosain), the full length cDNA of cdc2 (Sp-cdc2) and cyclin B (Sp-cyclin B) were cloned and characterized. The full length cDNA of Sp-cdc2 gene is of 1593 bp encoding a protein of 299 amino acids. Real-time quantitative PCR analysis revealed that the expression level of Sp-cdc2 in the ovary was higher than in other tissues (P<0.01); and its expression level was not significantly different in different stages of ovary development (P>0.05), meanwhile there was higher expression in T3 stage than in T1 and T2 stages (P<0.05). The full length cDNA of Sp-cyclin B is 1492 bp encoding a protein of 391 amino acids. The real-time PCR results showed that its expression level in the ovary was the highest in all examined tissues (P<0.01), and the gonad expression level in O5 stage was significantly higher than in previous 4 stages and the testis (P<0.05), and was also significantly higher in T2 stage than in T1 stage (P<0.05). In situ hybridization analysis showed that the expressions of Sp-cdc2 and Sp-cyclin B transcripts were presented in similar distribution patterns in different developing stages of ovary and testis. The positive signals of Sp-cdc2 and Sp-cyclin B mRNA were detected in the oocytoplasm of oogonia and pre-vitellogenic and primary vitellogenic oocytes, while these two genes had higher expression level in the spermatid and secondary spermatocyte following primary spermatocyte. These results suggested that Sp-cdc2 and Sp-cyclin B may play essential roles in the oogenesis and spermatogenesis of the crab.

SUMO-1 of mud crab (Scylla paramamosain) in gametogenesis

The small ubiquitin-related modifier-1 (SUMO-1) is a member of a family of ubiquitin-related proteins. SUMO pathway, which is involved in gene expression in eukaryotic posttranslational processing, plays important roles in gene expression, genomic stability and the occurrence of cells, development and other biological processes. Scylla paramamosain is one of the important economic breeding crabs in the southeast coast of China. To date, little is known about the distinct roles of SUMO in crustacean, especially in crabs. In the present study, we report the identification and characterization of mud crab, S. paramamosain SUMO-1 (SpSUMO-1) gene using an approach which combines expressed sequence tag (EST) and rapid amplification cDNA end (RACE). The full length cDNA of SpSUMO-1 gene (GenBank: HM581660) is of 732 bp, including a 282 bp open reading frame which encodes a protein of 93 amino acids. Tissue distribution analysis showed that SpSUMO-1 was expressed more abundantly in the ovary than in other tissues (P<0.01). And the expression profiles of SpSUMO-1 in the different gonad developing stages revealed that the highest expression of SpSUMO-1 occurred at proliferation stage, and then decreased gradually as the ovarian development progressed, while in the testis, the expression level of SpSUMO-1 was relatively stable at different stages of testis development. The distribution of SpSUMO-1 mRNA and its protein was observed in the crab gametogenesis by in situ hybridization and immunocytochemical method respectively. In oogenesis, SpSUMO-1 transcripts presented at the cytoplasm and nucleus of oocytes from proliferation stage to primary vitellogenesis stage, but only appeared in the nucleus of oocytes in secondary and tertiary vitellogenesis stages. Meanwhile, SpSUMO-1 protein was localized in the cytoplasm of oogonia and different developing oocytes. On the other hand, the SpSUMO-1 transcript was detected throughout the spermatogenesis, with the strong positive signals of SpSUMO-1 presented at the nuclei of primary and secondary spermatocytes, spermatids and spermatozoa. Interestingly, the positive signals of acrosomal tubules of spermatozoa were also detected. SpSUMO-1 protein was localized in spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid, but the positive signal was only detected in the nucleus of spermatozoa. All these results suggested that SUMO-1 may play essential roles in the gametogenesis of the crustacea.

Molecular cloning, characterization, and gene expression of the androgen receptor in the large yellow croaker, Larimichthys crocea.

Androgens mediate a wide range of physiological responses and developmental processes in vertebrates, involving both reproductive and nonreproductive systems. The activity of androgens is mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily. In this study, an AR gene was cloned from the large yellow croaker (Larimichthys crocea) for the first time. qRT-PCR revealed ubiquitous expression of AR in all adult tissues examined, with higher expression in the gonad and liver of both sexes and highest expression in the blastula stage of embryonic development. Using in situ hybridization, we detected positive signals of AR in the spermatogonium, spermatocyte, spermatid, and spermatozoon during spermatogenesis, in the cytoplasm of all oocytes during oogenesis and in the follicle cells of stage IV oocytes. Our findings support the important role that AR plays in gametogenesis, gonadal development, and the early stages of embryonic development.

Expressed sequence tag analysis for identification and characterization of genes related to Tributyltin (TBT) exposure in the abalone Haliotis diversicolor supertexta.

The analysis of expressed sequence tags (ESTs) is an efficient approach for gene discovery, expression profiling, and the development of resources for functional genomics. To analyze the transcriptome response to tributyltin of small abalone Haliotis diversicolor supertexta, a normalized cDNA library of hepatopancreas after exposure of animals to tributyltin was constructed. Three thousand and forty eight high quality ESTs were generated. After processing, a total of 2473 unigenes comprising 370 contigs and 2103 singlets were acquired. BLAST identified 1108 clones (45%) as known genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Simple Sequence Repeat (SSR) identification were also carried out to acquire more information on pathway and EST-SSR. According to GO categories, the total ESTs distribute across the various functional categories. In addition, 43 potential toxicology-related clusters were identified. This work provides the first set of genetic data for small abalone which is of great value for further exploitation of this species in functional genomics and toxicogenomics. Our interesting results will be mainly useful to choose biomarkers for ecotoxicological studies.

