Study On The Role Of ERK Signal Pathway-Related Genes In Ovary Of Muscovy Ducks At Different Stages

Muscovy duck broodiness is a quantitative character controlled by multi-genes and is a big hinder in poultry business.Although nesting behavior have been studied by some scholars,a certain gene on the specific stages of development and the role of the site are limited.In order to explore the role of ERK signal pathway related genes in the regulation of the ducks in the nest,this study uses SMART-RACE technology,relying on the degenerate primers and EST transcriptome database search from Muscovy Duck ovary successfully cloned the 2 involved in the ERK signaling pathway and genes related to ovarian growth:extracellular signal regulated kinase 2(ERK2)and growth factor receptor binding protein 2(GRB2),and using different bioinformatic methods of ERK2 and GRB2 genes were analyzed.In combination with Cytoscape software,the interaction network regulation models of these genes in different periods were constructed.The experimental results are as follows:1.The cDNA sequence of Muscovy duck ERK2 was 3037bp,including 5' noncoding region 194bp,3' noncoding region 1736bp and open reading frame 1107bp,which could encode 368 amino acids.The relative molecular mass was predicted to be 41.9 kDa and the isoelectric point was 6.36.The protein encoded by the secondary structure has 35.05%alpha helix,8.97%beta-fold and 55.98%random curl,belonging to a hydrophilic stabilizing protein,without signal peptide and transmembrane structure.The results showed that the gene had high homology with the mRNA of mallard and wild goose,and the evolutionary analysis showed that it had the closest genetic relationship with the green duck.The results of qPCR showed that the expression of ERK2 gene in ovary and hypothalamus of Muscovy duck was significantly higher than that in other tissues(P<0.01).In the level of Expression in ovary is highest in the gonadal axis of different age(adolescence,laying period and nesting stage)(P<0.01).Furthermore,the difference of hypothalamus and pituitary was similar to ovary.Compare with adolescence and nesting stage,the laying period was significantly high(P<0.05).2.The full-length of GRB2 gene of Muscovy duck is 3528bp,which includes 5 non encoding region 47bp,3 non encoding region 2827bp and the open reading frame 654bp,encoding 217 amino acids.Prediction of the relative molecular mass was 25.2 kDa and an isoelectric point was 5.78;structure prediction showed that the GRB2 protein contains three SH3-SH2-SH3 functional domain,SH2 consists of the alpha helix and intermediate antiparallel beta sheets,SH3 is mainly composed of beta sheets,which belongs to a hydrophilic-stabilized protein,without signal peptide and transmembrane structure;The results showed that the gene had high homology with the mRNA of the mallard duck,and the evolutionary analysis showed that it had the closest genetic relationship with the rhubarb.The results of qRT-PCR showed that the expression of GRB2 gene in Muscovy Duck ovary is significantly higher than that in other groups(P<0.01).GRB2 gene was the highest in laying and ovarian tissue of nesting phase,the youth decreased significantly,the difference between the three growth stages were significant(P<0.05).The expression in the hypothalamus from adolescence to broodiness period increased first and then decreased.However,the expression in the pituitary from adolescence to nesting period has been decreasing significantly(P<0.05).3.The other 20 ERK signaling pathway genes were screened from the EST database of the transcriptome group.These genes were detected in different stages of muscovy ducks ovary.The results showed that fifteen genes such as Mek2,Rap 16,MRAS,Elk],PLCB1,SRF,Raf1,Mos,Pak1,Crebl2,SOS1,SOS2,RASA2,ERK2 and GRB2 were found have significant differences in the expression of Muscovy Duck 23 weeks and 36 weeks,50 weeks of age in ovary(P<0.05).Seven genes like DUSP4,MAP2K1,MYC,PRKACB,GPR39,RPS6KA3 and Mknk1 were stably expressed in ovary of Muscovy ducks at different stages.The expression levels of GRB2,ERK2,SRF,SOS1,PLCB1 and RASA2 in the ovaries of laying stage were higher than those in adolescence and nesting.In addition,hierarchical clustering of all these genes under different periods was analyzed in different genes of Muscovy ducks.The results showed that the same gene had different degrees in different stages of ovary,and different gene expression patterns were initiated in different genes during the same period.4.In order to observe the expression profile of ERK signal pathway related genes in different periods,the expression pattern of egg laying gene was used as reference.The model of network regulation of adolescent and ovary was established by Cytoscape software.The results showed that the Elk1 gene was up-regulated in adolescence,and MYC,RASA2,SOS1,SOS2,MRAS and PLCB1 genes were down-regulated,suggesting that these genes may play an important role in the growth and development of Muscovy ducks.Rap16,MRB,Mek2,Raf1 and Mos genes were significantly up-regulated in the nesting period,and the GRB2,ERK2 and SRF genes were significantly down-regulated,indicating that these genes may play a regulatory role in the process of muscovy duck.The above two periods of network regulation pattern map to a certain extent revealed the ERK signal pathway related genes on the muscovy duck ovary development and nesting different effects.