Transcriptomic Analysis Of Eyestalk Involved In Ovary Development Of Scylla Paramamosain

Green mud crab Scylla paramamosain is one of the commercially important crustacean species inChina. Recently, researchers pay much more attention to the molecular mechanisms of ovarian development regulation. Eyestalk is an essential endocrinal organ of crustacean. X-organ-sinus gland(XO-SG) is one part of the eyestalk that synthesizes and secretes the neuropeptide of CHH family which regulates a lot of physiological process, such asmolt, reproduction and ingestion, etc. In this study, the transcriptomes of female and male eyestalks weresequenced by 454 pyrophosphate sequencing, then the transcrptome data from the two libraries were compared via bioinformatics analysis. On this basis, ESTs containing partial sequences of ovarian development regulation-related genes wereselected. SMART-RACE was used to clone the full-length cDNAs of guanine nucleotide binding protein subunit beta-1(Gnb1), crustacean hyperglycemic hormone-3(CHH3), VIH-like gene,14-3-3ζandMAPK activated protein kinase 2(MK2) gene. The resultsare shownas follows:1) 9897 female-specific transcripts, 21691 male-specific transcripts, and 6701(4308 are differential expression) common transcripts were obtained by 454 pyrophosphate sequencing. Part of them were confirmed by real-time quantitative PCR or semi quantitative PCR, and the results showed that: 11 transcripts were femle-rich expression; 13 transcripts were male-rich expression; and 3 transcripts were specific expression in female and male eyestalk respectively.2) The full length cDNA of fivegenes were cloned. The full length of Sp-CHH3 gene(GenBank: KC750206) is of 1671 bp, including a 375 bp open reading frame(ORF) which encodes a protein of 124 amino acids. The full length of Sp-VIH-like gene(GenBank: KF006815) is of 479 bp, including a 306 bp ORF which encodes a protein of 102 amino acids. The full length of Sp-Gnb1 gene(GenBank: JQ218936) is of 1487 bp, including a 1023 bp ORF which encodes a protein of 340 amino acids. The full length of Sp-14-3-3ζ gene(GenBank: JQ218935) is of 1092 bp, including a 747 bp ORF which encodes a protein of 248 amino acids. The full length of Sp-MK2 gene(GenBank: JQ316171) is of 1899 bp, including a 1095 bp ORF which encodes a protein of 364 amino acids.3)To obtain more information about Sp-VIH-like, Sp-Gnb1, Sp-14-3-3ζ, Sp-ERK2 and Sp-MK2 gene, tissue distribution and the expression profiles in ovary and eyestalk indifferent ovarydeveloping stages were revealed by real time quantitative PCR. Tissue distribution analysis showed that Sp-VIH-like is specific expression in eyestalk, none Sp-VIH-like was found in other tissues we tested. Sp-Gnb1 and Sp-14-3-3ζwere expressed more abundantly in ovary than in other tissues. With the development of the ovaries, the expression level ofSp-VIH-like was decreased from stage Ⅲ to stage Ⅴ.In eyestalk, the expression of Sp-Gnb1, Sp-14-3-3ζ, Sp-ERK2 and Sp-MK2 increase graduallywith development of ovary.In ovary, Sp-14-3-3ζ and Sp-MK2 genes gradually increased until to Ⅳ stage, then reducedwith development of ovary. Meanwhile, the level ofSp-Gnb1 fairly constantduring ovarydevelopment.