Differential expression of three estrogen receptors mRNAs in tissues, growth development, embryogenesis and gametogenesis from large yellow croaker, Larimichthys crocea.

The biological activity of estrogens in target organs is mainly mediated by estrogen receptors (ERs). Herein, we addressed the isolation and expression analysis of three nuclear estrogen receptors, namely LcERα, LcERβ1, and LcERβ2 from Larimichthys crocea by means of SMART-RACE, qRT-PCR, and in situ hybridization. Results in different tissues showed that both LcERα and LcERβ2 had the highest expression levels in female liver, followed by testis, but LcERβ1 expression level was significantly higher in testis and ovary than in other tissues. Expression of LcERα and LcERβ2 was significantly higher than LcERβ1 in female liver, and LcERβ2 was significantly higher than LcERα and LcERβ1 in male liver. Moreover, we analyzed the expression of LcERs in gonad and liver at three different growth stages during the same breeding season. Significant up-regulated expression of LcERα and LcERβ2 were found in female liver at 1000dph compared with at 270dph. The expression of LcERβ2 was prominently higher in male liver than LcERα, LcERβ1 and LcAR, while LcERβ1 was lower than other receptors in male and female liver at all the three stages. In ovary, LcERα at 270dph was lower than at 635dph and 1000dph, but had no significant change in testis. The two LcERβ subtypes and LcAR highly expressed in the early testis, and gradually decrease with the development of testis. In embryogenesis, a significant increase in the expression of LcERα and LcERβ2 were observed after appearance of optic vesicles phase (11.8hpf). LcERβ1 gradually decrease with the embryogenesis but increased dramatically at 1dph. Results of in situ hybridization showed that signals of LcERα and LcERβ1 mRNA were mainly detected in Stage I-Stage IV oocytes, as well as in follicle cells around the Stage II-Stage IV and degenerated oocytes. Signals of LcERβ2 were detected in the cytoplasm of Stage I and Stage II oocytes but not in the follicle cells of all oocytes stages. In parallel, LcERα and LcERβ1 were detected in all cell types of spermatogenesis, but in terms of LcERβ2, little or no signals were detected during spermatogenesis. Based on these results, we deduced that both LcERα and LcERβ2 play a major role in mediating the physiological effects of estrogen in female liver, and LcERβ2 maybe also play an important role in regulation of vitellogenesis in male liver. Differential expression of LcERs and LcAR imply their physiological functions during development and differentiation of gonad. The signals for LcERα and LcERβ1 in follicle cells suggested that the follicle cell maybe an important site of estrogen action, by which estrogens exert influences on the maturation oocytes and ovulation. Furthermore, the steroid hormones produced by follicle cells may be related to the differential distributions among ER subtypes. Besides, we deduced that LcERα and LcERβ1 rather than LcERβ2 may play a major role in spermatogenesis of croaker. However, the differential expression of LcERβ2 during gametogenesis also implicates its certain functions in mediating physiological process of estrogen action.

Molecular cloning, characterization and expression of Lc-Sox11a in large yellow croaker Larimichthys crocea.

Sox genes play important roles in various developmental processes such as sex determination, embryogenesis, oogenesis, neurogenesis, and larval development. In order to clarify the roles of Sox genes in the developmental process of large yellow croaker, the full-length cDNA of the Sox11a gene (Lc-Sox11a) was cloned for the first time. Bioinformatics analysis indicated that Lc-Sox11a contains a protein of 366 amino acids with a Ser-rich region, a C-terminal conserved region, and a high mobility group box. The expression of Lc-Sox11a in different tissues of both sexes and in different developmental embryonic stages revealed that Lc-Sox11a were expressed with tissue and gender specificity, of which the expression level in female was ovary>brain>eye>gill; in male was brain>testis>gill. The gender differences occurred in the brain and eye with the male brain>female brain, female eye>male eye. Moreover, the expression of Lc-Sox11a in the gonad and brain at different growth stages was detected. Significant up-regulated expression of Lc-Sox11a was found in the ovary and the male brain at 1000dph (days post hatching) compared with 270dph and 635dph. However, significant down-regulated expression of Lc-Sox11a occurred in the testis with growth. Besides, the expression of Lc-Sox11a in the female brain showed a trend of first rising then falling, with the highest peak in 635dph. The results of in situ hybridization displayed that Lc-Sox11a was widely distributed only in cytoplasm of oocytes at each stage in oogenesis. In early stage of oocytes, Lc-Sox11a was expressed weakly and evenly. As the appearance of vacuoles and synthesis of yolks, positive signals of Lc-Sox11a distributed intensively in the residual cytoplasm. In spermatogenesis, Lc-Sox11a was distributed in cytoplasm of all male germ cells except spermatozoon with spermatogonium>spermatocyte>spermatid. During embryogenesis, Lc-Sox11a was expressed in most embryonic stages, the highest expression occurred in the formation-of-eye-lens stage, closely followed by the closure-of-blastopore stage, then the beginning-of-heart-pulsation stage. The results of whole mount in situ hybridization showed that the expression of Lc-Sox11a began to increase beginning with the multiple-cell stage, with the major distribution of Lc-Sox11a in the brain and eye areas in the pre-hatching stage.