| 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. |
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