| The long non-coding RNA(lncRNA)is a non-translated RNA longer than 200 bp and is widely expressed in different tissues of mammals and birds,in which they are involved in various biological processes,such as cell proliferation,differentiation,chromatin remodeling,epigenetic regulation,genomic slicing,transcription,and translation and other key biological processes.Recently,studies have shown that lncRNA plays an important role of regulation on the reproduction of mammal and plants,and little information about lncRNA regulation on duck egg production related genes was reported.Duck as an oviparous,egg production related genes not only affects the reproductive performance of duck,but also have great effect on egg performance.Ovalbumin(OVL)is one of the main components of eggs and deciding the egg quality,and it also is the key gene to the research of poultry bioreactors.So both the mechanism of regulation lncRNA on egg production related genes and polymophsim issignifanceto poultry breeding of egg performance.The research contents transcriptome sequencing of ovary from different phrase ducks,lncRNA,coding genes identification and interaction analyze,small interfering RNA(siRNA)regulation on specific lncRNA expression,qPCR for lncRNA and coding genes and their association with egg performance in first laying duck,egg white analysis via protein mass spectrometry and their polymorphism among duck with other birds.This main obtains of this study showed as the follow:1.By transcriptome sequencing and sequencing analysis,15011 coding genes and 2905 lncRNA genes were identified in total from young duck(60 d),first-laying duck(160 d)and old ducks’(490 d)ovary.A total of 2240 differentially expressed genes and 135 different lncRNAs were identified with P<0.05.Among them,599 divergent transcription genes and 14 divergently expressed lncRNAs were identified between youth duck and first-laying ducks,and 518 divergent transcription genes and 93 divergently lncRNAs were identified in comparison of first-laying ducks and old ducks.Between old ducks and youth ducks existed 2004 and 71 divergently genes for coding genes and lncRNAs respectively.2.The clustering analysis were conducted between the divergently expressed genes in each two group of young duck,first-laying duckand old ducks.The results showed that divergently coding genes from each two group were clustered into three class with greater than 60% fitness,and lncRNAs clustered into four class with greater than 70% fitness.Based on the pattern of lncRNA and reproductive genes expression in three phase,there are similar expression pattern were found in duckovarian,such as follicle stimulating hormone(FSH).3.Based on the target for each lncRNAs in trans-acting and cis-acting patterns using correlation and binding free energy to predict the interaction between lncRNAs and coding genes.The results showed that 656 lncRNAs were predicted with trans function on 2949 mRNAs with correlated coefficient more than 0.90.Moreover,six lncRNAs were identified with cis function.The study screened the differentially expressed trans-acting and identified 52 lncRNAs strongly associated with 38 genes related to reproduction which were enriched to steroid hormone biosynthesis.4.The siRNA was designed based on sequence of the lnc-2531 which can regulate the FSH expression,and packaged into lentivirus and then injected into the abdominal cavity near the ovaries of peak-laying period ducks.After two week of treated,there was a significant decrease in the amount of laying eggs compared with the control group(P<0.05).The quantitative analysis of lnc-2531 and FSH-β expression in ducks’ follicle after two weeks of treated showed that the relative expression level of lnc-2531 or FSH-β was significantly decreased in the treated group(P<0.05),and the positive associated relationship was confirmed(P<0.05).There was also a significant association between the expression level of both with the amount of egg laying in the two week(P<0.05).5.By comparison duck egg white components with geese,chicken and pigeon,it was found that there is a high degree of similarity between duck and geese,especially the specific peptides of OVL gene expression products.In addition,the OVL gene transcriptional regulated region of duck,goose and chicken were aligned and showed ducks and geese had a high similarity,and found the diversity of the regulatory region in the ovalbumin gene is responsible for the higher expression of ovalbumin in the duck than in the chicken.All these results indicated that the lncRNA,which regulates duck FSH,can effect on duck reproduction trait via effect the expression level of FSH.The pattern of divergent lncRNA and mRNA,and the interaction net of lncRNA and divergent reproductive related mRNA were discovered.In addition,the lncRNA regulation on FSH was confirmed that supported the potential ability of lncRNA to be a molecular markers for selection a high-yielding ducks breeding or high-yielding ducks population.The polymorphism of the duck egg and ovalbumin gene regulatory regions with other poultry provided a basic structural support for the construction of a duck ovalbumin expression system as a bioreactor. |
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