中文摘要：
生殖细胞的发生对机体的延续和繁衍起关键作用,是复杂的分子调控过程,vasa、nanos和piwi基因是重要的生殖调控分子,对生殖细胞的发生起调控作用。与模式生物相比,十足目动物生殖调控的分子机制仍不清楚,因此,亟待开展十足目生殖基因的功能研究。本论文在前期研究基础上,以河南华溪蟹(Sinopotamon henanense)为研究对象,通过Western blot和荧光免疫组织化学揭示了河南华溪蟹VASA蛋白的表达模式,结合RNA干扰技术,对其功能进行了分析,在此基础上,通过检测镉暴露后vasa基因及其相关基因的变化,阐明了镉对河南华溪蟹生殖调控的毒性机制。研究结果如下:1.河南华溪蟹VASA蛋白的表达和定位本实验采用Western blot和荧光免疫组织化学技术,对VASA蛋白在河南华溪蟹卵巢发育不同时期的表达和卵母细胞发育中的定位情况进行了研究。Western blot结果显示,随着卵巢的发育,VASA蛋白量逐渐升高,大生长期开始降低,成熟期达到最低。荧光免疫组织化学结果显示,在卵原细胞中,VASA蛋白均匀分布于细胞质;卵黄发生早期的卵母细胞中,大量VASA蛋白在细胞质中分布,少量开始向细胞核周围聚集;卵黄发生中期、后期和成熟期的卵母细胞中,VASA蛋白主要分布在细胞核周围,且成熟期卵母细胞中VASA蛋白的信号最弱。2.镉暴露对河南华溪蟹VASA蛋白的影响本实验设置了对照组(0 mg/L)和五个镉浓度组(7.25 mg/L、14.5 mg/L、29 mg/L、58 mg/L、116 mg/L),采用急性毒性实验法,研究了镉暴露7 d对河南华溪蟹VASA蛋白表达的影响。Western blot结果显示:随着镉浓度的升高,增殖期和小生长期的卵巢VASA蛋白量均呈现出逐渐降低的趋势;大生长期的卵巢VASA蛋白量呈现出先升高后降低的趋势,镉浓度为14.5 mg/L,VASA蛋白量最高;而成熟期的卵巢VASA蛋白量随镉浓度升高而升高。在此基础上,利用荧光免疫组织化学方法检测了镉暴露后,VASA蛋白在卵母细胞中的变化。结果显示,镉暴露浓度为7.25 mg/L时,VASA蛋白量升高;镉暴露浓度为58 mg/L时,VASA蛋白量降低;浓度升至116 mg/L时,VASA蛋白表达量降至最低。3.河南华溪蟹nanos1、nanos2、piwi基因部分序列的克隆及表达特征研究表明,vasa基因对nanos1、nanos2和piwi基因的表达有调控作用,为深入研究vasa基因的调控途径,查阅相关资料找出nanos1、nanos2和piwi基因,并进行研究。本实验在前期研究基础上,克隆了nanos1、nanos2、piwi基因的部分cDNA序列。利用半定量PCR分别鉴定了河南华溪蟹nanos1、nanos2、piwi基因的组织特异性表达和在卵巢发育过程中的表达特征。结果显示,nanos1基因在卵巢、精巢、鳃、心和肌肉中均有表达;nanos2基因在卵巢、精巢、副性腺、心脏、肠和肌肉中有较高表达,且卵巢中表达量高于精巢中表达量;piwi基因特异表达于卵巢和精巢。卵巢发育中三个基因的表达时序研究显示,nanos1基因从卵巢发育的增殖期、小生长期、大生长期到成熟期表达量逐渐升高,与之相反,nanos2和piwi基因的表达呈现相反的趋势。4.镉暴露对vasa基因作用分子的影响本实验为验证vasa-siRNA的有效性,检测了vasa-siRNA的干扰效率。结果表明,与对照组相比,vasa-siRNA可显著降低vasa基因的转录水平(P<0.05),干扰效率达到67.4%。在此基础上,研究了镉暴露的效应。结果显示:与阴性对照相比,单独镉暴露7 d后,vasa和piwi基因的表达量显著升高,而nanos基因表达量显著下降;单独注射vasa-siRNA后,vasa、nanos、piwi基因的表达量均显著降低,且与空白对照(0.85%Nacl)无显著性差异;镉暴露同时干扰vasa基因后,与阴性对照相比,vasa和nanos基因的表达量反而显著上升,而piwi基因的表达量显著降低。综合以上研究结果,得出如下结论:(1)随着卵巢的发育,VASA蛋白量先升高后降低,并且随着卵母细胞的发育,VASA蛋白逐渐由细胞质向细胞核周围聚集,表明VASA蛋白在卵母细胞发生过程中发挥作用。(2)揭示了河南华溪蟹nanos1、nanos2和piwi基因的组织特异性及其在卵巢中的表达时序。(3)镉暴露影响了VASA蛋白的表达,且镉可能通过影响vasa及其作用分子nanos、piwi的功能引起河南华溪蟹的生殖毒性。
英文摘要：
The genesis of germ cells is a complex process of molecular regulation,involving the origin,migration and differentiation of primordial germ cells.Compared with the model organism,the regulatory mechanism of reproductive genes in Decapoda is still unclear.Therefore,it is urgent to study on the function of the reproductive genes of Decapoda.Based on the previous research,using Sinopotamon henanense as the research object,the expression and localization of VASA protein were revealed by western blot and immunohistochemistry.On this basis,RNA interference technology was used to further study on the effects of cadmium exposure on the expression pattern and molecular regulatory pathway of vasa gene.The results are as follows:1.Expression and localization of VASA protein in S.henanenseIn this study,western blot and fluorescence immunohistochemistry were used to study on the expression of VASA protein in different stages of ovarian development and its location during the oocyte development in S.henanense.Western blot showed that with the development of ovary,VASA protein increased gradually,and began to decrease at the large growth stage,the mature stage was the lowest.The results of fluorescence immunohistochemistry showed that VASA protein was evenly distributed in the cytoplasm of oocytes.At the early stage of vitellogenesis,a large number of VASA proteins were distributed in the cytoplasm,and a little proteins began to gather around the nucleus;VASA protein was mainly distributed around the nucleus of oocytes at the metaphase of vitellogenesis.At the vitellogenic and mature oocyte stages,VASA protein is distributed in plasma surrounding the nucleus,but the signal of VASA protein in oocytes is the weakest.2.Effect of cadmium exposure on the expression and localization of VASA protein in S.henanenseIn this experiment,the control group(0 mg/L)and five cadmium concentration