Screening Different Expression Genes In Metamorphosis Of Haliotis Divercolor And Analyzing The Function Of TH Signaling Pathway

Based on gene sequences from our research group and Gen Bank database for Haliotis diversicolor,186 candidate genes were screened by bioinformatics analysis.The metamorphosis expression profiles of thoses genes were obtained by q PCR.According to the expression of these different expression genes in veliger(VE),creeping larvae(CR)and juvenile(JU),a molecular network of these genes were established by Cytoscape.The network showed that TH signaling pathway may play an important role in metamorphosis.Then we explored the function of TH signaling pathway in metamorphosis by RNAi.The results were as follows:1)186 different expression genes in metamorphosis were analyzed by Gene Ontology.Results showed that proteins encoded by these genes mainly consist of ferritin,ribosomal protein,hormonal protein,metabolism-related enzyme,apoptosis-related protein,cyclin and transcription factor.Further analysis found that cellular component genes possess the most gene sequences.2)The expression stabilities of six candidate reference genes(EIF5A,OAZ1,YB1,RPL3,RPS9 and ACT)in metamorphosis and RNAi experiment were evaluated by Genorm,norm Finder and Ref Finder statistical algorithms.The results demonstrated that EIF5 A was validated as the most stable reference genes in VE,CR and JU;while it was RPS9 in the experiment of RNAi.3)The m RNA expression profiles of 186 different expression genes were analyzed by ANOVA.And we found that there were 106 genes whose expression had significant differences.And then to GO function classification for the 106 genes,showed that there are 23 genes participated in immune/stress response;13 genes involved in the translation process and cellular metabolic process;12 genes related to the cell cycle,10 genes coding membrane proteins;9 genes possessing transcription regulator activity;9 genes are associated with chromosome organization;8 genes related to calcium ion binding;7 genes related to the neurological system process,and 2 unknown genes.Then partial genes were established to a molecular network by Cytoscape.The above results show that the metamorphosis of H.diversicolor was a very complex biological processe,there were numerous genes and signaling pathways involved in,and formed a complex molecular network.Among them,there are some key signaling molecules,such as TH signaling pathway.4)After analysised the expression pattern of genes related to TH signaling pathway during VE,CR and JU in H.diversicolor,we found that the m RNA expression level of TPO was highest in VE,then it decreased;the expression level of TPO-1 increased greatly in CR,then decreased.The expression level of ID? in JU was highest,and significantly higher than the other two periods(p < 0.05);Integrin ?v shared same expression pattern with ID?;while the expression quantity of TR? appeared a downward trend,and showed significant difference respectively in all the three peroids(P<0.05).5)We dealed with the larvae in VE by three different concentrations of T3 solution(10-7mol/L,10-6mol/L and 10-5mol/L).The results showed that T3 could increase metamorphosis rate significantly at 10-6mol/L(P<0.05),10-7mol/L and 10-5mol/L did not significantly increase(P>0.05).6)We cloned the whole c DNA sequence of TR? by SMART-RACE technology.And the whole length of c DNA was 1829 bp,encoding 399 aa.It contains two important functional domains of nuclear receptor(DBD and LBD).Further more,the multiple comparison and phylogenetic analysis showed that DBD and LBD were highly conserved.7)TR?-ds RNA was synthesised in vitro and we chosed the time phase of VE to carry out RNAi experiment on H.diversicolor larvae.After 6 hours of interference,the metamorphosis rate of experimental group was down-regulated significantly compared to the negative control group(EGFP RNAi)and the blank control group(Non-treated).Besides,the death rate of experiment group was increased significantly compared to the control group.The results indicated that gene silencing on TR? had a great effect on larval metamorphosis in H.diversicolor.8)Using q PCR to detect the expression level of TH signal-related genes after TR? silencing.The results indicated that expression level of TR?,HIF-1?,STAT1 and CCND1 were down-regulated(P<0.05),and TR?,HIF-1? and STAT1 were down-regulated significantly(P<0.01)after TR? RNAi.There was no significant difference between experiment group and control group among other genes such as Integrin ?V,ID?,?-catenin,PI3 K,AKT,ERK,Src K,GSK,TRAP,TPO and TPO-1.The expression level of TR? was down-regulated after TR?-ds RNA,which indicated that the interference effect was remarkable.