Screening And Prokaryotic Expression Of Differentially Expressed Proteins In Hypothalamus Of Huoyan Goose During Different Laying Period

In recent years,the goose industry towards the development of large-scale,intensive and modern,and the slaughter,breeding is in the world.But geese low reproductive rate and overall productivity are very low which restrict the development of goose industry in China.However,Huoyan goose as the fine high-yielding varieties and the goose egg production in the world’s first,geese in the "national treasure" reputation,is ideal for cultivation for scientific research and breeding geese goose.Huoyan goose is a local breed,with a high egg production,strong reproductive performance,and resistance to crude feeding,cold resistance and other good traits.In recent years,with the increase of the number of introduced geese,the expansion of the other geese and hybridization,and to change gradually Huoyan goose its excellent characteristics,especially in high yield and high egg reproduction,resulting in raising economic benefits decline.Therefore,it is very important to use molecular biology methods to screen and identify the functional proteins related to egg laying performance,and to improve the egg laying performance by molecular breeding.In this study,we used Liaoning Huoyan goose as research object,to screen the differently expressed proteins in hypothalamus between pre-laying and laying period by iTRAQ,then to analyze these proteins using bioinformatics tools.Next,the expression of Complexin 1 and EAAT2 during pre-laying and laying period was detected using western blotting.At last,the prokaryotic expression vector of Complexin 1 and EAAT2 was constructed,and the expression of these two proteins was detected.Results are listed as follows:(1)34 differentially expressed proteins were screened,which include 18 up-regulated and 16 down-regulated.These proteins were mainly involved gonadotropin-releasing hormone,steroid hormones,progesterone synthesis and other physiological processes.(2)The Complexin 1 and EAAT2 proteins were detected in the hypothalamus of Huoyan geese,and the molecular weight of the protein was 15kDa and 60kDa.The protein expression of Complexin 1 and EAAT2 increased in the period from pre-laying to egg laying,and the egg laying period was significantly higher than that pre-laying.(3)The ORF coding region of the two genes of Complexin 1 and EAAT2 was obtained successfully,and the fragment sizes were 399 bp and 500 bp,respectively.(4)PMD-18T-Complexin 1 and pMD-18T-EAAT2,and prokaryotic expression vector pET28a-Complexin 1 and pET28a-EAAT2 were successfully constructed.Complexin 1 protein and EAAT2 protein were successfully expressed in E.coli.This study shows that the different expression proteins Complexin 1 and EAAT2 were successfully identified by iTRAQ technique and Western Blotting technology,which has certain relevance between the protein expression level and laying performance,in which the protein expression level of Complexin 1 and EAAT2 were up-regulated in egg laying period.These results lay the foundation for the further study of Huoyan goose molecular breeding work.