| The oriental river prawn,Macrobrachium nipponense,which mainly distributed in fresh water and low salinity areas,is one of the major species of freshwater aquaculture in China.Sexual maturity of M.nipponense is fast,it takes only about 45 days from metamorphosis to sexual maturity for female young shrimps.However,this characteristic results in the coexistence of multiple generations,high breeding density,increased feed consumption,and high risk of hypoxia,which greatly affected the economic benefits of shrimp culture.Therefore,the research on the mechanism of ovarian maturation in M.nipponense is of great significance.It has been proved that cyclin A gene plays an important role in reproductive regulation in vertebrates and insects,but the reproductive function of cyclin A gene in crustaceans has not been reported.In the early stage,by establishing the comparative transcription group of M.nipponense at different stages of ovarian development,it was found that there was a significant difference in the expression of cyclin A gene during ovarian development and according to the sequence of M.nipponense hepatopancreas,intestine and muscle transcriptional group,the unique type of cyclin A gene sequence was matched.In this paper,cyclin A gene was used as the research object,the gene was cloned by rapid amplification of c DNA ends,and phylogenetic analysis was carried out to clarify its evolutionary origin.The expression pattern of cyclin A gene in different developmental stages,adult tissues and ovaries of M.nipponense was studied by real-time fluorescence quantitative PCR(q RT-PCR),and the distribution of cyclin A gene m RNA in ovarian tissue was located by in situ hybridization(ISH)technique to explore the relationship between cyclin A gene and reproduction of M.nipponense.Finally,RNA interference(RNAi)technique was used to study the function of cyclin A gene in the reproductive process of M.nipponense.The full-length c DNA of cyclin A contained 2033 base pairs,encoding 456 amino acids.The encoded amino acid sequences of M.nipponense cyclin A has two unique conserved domains of the cyclin gene fam PLy,and there are three degradation motifs binding to APC/C in the 5 ’end sequence: KEN box,D box,ABBA motif,has two polyadenylate motifs in the3’ end sequence.Phylogenetic analysis showed that cyclin A of M.nipponense was successfully identified,and was designated as Mn-cyclin A.The analysis of the distribution and expression of Mn-cyclin A m RNA in male and female tissues during the breeding season showed that the expression level of Mn-cyclin A m RNA in male and female gonads(testis,ovary)was significantly higher than that in other tissues(eye,brain,heart,hepatopancreas,g PLl,muscle,androgenic gland,vas deferens)(P<0.01).Compared with testis,the expression of Mn-cyclin A m RNA in ovary was about 5times higher than that in testis.In the eye,brain,heart,hepatopancreas,g PLl,muscle and male gland and vas deferens of female and male shrimp,the transcription level of Mn-cyclin A m RNA in hepatopancreas of female and male shrimp was higher than that of other tissues(eye,brain,heart,g PLl,muscle,male promoting gland,vas deferens)(P<0.01).The expression of Mn-cyclin A m RNA in the hepatopancreas of male shrimp was about 1.5 times higher than that of female shrimp.At the same time,the transcriptional level of Mn-cyclin A m RNA in eyes,brain,heart,g PLl,muscle,androgenic gland and vas deferens was very low.The m RNA expression level of Mn-cyclin A in female and male shrimp showed obvious tissue specificity,and it was speculated that Mn-cyclin A may have the function of reproductive regulation in M.nipponense.Mn-cyclin A m RNA showed a complex expression pattern at different developmental stages of embryo,larva and young shrimp.In the stage of embryonic development,the expression level of Mn-cyclin A m RNA increased continuously from cleavage stage to inflexion stage,and the expression level on the first day of hatching larva was significantly lower than that in embryonic development stage(P<0.05).In the stage from larva to pre-metamorphosis,the expression level of Mn-cyclin A m RNA is low and there is a fluctuating pattern.On the 25 th day after metamorphosis(sex can be distinguished visually),the expression of Mn-cyclin A m RNA in female shrimp reached the peak,which was about 3.5 times higher than that in male shrimp.These results suggest that the transcriptional level of Mn-cyclin A may be regulated by development and is closely related to oogonia proliferation and oocyte formation.In different stages of ovarian development,the transcription level of Mn-cyclin A m RNA was the highest in stage IV(oocyte stage),but there was no significant difference in stage I(oogonia stage),stage II(primary oocyte stage),stage III(secondary oocyte stage)and stage V(degeneration stage).The transcription level of Mn-cyclin A m RNA in stage IV(oocyte stage)was about 1.8 times higher than that in the other four stages.These results suggest that the expression of Mn-cyclin A in ovary is positively correlated with ovarian development,and Mn-cyclin A may be involved in much more regulation of ovarian maturation in M.nipponense.The results of in situ hybridization showed that Mn-cyclin A m RNA was expressed on oogonia and oocytes in M.nipponense ovary,which also verified the expression prof PLe of Mn-cyclin A in different stages of ovarian development.In the continuous 19-day RNAi test,the expression of Mn-cyclin A m RNA in the experimental group was significantly lower than that in the control group(P<0.01).From the4 th to 19 th day after the first injection,the expression level of Mn-cyclin A m RNA in the ovary decreased by 72 % to 84 %,indicating that RNAi effectively reduced the expression of Mn-cyclin A m RNA in the ovary.Wh PLe interfering with Mn-cyclin A gene,the gonadal development index(Gonado somatic index,GSI)of the control group and the experimental group was detected.It was found that there was no significant difference in the value of GSI between the control group and the experimental group from the first day to the 10 th day after the first injection.On the 13 th day after the first injection,the GSI value of the control group was significantly higher than that of the experimental group.The ovaries of the control group developed to III stage(secondary oocyte stage)and the experimental group developed to stage II(primary oocyte stage).On the 16 th day after the first injection,the ovaries of the control group entered the second round of IV(oocyte stage)and the experimental group developed into III stage(secondary oocyte stage).On the 19 th day of the first injection,the ovaries of the control group entered the second round V(degenerative stage)and the experimental group entered the second round of IV(egg stage).The results showed that Mncyclin A RNAi could delay the gonadal development cycle and confirmed that Mn-cyclin A gene played an important role in the development and maturation of ovary.The results of this experiment are of great significance for the study of the mechanism of egg maturation of M.nipponense.Through the study of the function of cyclin A gene,it provides a theoretical basis for the study of molecular regulation mechanism in the process of crustacean oogenesis. |
PDF Full Text Request