IGFBP4?5?7 And Insulin-related Peptide Roles Larval Settlement And Metamorphosis In The Small Abalone Haliotis Diversicolor

Larval settlement and metamorphosis in small abalone is the very vital stage in its life history.It is the high mortality rate that directly affects its population distribution and population dynamics,which is on the account of the fragile and sensitive physical condition of larvals.Partial sequence of IGFBP4,IGFBP5 and MIRP were obtained from our EST database of small abalone,and the full-length cDNA sequences of the three genes were cloned by SMART RACE techniques,which were denoted as hdIGFBP4 ? hdIGFBP5 ? hdMIRP.Real-time quantitative PCR(RT-qPCR)and RNA interference(RNAi)were applied to reveal the role of hdIGFBP4?hdIGFBP5 and hdMIRP,together with hdIGFBP7,a gene cloned full-length cDNA before in the same lab.The main results of this study were reported as follows:1)The full-length cDNA of hdIGFBP4 is 584 bp,and the open reading frame encodes a protein of 137 amino acids.This is the first time to obtain the IGFBP4 gene of invertebrates sofar.The predicted protein contains a IB domain,a conserved motif CGCCXXC and 10 high conserved cysteine residues.Compared with IGFBP4 in vertebrates,hdIGFBP4 is in the lose of the conserved TY C-terminal domain and the characteristic Linkage midregion.RNAi was applied on larvals during the the late veliger stage and the result shows: compared with blank control,within 4 hours after RNAi,the mortality rate had significant rise(P<0.05),settlement rate had significant drop(P<0.05),while the floating rate had no obvious difference(P>0.05).Real-time PCR shows: the quantity of hdIGFBP4 mRNA down-regulated(P<0.01)after RNAi,meanwhile,the mRNA quantities of 4 related pathways down-regulated highly significantly as well.2)The full-length cDNA of hdIGFBP5 is 921 bp,and the open reading frame encodes a protein of 136 amino acids.The predicted protein contains a IB domain,a conserved motif CGCCXXC and 11 high conserved cysteine residues.Compared with IGFBP5 in vertebrates,hdIGFBP5 is in the lose of the conserved TY C-terminal domain and the characteristic linkage midregion.RNAi was applied on larvals during the the late veliger stage and the result shows:compared with blank control,within 4 hours after RNAi,the mortality rate had a high significant rise(P<0.01),settlement rate had a high significant drop(P<0.01),while the floating rate had no obvious difference(P>0.05)as dIGFBP4 did.Real-time PCR shows: the quantity of hdIGFBP5 m RNA down-regulated(P<0.01)after RNAi,meanwhile,the mRNA quantities of 4 related pathways down-regulated highly significantly as well.3)The full-length cDNA of dIGFBP7 is 1812 bp,obtained years before in the lab,and the open reading frame encodes a protein of 239 amino acids.The predicted protein contains a IB domain,a KI domain and a IGc2 domain,a conserved CGCCXXC and CGXDXXTYXN motifs and 11 high conserved cysteine residues.RNAi was applied on larvals during the the late veliger stage and the result shows: compared with blank control,within 4 hours after RNAi,the mortality rate had a high significant rise(P<0.01),settlement rate had a high significant drop(P<0.01),and the floating rate had a high significant rise(P<0.01),which is quite different from hdIGFBP4 and dIGFBP5.Real-time PCR shows: the quantity of hdIGFBP7 mRNA down-regulated(P<0.01)after RNAi,meanwhile,the mRNA quantities of 4 related pathways down-regulated highly significantly as well.4)The full-length cDNA of hdMIRP is 935 bp,and the open reading frame encodes a protein of 118 amino acids.The predicted protein contains a Insulin-like domain,and 6 high conserved cysteine residues.RNAi was applied on larvals during the the late veliger stage and the result shows: compared with blank control,within 4 hours after RNAi,the mortality rate had a high significant rise(P<0.01),settlement rate had a high significant drop(P<0.01),and the floating rate had a significant rise(P<0.05),which is similar with hdIGFBP7 but quite different from hdIGFBP4 and hdIGFBP5.Real-time PCR shows: the quantity of hdMIRP mRNA down-regulated(P<0.01)after RNAi,meanwhile,the mRNA quantities of 4 related pathways down-regulated highly significantly as well.