| Daphnia is a small type of planktonic crustacean with both parthenogenesis and sexual reproduction.Parthenogenetics is usually carried out when the external conditions are more suitable;When daphnia are under the adverse environmental conditions,the sexual reproduction produces dormant eggs to overcome the adversity.The research on the reproductive transformation of cladocerans mostly focuses on the description of environmental inducing factors and the reproductive toxicity of chemical substances.There are few reports on its intrinsic molecular mechanism.This study begins with the developmental biology of Ceriodaphnia quadrangula,and detects and screens environmental factors affecting the reproductive pattern of the cladocerans;then Daphnia magna is used as the research object,and the important inducing factor-population Qualitative analysis of density,detection of chemical components in high-density D.magna culture water;establishment of a method for efficient induction of reproductive conversion by hormones(Fenoxycarb),and transcriptome sequencing analysis of D.magna during induction;The differentially expressed genes in the transcriptome were analyzed and identified,and some of the potentially related genes screened were functionally verified.The main results of this study are as follows:1.Using Ceriodaphnia quadrangula as experimental material,the effects of temperature,photoperiod and population density on reproductive transformation were analyzed.Experiments have found that these factors can affect the transformation of reproductive patterns between parthenogenetic and sexual reproduction of Ceriodaphnia quadrangula,and population density is the most important factor.2.Using Daphnia magna as experimental materials,it was verified that high-density breeding is a highly effective sexual reproductive inducing factor;High Performance Liquid Chromatography(HPLC)and Gas Chromatography-Mass Spectrometry(GCMS)were used to identify high-density Daphnia magna breeding water.A juvenile hormone analogue,fenoxycarb;a parthenogenetic individual treated with fenoxycarb greater than 2?g/L can induce almost all offspring to be male.3.Treatment of 1?g/L fenoxycarb in the female of Daphnia magna whose brood chamer with specific eggs,then sampling at 6 h,24 h and 36 h after stimulation,and RNA-seq using the Illumina HiSeq~TMM 2500 platform.The group determined that 195,869 unigenes were obtained with an average length of 793 bp.Through the Blastx algorithm,seven publicly available databases,such as Nr,Nt,KO,SwissProt,PFAM,GO and KOG,have a total of139042 annotated genes,accounting for 66.85%of the total unigenes of the transcriptome.The expression profiles of unigenes were summarized,and the types and quantities of expressed genes were elucidated.The classification,function and metabolic pathways of some encoded proteins were analyzed.4.To analyze,screen and re-annotate differentially expressed genes in Daphnia magna transcriptome,we identified Doublesex(Dsx)containing DM domain and HLH domain from the differentially expressed genes of Daphnia magna after 6 hours of hormone treatment.Daughterless(Dal)and histone demethylases containing the JmjC domain may be related to the sex differentiation and reproductive development of the organism.After preliminary analysis and inference,the sex determination of Daphnia magna should have been determined at the beginning of embryonic development.5.Verifying the gene function of the three selected genes.The expression of DmDsx,DmDal and DmJmjC genes in Daphnia magna was effectively knocked down by RNAi technology.At this time,treatment with fenoxycarb reduced the proportion of males in their offspring,indicating that these three genes involved in the processes of Daphnia magna,such as sex differentiation and reproductive development. |
PDF Full Text Request