Studies On The Screening And Expression Regulation Of Sex Related Genes In Shrimp

Monosex culture has a great priority on increasing production efficiency andyield in shrimp aquiculture. Mature technologies on sex control are the basis forsuccessful development of monosex culture. However, it is still difficult to developsuch effective technologies to implement large-scale cultivation due to lack ofunderstanding on the molecular mechanism of sex determination and differentiation inshrimp. In the present dissertation, suppression subtractive hybridization (SSH)library and full-length cDNA library of shrimp androgenic gland related tissues(including androgenic gland and part of the spermatophore sac) were constructed andprimary studies were performed on the molecular mechanism of sex determinationand differentiation in shrimp. Molecular markers were also tentatively developed toidentify shrimp gender at early developmental stages. These data provide theoreticalbasis and guidance for illustrating the molecular mechanism of sex determination anddifferentiation and developing effective technologies on sex control in shrimp. Themain progresses are as follows:Firstly, in the full-length cDNA library,745unigenes were obtained and455ofthem had homologous annotations (E<1×e-5). Full-length analysis showed that334unigenes were completed cDNA sequences, which took part of73.4%(334/455) ofthe annotated unigenes. the SSH library and full-length cDNA library of shrimpandrogenic gland related tissues were constructed, respectively.402expressedsequence tags (ESTs) were obtained from the SSH library.48contigs and104singletswere assembled based on the ESTs.11differentially expressed transcripts wereselected to detect their expression profiles in androgenic gland and other tissues inshrimp via semi-quantitative RT-PCR. Results revealed that all selected transcriptswere exclusively expressed in the androgenic gland related tissues except for one candidate, which was also detected in testis. These results indicated an high efficiencyof the SSH library. Two round RT-PCR were carried out on the11selected candidatesand one of them was proved to be an molecular marker distinguishing shrimp genderat the early developmental stages. In order to investigate their biological function inshrimp, a serial interesting genes including transformer2(FcTra-2), sex lethal(FcSxl), doublesex (FcDsx), insulin-like androgenic gland hormone (FcIAG) andcrustacean hyperglycemic hormone (FcCHH) were further analyzed, which laid afoundation for exploring the molecular mechanism of sex determination anddifferentiation in shrimp.Secondly, Fctra-2, FcSxl and FcDsx gene were successfully obtained. Threemature variants of FcTra-2generated by alternatively spliced were isolated, all ofwhich exhibited higher expression level in gonad than in other tissues. One of theFctra-2variants (Fctra-2c) showed significantly higher expression level in ovary thanin testis, and its expression level sharply increased at mysis stage before gonaddifferentiation, which indicated that Fctra-2c might be involved in the sexdetermination process in shrimp. Transcription ananlysis revealed that FcSxl andFcDsx also displayed sexual dimorphism on the expression level.Thirdly, full length cDNA sequence and genomic DNA sequence of Fc-IAG wereobtained. Two alternatively spliced variants (Fc-IAG1and Fc-IAG2) of Fc-IAG wereisolated and they all possessed the conserved sequence structure with IAGs from othercrustacean species. Further study revealed that Fc-IAG1and Fc-IAG2should performdifferent biological function. Transcription factor binding sites were predicted on the5'-flanking sequence of FcIAG gene, which primarily revealed the possiblytranscriptional regulation mechanism and laid a foundation for gene function study.Fourthly, two isoforms of FcCHH gene (FcCHH1and FcCHH2), whosededuced amino acid sequences had a similarity of78%, were isolated fromFenneropenaeus chinensis. Sequence analysis revealed they were two different gene copies from the genomic DNA. In contrast with previous studies, FcCHH transcriptswere exclusively expressed in the endo-epithelium cells of spermatophore sac.Expression analysis during shrimp developmental stages showed that FcCHHtranscripts started to turn up along with the appearance spermatophore sac.Prokaryotic recombinant protein of FcCHH (His-CHH) was expressed in vitro and itsrabbit polyclonal antibody was prepared for investigating the gene function on theprotein level.