Identification And Functional Analysis Of P. Cerevisiae-like Parathyroid Hormone-like Receptors (PTHRLs) And Insulin-like Receptors (ILRs)

As a seriously harmful agricultural pest distributed all over the world,Tribolium castaneum is a representative insect for coleoptera and a new model insect,which completed the whole genome sequencing and effectively RNA interference.Parathyroid hormone receptors and insulin receptors are membrane receptors and transfer the signal into the intrecellular after binding with the ligand.And then the signal progressively enlarge by the second messanger and produce physiological and biochemical effects.Parathyroid hormone receptors play important role in the growth and development of the mammals.Parathyroid hormone receptors was originally identified in rat,human for the regulation of blood Ca2+ concentration,bone formation and mineral ion homeostasis in the vertebrates.Surprisingly,this signal system did not discovered from any model insects neither D.melanogaster nor A.gambiae.Until very recently,we have mined out two putative parathyroid hormone receptors like(PTHRL)receptors from Tribolium castaneum for the first time,and further examined out that PTHRL also existed in several other insects,Apis mellifera,Nasonia vitripennis,Pediculus humanus corporis and ants.The function in insects is unknown.There are only one insulin like receptor in the Drosophila melanogaster,Anopheles gambiae and Bombyx mori.In the social insects,honey bee or ants,there are two insulin-like receptors.And in the Tribolium castaneum,which is not the social insects,there are also two insulin-like receptors.Whether function of these two receptors is different from each other is unknown.Thus,two putative pthrl and ilr genes,named as,T.cas-pthrll and T.cas-pthrl2,T.cas-ilrl and T.cas-ilr2 have been cloned from Tribolium castaneum.Firstly,we analysis the phylogenetic of these recetors and their expression pattern in different development stages of the Tribolium castaneum.We preliminary analysis the function of these receptors by RNAi.Secondly,we make use of the RNA-seq and proteomics technology combined with the RNAi to study the change of the downstream genes in the mRNA or proteins level.The main findings are as follows:Phylogenetic analysis revealed that the PTHR happened two duplications in the vertebrates and have three receptors:PTHR1,PTHR2 and PTHR3.It is happened deletion in the birds and mammals to lost of PTHR2 and PTHR3 respectively.In insects,it is lost(eg:Drosophila melanogaster,Anopheles gambiae,Bombyx mori and Acyrthosiphon pisum,there are two clades:PTHRL1 and PTHRL2.But in Apis mellifera,Nasonia vitripennis and Pediculus humanus corporis there is only one parathyroid hormone like receptors which are claded in the PTHRL2 and may be lost the PTHRL1.The expressions pattern of T.cas-pthrls research showed that T.cas-pthrl1 highly expressed in every late period than the early stage of egg,larvae,pupae and adults and reached peak in the late larvae.The expression level of T.cas-pthrl2 is the highest in the late adults.This results showed that the two receptors may be happened function differentiation.Larval RNAi knocked down the expression of T.cas-pthrll caused 81.34%insects not entering into the adult,while T.cas-pthrl2 RNAi only caused 27.33%.Furthermore,parental RNAi only made ds-pthrll insects only lay 2.63 ± 0.07 eggs/day/female and?10%eggs could survivor compared with wild-type insects.However,there is no significant defect for eggs reproduction for ds-pthrl2 insects.And the hatching rate is declined.These results illustrated that the function of these two T.cas-PTHRLs divergent between each other and T.cas-pthrl1 play a role in the development and reproduction.RNA-seq reveals that there are 1084(ds-pthrl1)? 1351(ds-pthrl2)different expression genes(DEGs).In both ds-pthrls there are 295 genes up and 449 down.There are 235 genes up-regulation in ds-pthrl1 and 105 down-reguation,while there are 219 up-regulation and 388 down-regulation.Down of T.cas-pthrls showed that it play important role in the ecdysis,epidermis development and reproduction.In ds-pthrl1 the ricket or ccapr-2 genes changed which maybe affect the ecdysis in Tribolium castaneum.In addition,the change of many cytochrome P450 genes in ds-pthrls cause the change of 20E,leading to the ecdysis.T.cas-pthrls play a key role in the epidermis development by effect the chitin synthase and chitinase.There are many chitinase changed in the ds-pthrls.Nanos and GTP binding protein are up-regulation in ds-pthrl1.Thus,T.cas-pthrl1 may be infulence the reproduction by nanos.Proteomics reveals that after T.cas-pthrls RNAi,there are 25 proteins identified.It is 10 proteins(cuticular protein,desiccation stress protein and H+-transporting ATPase)down-regulation while 5 including adenylate kinase up-regulation in ds-pthrls.H+-ATPase was down-regulation after T.cas-pthrls RNAi and ds-pthrll is lower.The up-regulation of Adenylate kinase 2 after RNAi may be retrieve the defect of the larvae development by T.cas-pthrls RNAi.In these proteins 4 proteins in ds-pthrll up and ds-pthrl2 down while 5 proteins in ds-pthrll down and ds-pthrl2 up.Hexamerins protein showed down-regulation in ds-pthrll and up-regulation in ds-pthrl2,which may be one of the reason in ecdysis and reproduction defect.It was found that H+-ATPase,Adenylate kinase 2 and hexamerins may be involved in the process of parathyroid hormone like receptors pathway in Tribolium castaneum.GO analysis showed that the structural constituent of cuticle included the most different expression proteins,which was consistent with the RNA-seq results.Thus,the research on parathyroid hormone like receptors in T.castaneum showed that in genes level T.cas-pthrls play a key role in the ecdysis by the change of ricket,ccapr-2 and CYP family.The change of chitin synthase and chitinase lead to the defection of epidermis development after knocking down of T.cas-pthrls.The down of T.cas-pthrls affected the nanos leading to the reproduction descent.In proteins,there are many proteins changed by the down of T.cas-pthrls.Insulin receptors have been duplicated at least twice among vertebrates:IGFR,IRR and IR,and in the reptiles the IR is lost.In insects,insulin like receptors is one in the Diptera,Lepidoptera,and Phthiraptera.But there are two receptors in Coleoptera,Hymenoptera and Hemiptera except the Nasonia vitripennis.Maybe there is one duplication occurred before three category differentiation,but one was lost in the Nasonia vitripennis.The expression of T.cas-ilr1 reached the peak in the late adult.T.cas-ilrl is lowest in the early larvae and gradually increased into the eraly pupa then decreased.While T.cas-ilr2 is reach the peak at late larval stage,and followed by early larvae and late adult stage.In the other stages T.cas-ilr2 showed lower.Larval RNAi caused 100%and 41.2%insects arrested at larval stage for ds-ilrl and ds-ilr2 insects,respectively.Parental RNAi suggested that the suppression of T.cas-ilr1 and T.cas-ilr2 caused 100%and 31.5%eliminationon the fertility,respectively.And there are only 62.9%eggs hatching rate for ds-ilr2 insects.These results showed that the function differentiation exist in the two insulin like receptors.Moreover,it will up-regulated pkc,foxo,jnk,cdc42,ikk and mekk,but down-regulate erk gene expression at both cases after RNAi of T.cas-ilr1 and T.cas-ilr2.Thus,the insulin like receptors may be affect the development and reproduction by JNK,ERK and PI3K pathway.There are ac,akt,ask and pi3k up-regulation after ds-pthrl1 and pka up-regulation after ds-pthrl2.The difference for the down-stream implys the function differentiation.