| Penaeus monodon,commonly known as black tiger shrimp,belongs to Penaeus,Penaeidae,Decapoda,Crustacea,Arthropoda.It is the largest species in the genus Penaeus,which is an important economic species in China and Southeast Asia.During the shrimp cultivation,differences of growth rate of body and gonads between female and male were found,it is important to carrying on research on sex determination and differentiation of P.monodon for that.Besides,it is of great reference for developing the controlling techniques of gonadal maturation.Micro observation,ultramicroscopic observation,sequence alignment and detection of In Del Markers were performed to identify and detect the sex of P.monodon larvae,thus a sex discriminating method for P.monodon in embryonic and early postembryonic development based on an In Del molecular marker was established.Besides,the Sox9 gene in mammals[1]and fem?feminization-1?gene in nematodes[2]were thoroughly studied and proved to be the key genes in sex-determining pathways,so characterization and functional analysis were performed for the homologous gene in P.monodon.The details are shown as followed:In order to distinguish the sex of P.monodon larvae in the embryonic and early stage of postembryonic development,the individuals of fertilized eggs,Nauplii,Zoea,Mysis,PL1?1 day post-larvae?,PL30 and subadults were collected.The external genitalia of the larvae of P.monodon in different periods was observed by anatomical microscope and electron microscope in order to find the male and female specific external characteristics in the early stage of development.Bisedes,a series of primers were designed for amplifying the Insertion/Deletion?In Del?marker.Finally one of these primers was selected after doing a series of experiments in different condition to be applied to a detecting method using the q RT-PCR?quantitative real-time polymerase chain reaction?with SYBR Green dye—A sex detecting method for P.monodon according to sequencing and melting curve analysis of the q RT-PCR was established.The results showed that the sex of P.monodon was unable to distinguish before PL30 depending on the appearance differences,the sex-specific sequences can be obtained by PCR amplified with the sex-In Del primers.The result of sex discriminating of subadult shrimp by sequencing method is consistent with that of observing method,and the correct rate is 100%.Sex-specific sequences can be amplified from the samples of fertilized eggs,nauplius,zoea larvae,Mysis larvae and post-larvae.Furthermore,a rapid discriminating method based on the melting curve of q RT-PCR?quantitative real-time Polymerase Chain Reaction?with SYBR Green was established,in which the value of Melting Temperature?Tm?of sex-specific sequences is 79.10±0.10?for male and78.45±0.20?for female.The sex of individuals of PL30,subadults and the sixth stage of Nauplii can be accurately distinguished by the specific melting curve,the correct rate is above 96.3%.According to the EST?Expressed Sequence Tag?in the transcriptional library,a homologous gene of Sox family was amplified by RACE technique,which was named Pmsox9 gene.Its c DNA is 1599 in length and encodes 344amino acids in total.After a series of bioinformatics analysis,a HMG-box domain was predicted to be among the Pmsox9 gene,which was confirmed to be a member of HMG?high mobility group?protein family.The results of tissue expression profile showed that Pmsox9 was expressed in all tissues,with a highest expression in nerve tissues such as eyestalk ganglia and brain ganglia,as well as testes,which was significantly higher than that in ovary?P<0.05?.After exposure of P.monodons larvae to 17?-methyltestosterone?17?-MT?,it was found that a lower dose?5?g/L?of 17?-MT could significantly increase the expression of Pmsox9 since it was early larvae?late nauplii?,while a higher dose?50?g/L?could reduce the expression of Pmsox9,but the effect was not significant?P>0.05?.Also,the expression of Pmsox9 showed no significant difference among the groups?P>0.05?as it was exposed to 17?-MT since PL15.RNAi?RNA interference?with synthetic long-stranded double-stranded Pmsox9 RNA could maintain a silencing efficiency of 86%of Pmsox9.The paraffin sections with HE staining of testes tissue were observed under microscope,and the number of sperm in the spermatophore sac was counted.The paraffin sections showed that the sperm count of testes tissue after dssox9 injection was lower than that of PBS and ds GFP group on 7 dpi?7 days post injection?and 28 dpi.The statistical results of sperm count of spermatophore sac showed no significant difference among groups?P>0.05?on 28 dpi while the sperm count of dssox9group was significantly higher than the control group on 7dpi?P>0.05?,which showed that Pmsox9 was closely related to the maintenance of gonads for male P.monodon.According to the existing EST sequence of P.monodon and the full length of m RNA of fem gene of the Pacific white shrimp?Litopenaeus vannamei?,primers were designed to amplify the ORF region of fem gene of P.monodon.The total length of the sequence after splicing is 2137 nt,which can encode 639 amino acids,which was named Pmfem gene according to the result of BLASTx.It was predicted that the Pmfem gene contained six consecutive anchor protein?ANK?repeats.The results of expression analysis of gonads showed that Pmfem was expressed in both sexes and the expression in testes was significantly higher than that in ovaries.The results of exposure to 17?-methyltestosterone were consistent with those of Pmsox9,but the range of change was small,indicating that 17?-methyltestosterone had less effect on the expression of Pmfem gene.Synthetic long-chain ds Pmfem could reduce the expression of Pmfem significantly?P<0.05?;the paraffin sections with HE staining showed that the sperm density of testes tissue of dsfem group was lower than that of PBS and ds GFP group after ds RNA injection,while statistical results showed that there was no significant difference in sperm count of spermatophore sac among groups?P>0.05?. |
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