【作者】 魏莹莹；

【导师】 王德寿；

【作者基本信息】 西南大学 ， 水生生物学， 2011， 硕士

【摘要】 翻译延伸因子1A(eukaryotic translation elongation factor 1 A, eEFIA)是广泛存在于所有生物许多类型细胞的一类含量丰富且高度保守的蛋白质家族。它属于G蛋白家族,在肽链延伸过程中作为翻译延伸复合物的一部分,促使GTP依赖的氨酰tRNA转移至核糖体。42Sp50是eEFIA家族的特殊成员,目前仅在鱼类和两栖类卵黄发生前期卵母细胞中发现。大量研究表明eEFIA家族通常含有多个成员；在不同物种的不同发育过程中,eEFIA存在许多组织和时期特异表达的亚型,如鸟类、哺乳类等有两个eEFIA:eEFIA1禾(?)eEF1A2,两栖类则有三个：eEF1A1、eEF1A2和42Sp50,但关于真骨鱼类有几个eEF1A家族成员、eEF1A家族的系统进化分析及其在性腺中的表达、作用等鲜有报道。为解决上述问题,本研究从尼罗罗非鱼中克隆了42Sp50基因,从3月龄罗非鱼雌、雄性腺转录组序列中分离得到另外三个不同基因编码的eEF1As,分析了eEFIA家族的系统进化,并通过RT-PCR、Realtime-PCR和原位杂交等方法重点研究了42Sp50和eEF1Ab的表达模式,其结果如下：42Sp50 cDNA全长为1718bp,其开放性阅读框(ORF)长1371bp,编码456个氨基酸；三个eEF1As的ORF长均为1389bp,编码462个氨基酸。通过比对从GenBank下载到的各代表物种的eEF1As序列,我们构建了系统进化树,发现斑马鱼中有五个eEF1As (eEF1A1、eEFA2、eEF1A3、eEF1A4和42Sp50),罗非鱼中也有五个eEF1As (eEFlAla、eEF1Alb、eEF1A3、eEF1A4和42Sp50),而其他真骨鱼类中只找到四个eEF1As (eEF1A1、eEFA3、eEF1A4和42Sp50)。进化树表明来自不同脊椎动物的eEF1As序列聚为明显的两枝：42Sp50和其他eEF1As,其他eEF1As又分别聚为四类：eEF1A1、eEF1A2、eEF1A3禾PeEF1A4。其中,eEF1A1禾PeEF1A2存在于脊椎动物中,而eEF1A3和eEF1A4只在真骨鱼类中发现。42Sp50目前仅在两栖类和真骨鱼类中找到,它与其他eEF1As分开,单独聚为一枝且位于系统进化树的基部。这表明42Sp50可能在进化早期即已与其他eEF1As分离,摒弃了导致eEF1A和42Sp50分离的基因复制发生时间相对较近,随后编码42Sp50的基因快速进化的观点。通过RT-PCR发现eEF1A1b在卵巢和精巢中均有表达,且在精巢中的表达明显高于卵巢,42Sp50则特异地表达于卵巢中。同时,我们发现42Sp50在雌激素诱导的雄鱼卵巢中高表达,而在芳香化酶抑制剂处理后的雌鱼精巢中完全被抑制。eEF1A1b则在雌鱼精巢中表达高于雄鱼卵巢。表明42Sp50和eEF1A1b的表达与基因型无关,只与表型(雌性卵巢或雄性精巢)相关。进而,通过Real-time PCR研究了42Sp50和eEF1A1b基因在罗非鱼个体发生过程中雌、雄性腺的表达模式,结果表明42Sp50从孵化后10天开始在卵巢表达,随发育进程逐渐升高,至孵化后60天表达量达到最高,此后逐渐降低。相反,在雄性性腺发育过程中其表达始终保持在本底水平。罗非鱼雌、雄性腺中,孵化后10天至70天,eEF1A1b仅有微量表达。从孵化后90天开始,eEF1Alb在精巢中的表达剧增,并于孵化后120天达到最高,随后逐渐降低,在孵化后240天又一次增高,但这期间它在卵巢中始终保持较低水平的表达。用原位杂交的方法检测了42Sp50, eEF1A1b以及eEF1A4在罗非鱼性腺表达的细胞类型,结果显示在卵黄发生前期卵母细胞细胞质中,42Sp50均一地表达,而eEF1A4则呈单一小球状集中表达,eEF1A1b没有表达。eEF1A1b和eEF1A4在精巢的初级和次级精母细胞中表达,且eEF1A4的表达量明显高于eEF1A1b, eEF1A1b在次级精母细胞中的表达量高于初级精母细胞,42Sp50则没有表达。本研究进一步采用荧光素酶报告基因研究了HEK293和CHO细胞中雌激素受体(ERα、ERβ1和ERβ2)对42Sp50启动子的影响。结果发现,两种细胞系中,三种雌激素受体都抑制了42Sp50的启动子活性。此外,本研究用pCold I载体在大肠杆菌中表达、纯化了N端带有His标签的42Sp50重组蛋白,并免疫小鼠制备了多克隆抗体,Western blot检测证实抗体工作良好。综上所述,本研究发现罗非鱼中共有五个eEFIA家族基因：42Sp50、eEF1A1a、eEF1A1b、eEF1A3和(eEF1A4已报道)；对eEF1A家族的系统进化分析表明eEF1A1b是eEF1A基因家族的一个新成员；42Sp50和eEF1A1b在性腺中的表达存在明显的性别二态性,42Sp50在卵黄发生前期卵母细胞中特异表达,而eEF1A1b主要在初级和次级精母细胞中表达；荧光素酶分析发现三种雌激素受体对罗非鱼42Sp50启动子活性都有抑制作用。本研究为初次发现雌雄性腺可能分别通过不同的蛋白质翻译延伸因子进行性别特异的蛋白质合成。更多还原

