Identification,Expression And Regulation Of Key Genes For Liver Lipoprotein Transport In Chicken

Microsomal triglyceride transfer protein(MTTP)is a member of lipid transfer protein family(LLTPs).In mammalian,MTTP is an indispensable regulatory protein in liver lipid metabolism,which was mainly involved in the VLDL assembly and secretion.Defective MTTP would result in β-hyperlipoproteinemia.However,up-regulation of MTP in the liver is not occured for the increased VLDL assembly during egg production in the chicken.It implies that there must be other gene(s)that regulate(s)the formation and transport of lipoprotein in liver in chicken.In this study,transcriptome of livers of 20-week juveniles(3 individuals)and 30-week laying hens(3 individuals)were firstly implemented by RNA-seq technology.The significantly differently expressed(SDE)genes between the juveniles and laying hens were screened out.All of the novel genes,which were no annotations both in NCBI Gen Bank and Ensemble currently,were picked out from the SDE genes.The structural and functional domains of the novel genes were analysed by bioinformatics tools.A gene,named MTTPL gene,was singled out.The CDS sequence of MTTPL was cloned,and the expression pattern of MTTP in different tissues and developmental stages were analysed.The relationship between MTTPL and lipid metabolism was investigated both in intro and in vivo.The results was as follows:1.A novel gene,named MTTPL gene,which contains the functional domains for VLDL assembly and transport,was singled out by transcriptome data mining and bioinformatics analysis.2.According to the predicted m RNA sequence of MTTPL in NCBI database,cloning primers of MTTPL were designed.The CDS of MTTPL was cloned,sequenced and uploaded to NCBI database(Genbank accession number: KT899704).Comparative analysis of amino acid sequences of the MTTPL,MTTP and human MTTP indicated that the chicken MTTPL and human MTTP possess the samilar domains,motifs and specific amino acid residues including the signal peptide,LPN domain and LCR domain.However,there was no LCR domain in chicken MTTP.The phylogenetic analysis of MTTPL indicated that the MTTPL and MTTP might originated from the same ancient gene.These results suggest that the chicken MTTPL might have similar functions to the human MTTP.3.To analyse the function of MTTPL,we expored the tissue expression pattern of MTTPL in 30-week Lushi blue-shelled-egg chicken and detected the expression changes of MTTPLand MTTP in the specific tissues of 1-day,10-week and 30-week chicken.The results showed that MTTPL was only expressed in liver and kidney tissues.And the expression level of MTTPL in liver was higher than that in kidney.Along with the increasing of chicken age,the expression of MTTPL rose significantly(P <0.05),while there was no significant change happened in the expression level of MTTP in between 10-week and 30-week chicken liver(P >0.05),which implied that the gene involved in liver lipid transport in laying hens might be MTTPL rather than MTTP.4.To further study the function of MTTPL,we constructed the model of estrogen-treated hens and emboryonic primary hepatocytes and detected the expressed change of MTTPL,MTTP and Apo B with different treated conditions.The results showed that estrogen could significantly up-regulate the expressions of Apo B and MTTPL in chicken liver tissue and emboryonic primary hepatocytes(P <0.05),but not significantly change were founded in the expression of MTTP.In summary,along with the hen’s sexual maturity,the concentration of VLDL was significantly increased in chicken blood.In the meanwhile,the expression of MTTPL was also gradually up-regulated in hen’liver,while the expression of MTTP was no significant changed.The in vetro and in vivo experiments with estrogen-treated hens and emboryonic primary hepatocytes indicated that the expression of MTTPL in hen’liver was positively regulated by estrogen,but the expression of MTTP was not influced remarkably by estrogen.In addition,combining the protein structural analysis of MTTPL and MTTP,we speculate the key gene involved in liver lipid transport of hens may be MTTPL rather than MTTP.