| Chinese mitten crab(Eriocheir sinensis)is one of the most important aquaculture species that is widely distributed in freshwater and low-salinity estuarine regions in China.The male mitten crab grows faster and is more aggressive than females in the dimorphic sexually culture system at present,which may result in high mortality of the females and thus influence the production of crab.It is obvious that culture of monosex all-male or all-female populations would be more economically advantageous to crab aquaculture industry.In this study,high-throughput sequencing technique is used to obtain the transcriptome data regarding androgenic gland of the Chinese mitten crab in both proliferative and secretory stages.By means of comparative transcriptomics analysis,genes are screened which are related to sex development of the Chinese mitten crab and maintenance of the male secondary sexual characteristics as well as their fighting and mating behaviors.The androgenic gland hormone gene(Es-IAG)is cloned and examined about its expression in different development stages of the crab.The critical period of sex differentiation is also analyzed.The expression of Es-IAG was detected by qRT-PCR.After in-vivo injection of dsRNA,the interference effect is acquired by testing the Es-IAG genes.The test is conducted on the small RNA in androgenic gland at both proliferative and secretory stages,followed by analysis of functions of differentially expressed miRNA,and then initial screening of miRNAs with the Es-IAG as the target genes.Using Illumina Hiseq platform,androgenic gland transcriptome de novo sequencing is conducted at respectively proliferative and secretory stages of Chinese mitten crab.A total of 4027 differentially expressed genes were found in 72,000 genes by comparison of the transcriptome analysis.According to the results of GO analysis,the differential genes mainly concentrate in biological processes and metabolic pathways related to protein synthesis and secretion,such as transcription,translation and signal transduction and so forth.Furthermore,the genes,includingEs-IAG,Tra-2,Cytochrome p450,SRY and FTZ-F1,are screened which may possibly play a part in the sex differentiation and determination of the gender.Relevant genes involved in spermatogenesis,such as Ubiquitin,serpin,CathepsinA and CathepsinD,are also analyzed.By analyzing insulin signaling pathway,it is found that 3 insulin receptor plays a critical part in the insulin pathway.They regulate cell proliferation and differentiation,apoptosis,and protein synthesis through different signaling pathways.Es-IAG hormone,as the only insulinoid in Chinese mitten crabs,is speculated to regulate testis development and spermatogenesis through insulin receptor.What is also analyzed includes the neuroactive ligand receptors interaction pathway.In secretory phase,there is a significant change in the expression quantity of melatonin,5-hydroxytryptamine and octopamine receptors in androgenic gland,which indicates that melatonin,5-hydroxytryptamine and octopamine,with the help of androgenic gland,may play a regulatory role in the gonad development,spermatogenesis as well as fighting and mating behavior of Chinese mitten crab.Using the Es-IAG fragment acquired in transcriptome,taking advantage of PCR to clone Es-IAG genes 3' and 5' end and also conduct bioinformatics analysis,it is found that there is greatest uniformity between Chinese mitten crab IAG and blue crab(C.sapidus)in terms of amino acid.And scylla paramamosain(S.paramamosain)comes the second.The secreting signal peptide sequence exists in the Polypeptide amino acid sequence of the Es-IAG in the Chinese mitten crab,which suggests that Es-IAG hormone is secreted protein.No transmembrane domain is found.There exists a typical R**R protease cleavage site between Chain C and Chain A,Chain C and Chain B,suggesting that the Es-IAG precursor polypeptide becomes a mature IAG hormone after removal of Chain C.The six conservative Cys residues in mature peptide chain make up two inter-chain disulfide bonds and a disulfide bond within the chain.The glycosylation sites in the mature peptide chain are the same as 0those of other crustacean IAGs.The uniformity in three-dimensional structure with other crustacean IAG hormones shows that the crustacean IAG polypeptide structure is highly conserved in evolution.The phylogenetic analysis of the reported crustacean IAG was carried out.It was found that Chinese mitten crab has the closest evolutionary relationship to blue crab and scylla paramamosain,and forms a branch together with Drosophila melanogaster and Stomoxys calcsitrans.Fluorogenic quantitative PCR is employed to inspect the expression of in-vivo Es-IAG genes at different growth stages of Chinese mitten crab's development from zygote to V larva crab,involving zygote,blastocyst,gastrula,eye spot and heart-beating phase,as well as zoea,megalopa and larva crabs and so forth amounting to 16 different stages of development.The results shows,at phase ? of megalopa metamorphoses,the expression of Es-IAG genes increased sharply,and through the morphological observation,it is found that the differentiation of sex-related appendages began in phase ?,which indicates that phase ? of the larva crab may be the key period of gender differentiation.3 dsRNAs are designed according to Es-IAG gene sequence and in-vivo transfection occurs to Chinese mitten crab.24 hours after injection,qRT-PCR is used to inspect the expression of Es-IAG genes.The results show interfering efficiency of one of the dsRNAs could reach 67%.Soaking of the megalopa interfering Es-IAG gene expression can significantly inhibit the growth of male appendages in ? period crabs,which can be helpful in the further study of sex reversal.HiSeq high-throughput sequencing technique is used to detect the expression of small RNA in androgenic gland at proliferative stage and secretory stage of Chinese mitten crab.3719 already-known miRNAs and 2246 pieces of differentially expressed miRNAs were screened out,of which 1780 pieces show a significant decline in expression in androgenic gland during secretory stage,and of which 466 pieces show a significant rise,and Let-7 might be involved in the development of the gonads of Chinese mitten crab.Among 56 new pieces of miRNA,37 show a significant decline in expression and 19 pieces show a significant rise.27 miRNAs have Es-IAG as their target gene,among which miR-197,miR-2443 and miRNA-6368 might be involved in the development and regulation of androgenic gland.Target prediction is done to miRNA.Based on GO analysis,it was found that many biological functions concentrate in target genes,mainly including the synthesis,metabolism,signal transduction,cell fraction and binding of biological components.These functions are closely related to the proliferation and secretion of androgen gland,and the regulatory function.Among the target genes corresponding to differentially expressed miRNA,miRNA corresponding to many functional genes related to sex of Chinese mitten crabs,e.g.IAG.FTZ-F1 and TRA-2 and etc.might be the key miRNAs involved in sex development.65 new miRNAs are screened out with their functions concentrating on physiological regulation,cellular processes,metabolic processes,signal transduction and so on,which lays a foundation for study on expression regulation and formation mechanism of character of Chinese mitten crab genes. |
PDF Full Text Request