groups(7.25 mg/L,14.5 mg/L,29 mg/L,58 mg/L,116 mg/L)were set up.The changes of the expression levels of VASA protein in ovaries at different developmental stages of S.henanense were studied by acute toxicity test after cadmium exposure.The results of western blot showed that: with the increase of Cd concentration,the VASA protein content at proliferation and small growth stages decreased gradually.The amount of VASA protein increased first and then decreased at large growth stage.When the concentration of cadmium was 14.5 mg/L,the amount of VASA protein was the highest.The amount of VASA protein at mature stage increased with the increase of cadmium concentration.Furthermore,the localization of VASA protein in oocytes after cadmium exposure was detected by fluorescence immunohistochemistry.The results showed that the VASA protein increased when the cadmium exposure concentration was 7.25 mg/L.When the concentration of cadmium was 58 mg/L,VASA protein decreased.When the concentration was 116 mg/L,VASA protein expression decreased to the lowest level.3.Cloning and expression characteristics of some sequences of nanos1,nanos2 and piwi genes of S.henanenseBased on the previous research,the partial cDNA sequences of nanos1,nanos2 and piwi genes were obtained.Semi-quantitative PCR was used to identify the tissue-specific expression of nanos1,nanos2 and piwi genes in S.henanense and their expression patterns during ovary development.The results showed that nanos1 gene was expressed in ovary,testis,sputum,heart and muscle;the nanos2 gene was highly expressed in ovary,testis,accessory gonad,heart,intestine and muscle,andespecially the expression level in the ovary was higher than that in the testis;piwi gene was expressed specifically in ovary and testis.Furthermore,the detection of the expression pattern during the ovary developmental showed that the expression level of nanos1 gene was increased gradually in ovary from proliferative stage,small growth stage,large growth stage to mature stage.Comparatively,the expression trends of nanos2 and piwi genes showed the opposite patterns.4.Effects of cadmium exposure on vasa gene and its regulation moleculesIn order to verify the effectiveness of vasa-si RNA,the interference efficiency of vasa-si RNA was tested.The results showed that compared with the control group,vasa-si RNA significantly reduced the transcription level of vasa gene(P< 0.05),and the interference efficiency reached 67.4%.On this basis,The effects of cadmium exposure were investigated.The results showed that the expression levels of vasa and piwi genes increased significantly but that of nanos decreased significantly,after exposure to 14.5mg/L cadmium alone for 7 days.After single injection of vasa-si RNA,the expression levels of vasa,nanos and piwi genes decreased significantly compared with the negative control group,and there was no significant difference with the blank control group(0.85%Na Cl).With both cadmium and interference of vasa gene,compared with the negative control,the expression levels of vasa and nanos genes increased significantly,while the expression level of piwi gene decreased significantly.Based on the above results,conclusions are as follows:(1)With the development of theovary,the amount of VASA protein first increased and then decreased.With the development of oocytes,VASA protein gradually accumulates from the cytoplasm to the periphery of the nucleus.It shows that VASA protein plays an important role during oogenesis.(2)The tissue specificity of nanos1,nanos2 and piwi genes and their expression sequence in ovary of S.henanense were revealed.(3)Cadmium exposure affected the expression of VASA protein.Cadmium may be caused reproductive toxicity of S.henanense by affecting the function of vasa and its acting molecules,nanos and piwi.