【Abstract】 Eukaryotic translation elongation factor 1A (eEFIA) is a ubiquitous protein family present in abundance in many types of cells, promoting guanosine triphosphate (GTP)-dependent binding of aminoacyl-transfer ribonucleic acid (tRNA) to ribosomes during peptide chain elongation as part of the translational elongation complex.42Sp50 is a special member of eEFIA family which has been found only in the previtellogenic oocytes of teleosts and amphibians so far. Many studies of eEFIA revealed that it has several isoforms that are expressed in a tissue- and stage-specific manner during development. For example, two eEFIA isoforms exist in birds and mammals, designated as eEF1A1 and eEF1A2, while three were found in amphibians, i.e. eEFIA1, eEF1A2 and 42Sp50. There are few reports on phylogenetic analysis of eEFIA family, and there is no report on their expressions and roles in gonads. In order to investigate eEF1As expression patterns and functions in gonads,42Sp50 was cloned from the Nile tilapia using RT-PCR and RACE technology, and other three eEF1As were identified using sequences from the 3-month Nile tilapia gonads transcriptomes. Moreover, a complete phylogenetic analysis was performed, and the expression patterns of 42Sp50 and eEF1A1b were investigated by RT-PCR, Realtime-PCR and ISH. The results are as follows, The full-length cDNA of 42Sp50 was 1718 bp, with an open reading frame (ORF) of 1371 bp, encoding a protein of 456 amino acids; and all the ORFs of three eEF1As had 1389bp, encoding proteins of 462 amino acids. Based on the alignment of eEF1A sequences isolated from GenBank, a phylogenetic analysis was performed. The phylogenetic tree showed that eEF1A sequences from various vertebrates are clustered into five separate clades:eEF1A1, eEFlA2, eEF1A3, eEF1A4 and 42Sp50. eEF1A1 and eEF1A2 existed in vertebrates, while eEF1A3 and eEF1A4 existed only in teleosts. 42Sp50 which was found in teleosts and amphibians clustered into one clade different from the other eEF1 As clades.By RT-PCR, eEF1A1b was found to be expressed in both testis and ovary with higher expression in the testis, whereas 42Sp50 was only detected in the ovary. Moreover,42Sp50 was found to be strongly expressed in 17(3-estradiol induced XY ovaries, but completely inhibited in Fadrozole induced XX testes. The expression of eEF1Alb was higher in XX testes than that in XY ovaries. By Real-time PCR, ontogenic expression of 42Sp50 was detected from 10 dah (days after hatching) in the ovary, peaked at 60 dah, and then decreased onwards, while only faint signals were detected in all stages of testicular development. As for eEF1Alb only faint expression was detected from 10 to 70 dah in the testis, increased sharply from 90 dah onwards, peaked at 120 dah, and decreased gradually, then peaked again at 240 dah, while in the ovary, it remained low during the whole period. ISH (in situ hybridization) was performed to determine the cell types which express 42Sp50. eEF1Alb and eEF1A4. In previtellogenic oocytes,42Sp50 signal was found to be uniformly distributed, while eEF1A4 appeared to be concentrated in the small area. Both eEF1Alb and eEF1A4 were expressed in primary and secondary spermatocytes. Moreover, promoter analysis of 42Sp50 was performed using three estrogen receptors (ERa, ERβ1 and ERβ2) in HEK293 and CHO cells. The results showed that all three ERs suppressed the promoter activity of 42Sp50 in both cell lines. Recombinant protein with His-Tag at the N-terminal (expressed using pCold I vector in E. coli) of 42Sp50 was obtained and purified, and then its polyclonal antibody was produced in mouse. Western blot analysis was performed to check the quality of the antibody. Our antibody could recognize the same recombinant protein as recognized by His-Tag antibody.In summary, five eEF1As named as 42Sp50, eEF1A1a. eEF1A1b, eEF1A3 and eEF1A4 were identified and cloned in tilapia by transcriptome analysis and gene cloning (eEF1A4 had been reported before). A thorough phylogenetic analysis of eEFlAs from animal kingdom revealed that eEF1A1b is a new member of eEF1A family. Sexual dimorphic expression of 42Sp50 and eEF1A1b were detected in gonads. 42Sp50 is specifically expressed in the previtellogenic oocytes while eEF1A1b mainly in primary and secondary spermatocytes. Moreover,42Sp50 promoter activity was found to be suppressed by all three ERs in the luciferase assay. Our findings suggest for the first time that sex specific protein synthesis in male and female gonad might be undertaken by different translation elongation factors,更多还原