| Sex determination and sex differentiation of fish are influenced by both genetic and environmental factors.Sex steroid hormone of environment can completely change the direction of sex differentiation determined by genetic factors.In this study,yellow catfish larvae(Pelteobagrus fulvidraco)were treated by different doses of EE2 in different periods,carried by crushed yellow eatfish feed and artemia respectively,to study the role of different carrier,different doses and different periods during sex reversal by measuring female rate,proportion of gonad undeveloped individual and survival rate;weight gain rate,specific growth rate and condition factor of all-male yellow catfish larvae(Pelteobagrus fulvidraco)were measured to study the role of EE2(17a-ethinylestradiol)of growth of male yellow catfish(Pelteobagrus fulvidraco).According to the above results,suitable carrier,suitable dose,suitable period of EE2 expose were identified.All-male yellow catfish larvae(Pelteobagrus fulvidraco)of 9 dah were treated with EE2(200 ?g/L)by artemia for 40 days,the transcripts in gonad during 0 d,20 d and 40 d were sequenced by RNA-sequencing,and mRNA expression of Cyp11b2,Cyp19a1a ?Cyp17al were measured by RT-PCR,to identify the predicted key genes in gonad during sexual reversal by EE2 treat,meanwhile,further study the molecular mechanism of sex determination and sex maintenance of yellow catfish(Peltebagrus fulvidraco),provide theoretieal and technical support for genetic breeding of yellow catfish(Pelteobagrus fulvidraco).The results were as follows:1.In the study of yellow catfish larvae(Peleteobagrus fulvidraco)treated by different doses of dietary EE2 carry by feed,female rate between groups had no significant differences.Proportion of gonad undeveloped individual was relatively larger and turned out ascend trend along with EE2 dose.Survival rate in control group was significantly higher than each treated group(P<0.05).In the study of yellow catfish larvae(Peleteobagrus fulvidraco)treated by different doses of dietary EE2 carry by artemia,female rate reached more than 90%during EE2 treated groups,and had significant differences between control group(P<0.05),proportion of gonad undeveloped individual was relatively smaller and had no significant differences between groups.Survival rate of control group was significantly higher than each treated group(P<0.05).2.In the study of all-male yellow catfish larvae(Peleteobagrus fulvidraco)treated by different doses of dietary EE2 carry by artemia,weight gain rate and specific growth rate between treated groups and control group had significant differences(P<0.05).With the increase of EE2 dose,the weight gain rate and specific growth rate of each group showed a decreasing trend.Weight gain rate and specific growth rate between 100 ?g/L treated groups and 200 ?g/L treated groups had no significant difference.Condition factor between groups had significant difference(P<0.05).3.In the study of yellow catfish larvae(Peleteobagrus fulvidraco)treated during different periods carried by artemia,female rate in all groups reach more than 90%except 21 dah treat group.Proportion of gonad undeveloped individual was relatively smaller and had no significant differences between groups.Survival rate decreased with the delay of period of EE2 treat.In the study of all-male yellow catfish larvae(Peleteobagrus fulvidraco)treated during different periods carried by artemia,weight gain rate of 9 dah treat group had significant differences with other groups(P<0.05)except 12 dah treat group.Specific growth rate among three groups 9 dah treat group,12 dah treat group and 15 dah treat group had no significant differences,but had significant differences with other treat groups(P<0.05),Weight gain rate and specific growth rate decreased with the delay of period of EE2 treat.Condition factor between groups had no significant difference.4.Transcriptome sequencing of a mixture of gonad samples treated for 0 d,20 d and 40 d was filtered,stitched and assembled to obtain 192851 transcripts,GC content 45.06%,N50 1882 bp,unigenes 79715,GC content 44.12%,N50 1782 bp.The quality of unigenes was good.5.Function annotation and classification for 79715 unigenes,30785 unigenes annotations succeed in Nr data base,38.62%of all unigenes.Classification by GO annotation was divided into 3 major categories of 50 biological functions,most of the annotations was in member.Posttranslational modification,protein turnover,chaperones was the largest of the 50 functional groups in the eggNOG classification.KEGG analysis of unigenes was for the 6 major branches,40 small branches,317 pathways,metabolize was the largest of the 6 functional groups.6.The transcripts of EE2 treat group and control group during 20 d and 40 d of EE2 treat were compared and screened to get differentially expressed genes.There were 5328 differentially expressed genes between EE2 treat groups and control group during 20 d of EE2 treat,4513 up,815 down.There were 3021 differentially expressed genes between EE2 treat group and control group during 40 d with EE2 treat,2642 up,379 down.Then performed KEGG enrichment for these genes,obtained a large number of genes of steroid hormone synthesis pathway.7.Differentially expressed genes were analyzed eombined with the result of RT-PCR.During 20 d of EE2 treat(in the process of sex reversal),the expression of Star(insignificantly),3?-HSD7(insignificantly),11?-HSD2(insignificantly),Cyp19a1b(insignificantly),Cyp11b2(insignificantly),Foxl2(significantly)and Cyp19a1a(significantly)increased compared with control group,the expression of 17?-HSD7(insignifieantly),Cyp17al(insignificantly)and Cyp26b1(significantly)decreased.During 40 d of EE2 treat(when sex reversal is completed),the expression of Star(insignificantly),3B-HSD7(insignificantly),Cyp11b1(insignificantly),Foxl2(insignificantly),Cyp26b1(insignificantly),11?-HSD2(significantly),Cyp17a1(significantly),Cyp19a1b(significantly)and Cyp19a1a(significantly)increased,the expression of 17?-HSD2(insignificantly)decreased.In conclusion,compared with feed,artemia is more suitable as carrier of sexual reversal of yellow catfish larvae(Pelteobagrus fulvidraco)by EE2 treat,the EE2 doses between 100?g/L and 200 ?g/L is suitable,periods between 9 dah and 12 dah is suitable.During 20 d of EE2 treat(in the processing of sex reversal),the expression of Foxl2 and Cyp19a1a increased significantly while the expression of Cyp26b1 decreased significantly in gonad compared with control group.Therefore,iconic gene of ovarian formation Foxl2,ovarian aromatase gene Cyp19a1a and RA degradation enzyme gene Cyp26b1 might be the key genes during sexual reversal by EE2 treat of yellow catfish larvae(Pelteobagrus fulvidraco). |
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