摘要
在实验室所构建的拟穴青蟹（Scylla paramamosain）EST文库基础上筛选出泛素基因（Ubquitin,Sp-Ub）片段,利用RACE技术克隆其全长cDNA序列,并对其结构进行分析;利用实时定量PCR技术检测该基因在雌蟹各组织及性腺不同发育阶段中的表达情况。结果显示,Sp-Ub基因全长555 bp,编码154个氨基酸,其第1~76个氨基酸为高度保守的泛素结构域,第101~147的氨基酸残基为典型的核糖体蛋白S27a的保守区域;预测的泛素结构域二级结构由5个延伸链,1个α-螺旋及其连接的无规卷曲构成,Sp Ub三维结构由1个α-螺旋以及5个β片层。多重比对表明不同物种的泛素结构域和核糖体蛋白S27a结构域在进化过程中高度保守。定量PCR结果显示,Sp-Ub在鳃中的表达水平最高,卵巢次之,各组织间的表达无显著性差异,说明Sp-Ub基因在各组织中的表达恒定,且具有普遍作用;在卵巢发育过程中,Sp-Ub在O5期的表达水平最高,O1、O4期次之,O2期表达量最低且与O5期具有显著差异（P〈0.05）;在精巢发育过程中,Sp-Ub在T2期的表达量最高,其次为T1期,T3期表达最低且与T2期具有显著差异（P〈0.05）。本研究结果说明了Sp-Ub对拟穴青蟹生殖细胞的生长和发育可能具有重要作用。本研究将为甲壳动物性腺发育调控的分子机制奠定基础。
Scylla paramamosain is an economically important breeding crab found along the southeast coast of China.Although extensive research on reproductive physiology has been carried out for this species,description of the regulatory mechanism of the gonad development at a molecular level is limited.Within all tissues,the majority of intracellular proteins are degraded by the ubiquitin（Ub）–proteasome pathway（UPP）.In this pathway,Ub is conjugated to proteins that are destined for degradation by an ATP-dependent process that involves three enzymes.To date,some studies have discovered that components of the ubiquitin system may be involved in various steps and processes during gametogenesis,modulation of steroid receptor concentrations,placental development and endometrial modification at the beginning of pregnancy.However,research on the action of UPP in gonad devel-opment in crab species remains largely unknown.In this study,the S.paramamosain Ub gene（Sp-Ub） was identi-fied and characterized using an approach which combines the expressed sequence tag（EST） method and rapid amplification of cDNA end（RACE）.The expression pro？le of Sp-Ub in the various tissues and different develop-ing stages of the gonad was determined by real-time quantitative PCR.The full length cDNA of the Sp-Ub gene is of 555 bp.It encodes a protein of 154 amino acids,in which the 1-76 amino acids fall within the ubiquitin domain,and the 101–147 amino acids fall within the ribosomal_S27a domain.The second structure prediction revealed that the ubiquitin domain of the Sp-Ub protein contains five extended strands,an α-helix and some random coils.Moreover,the three-dimensional structure predicted by the SWISS-MODEL showed that it contains an α-helix and four β-pleated sheets.Alignment of the amino acid sequences within different Ub proteins indicated that the ubiq-uitin domain and the ribosomal_S27a domain among different species are highly conserved during evolution.Real-time quantitative PCR results showed that Sp-Ub was expressed by the ubiquitin domain in all tissues exam-ined（P0.05） which illustrated that the Ub gene plays a wide variety of roles in biological processes.The expres-sion profiles of Sp-Ub in the different developing stages of the ovary revealed that its expression level was the highest at stage O5 following stage O1,O4 and O3.It was the lowest level at stage O2 which was significantly lower than stage O5（P0.05）.Within the developing stages of the testis,the expression level of Sp-Ub was higher in stage T2 following stage T1 and T3,and its expression level was significantly higher in stage T2 compared to stage T3（P0.05）.During gametogenesis,the oogonia and spermatogonium undergo active mitosis proliferation for increasing their number at the proliferation stage.Previous research showed that cyclin/Cdks work closely with the ubiquitin-proteasome pathway during the mitotic cycle.Thus,it is reasonable to observe that the expression level of Sp-Ub was relatively high at stage O1.The reduction of the Sp-Ub expression level at stage O2 may be due to the initiation of formation of primary oocytes,and initial vitellogenesis with a low level of proliferation.Moreover,the expression level of Sp-Ub gradually increased as the yolk proteins were accumulating in the grow-ing oocytes during vitellogenesis.A mass of proteins needed to be synthesized and degraded during gametogenesis,and the evidence of ubiquitin –conjugated vitellin polypeptides in insect oocytes had been reported.These results indicated that Sp-Ub may be involved in vitellogenesis.Furthermore,the testis of crab contains germ cells of various development stages within the seminiferous tubules.Hence,the higher expression level of Sp-Ub at stage T2 of testis development may be necessary for fast increasing the number of spermatocyte cells,which indicated that UPP was more active in these cells.Conclusively,our studies are the first report of cloning and expression profiling of Ub in crustaceans.All of these results can provide basic data showing the role of Sp-Ub within the gonad development of the crab.
出处
《中国水产科学》
CAS
CSCD
北大核心
2012年第6期946-955,共10页
Journal of Fishery Sciences of China
基金
国家自然科学基金资助项目(31072200)
福建省自然科学基金资助项目(2009J01179)
集美大学创新团队基金资助项目(2010A001)
关键词
拟穴青蟹
泛素基因
组织表达
性腺发育
Scylla paramamosain
Sp-Ub
tissues expression
gonad development
