The Expression Of Four Nuclear Hormone Receptors In Caenorhabditis Elegans And The Roles In Lifespan Regulation

Nuclear hormone receptors(NHRs) are important regulators of development and metabolism in animal species.They are characterized by the ability of regulate gene expression in response to the binding of small hydrophobic molecules,hormones, metabolites,and xenobiotics.Caenorhabditis elegans genome contains 284 NHRs sequences that share homology to vertebrate and insect NHRs.The majority of C.elegans NHRs is nematode-specific and referred to as supplementary nuclear receptors(supnrs) that are thought to have originated by duplications of an ancient homolog of vertebrate HNF4.Among these 284 NHRs genes,there are 15 genes conserved among the Metazoa.Four nuclear hormone receptors nhr-51,nhr-101,nhr-168,nhr-203 were studied in this paper.Knock down these genes by RNAi,the lifespan of N2 and daf-16(-),daf-2(-) mutants were affected.The lifespan of N2 was shortened after knock down nhr-51 by RNAi,and the same consequence was happened in daf-2(-) by nhr-51 RNAi;but the lifespan of daf-16(-) was extended by nhr-51 RNAi.The lifespan of N2 and daf-2(-),daf-16(-) mutants were all shortened by nhr-101 RNAi. The lifespan of N2 and daf-2(-) mutant were shortened after knock down nhr-168 by RNAi,but the lifespan of daf-16(-) mutant had no significiant change by nhr-168 RNAi.The same consequence happened in N2,daf-2(-) and daf-16(-) mutants by nhr-203 RNAi as these stains by nhr-51 RNAi.At the same time,the four NHRs also affected the distribution of DAF-16::GFP in Caenorhabditis elegans,the distribution of DAF-16::GFP appeared in gonad by nhr-51 and nhr-203 RNAi,and in oocyte by nhr-101 RNAi.In order to study the function of NHR-51,NHR-101,NHR-168 and NHR-203, the cDNAs encoding NHR-51,NHR-101,NHR-168,NHR-203 were amplified by RT-PCR from Caenorhabditis elegans N2 mRNA.The fragments of cDNAs were about 1200bp(nhr-51),1200bp(nhr-101),1400bp(nhr-168) and 1300bp(nhr-203) respectively,and the cDNA products were cloned into pMD18-T vector then transformed into E.coli DH5αand cultured on LB plus ampicillin(100μg/ml) plates. Colonies containing the recombinants were selected by PCR and the plasmids DNA were extracted and digested with enzymes.Plasmids containing the right insert were sequenced to confirm their identities,and the right recombinants were digested and cloned into the expression vector pET-32M then transformed into E.coli BL21(DE3) strain.Bacterial lysates from cultures induced by IPTG were loaded onto SDS-PAGE gel,and a distinct band of 44KDa(NHR-51),40KDa(NHR-101 ),50KDa(NHR-168) and 44KDa(NHR-203) respectively was detected in the SDS-PAGE gel.The purification of recombinant proteins and the immunization of mice were undertaken, the antibodies were obtained.The expressions of four NHRs were detected by immunofluorescence antibody staining.NHR-51 distributed in mouth,esophagus, intestine and spermatheca of adult worm,and also in L4 larvae intestine,gonad and the pharynx muscle as well as in the posterior end of the embryo,also in the centromere of division cell of embryo.NHR-101 distributed in nerve ring,intestine, spermatheca and hypoderm cells of adult worm,also in larvae intestine,gonad and pharynx as well as in the embryo.During the embryo development,NHR-101 expressed in the cytoplasm of the cell,some around nuclear liked a ring.NHR-168 expressed in the adult intestine,spermatheca,gonad and tail,also in the cytoplasm of 2-cell,4-cell and 8-cell embryos.In late embryo,NHR-168 expressed in the posterior cell of the embryo around nuclear like a ring,also in the larvae stage,NHR-168 was detected in intestine,gonad and the pharynx muscle.NHR-203 was detected in the intestine and gonad,also in the cytoplasm of 2-cell embryo till to the late embryos. NHR-203 also expressed in intestine and hypodermis cells in larvae stage.The interaction between four NHRs and DAF-16,DAF-2 were detected using Western Blot.Co-Immunoprecipitation combined with MALDI-TOF analysis was used to identify the proteins interact with NHR-51,NHR- 101,NHR-168 andNHR-203 respectively in wild type N2 and daf-16(-),daf-2(-) mutants,the results showed that several molecules might interact with four NHRs.