| The Silurus asotus belongs to Siluriformes,Siluridae and Silurus.It is one of the most important indigenous economic fishes in China.In the course of natural growth or breeding,the female and male ratio of S.asotus is 1:1.However,female S.asotus tend to grow faster and larger than males.In the same age,the individual quality of the female S.asotus is usually 1-2 times than that of the male S.asotus.In the industrialized production,the growth rate of male S.asotus has seriously affected the increase of the overall production of S.asotus.Therefore,the experiment carried out that the screening of Tamoxifen and Letrozole induced S.asotus sex reversal and sex specific gene.The aim of this study was to provide theoretical support for the acquisition of "pseudomale fish" and the technique of identifying "pseudomale fish" in vivo.In this experiment,the use of exogenous estrogen receptor antagonist Tamoxifen(Tamoxifen,TAM)and aromatase inhibitor Letrozole(Letrozole,LT)to deal with sex differentiation of larve S.asotus.In this experiment,the exogenous estrogen receptor antagonist Tamoxifen(TAM)and aromatase inhibitor Letrozole(LT)were used to treat S.asotus larvae which was in the early stage of sex differentiation,inducing a sexual reversal and the effects of two drugs on the growth and gender differentiation of S.asotus were observed;At the same time,Illumina was used to sequence the transcriptome of females and males in different stages of gonadal development.According to the sequencing results,the genes related to S.asotus sex were selected,some candidate sex specific genes were verified by qRT-PCR,analysis of their expression in different genders of S.asotus.(1)The fish were treated with Tamoxifen after 9 days of incubation with a concentration of100?g/L(early zooplankton feeding)+ 25mg/kg(late feed feeding)for 50 days.The results showed that theTAM treatment had no inhibitory effect on the growth and development of the S.asotus or the formation of intersex glands and the atrophy of gonadal glands were not found in gonad development compared with the control.In the 4 months of age,about the testis morphology,there were no significant differences between the TAM and the controls.However,the increase in the number of primary ovarian oocytes was inhibited.The male rate increased from 50% to 75%,significantly higher than the control group(P<0.05),at the age of 5 months.(2)The usage of Letrozole is the same as above,with a concentration of 100?g/L(early-stage zooplankton feeding)+ 50mg/kg(late feed feeding).Results showed that the drug treatment of S.asotus did not inhibit growth and development.The testis development process is synchronized with the control,but some testicular morphology has a little change in the 28 days old.Some individual testis were small and no ovarian tissue was observed at the age of 4 months,it may be that the ovaries degenerate in the process of gonad differentiation and form a smaller testis.The male rate after Letrozole treatment was 100%,significantly higher than that of control group and Tamoxifen group(P<0.05).(3)Using Illumina sequencing technology,the transcriptome sequencing was performed on nine samples from three stages,which were the whole fish,adult female S.asotus,adult fish ovary,adult male S.asotus,adult fish sperm nest,young female S.asotus,young fish ovary,young male S.asotus mix,young fish testis and the mixed samples were mixed samples of 8 tissue RNA of brain,gill,heart,liver,spleen,kidney,intestine and muscle.A total of 208,740,585 clean reads were produced and 74,840 usable transcripts were assembled.The transcripts obtained from the assembly were mapping to the Nr database,and 60477(80.81%)were annotated.The expression of the gene was calculated using the RPKM value.If the RPKM is less than 1,the gene is not expressed.The number of transcripts with RPKM greater than 1 in different sequencing samples was 28015,23183,27901,34216,31468,31415,30606,33606,27964,respectively.A total of 138 female-specific transcripts and 707 male-specific transcripts were screened.(4)We selected11sex-specific genes that were only expressed in males based on the result of transcriptome sequencing.Sequencing was carried out by RT-pcr and TA cloning,and the sequencing results showed that only eight genes were correct.qRT-pcr was used to detect the expression differences of these 8 genes in different genders of S.asotus,and the results were the same as those obtained from Illumina sequencing data.All the confirmed genes were expressed in adult male S.asotus,but only gene 2had male sex expression specificity.Eight genes were expressed in all tissues of adult male S.asotus,and the expression trend of each gene in tissue was similar,and the expression level was higher in liver,spleen and kidney.We speculate that these genes may be the same type of gene that is structurally and functionally similar.In addition to gene 2,other confirmative genes are expressed in females,but the expression is very low,and the expression in males is several hundred to several thousand times that of females. |
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