Gene Expression Profile And DNA Methylation Of ?-defensins In Crested Ibis Population In Zhejiang Province

The crested ibis(Nipponia nippon)is a class I protected bird in China,and it is an endangered species all over the world.At present,it mainly exists in part of China,Japan and South Korea in the form of artificial captive population.Despite the increasing number of the crested ibis,the factors of scattered population distribution,low fertilization rate in artificial breeding and low survival rate of nestlings still exist,which restricts the reintroduction of the crested ibis.Therefore,in order to better guide the captive breeding of the crested ibis,it is necessary to do further research on immunity and breed of the crested ibis from the molecular level.?-defensins is a kind of important immune effector molecule,which is of great significance for organisms to resist bacteria and adapt to the environment.However,there is no study on the expression of ?-defensin genes in the crested ibis population.Therefore,the study on the expression and its regulation of ?-defensin genes of the crested ibis is necessary,which is helpful to clarify the immune function of ?-defensins in the crested ibis population.In this study,we selected 74 crested ibis of diffferent ages as object from the crested ibis-breeding base in Xiazhu Lake,Deqing County,Zhejiang Province and their peripheral blood was taken as the research sample.Firstly,we used real-time fluorescent quantitative PCR to detect the blood expression level of 11 ?-defensin genes in the crested ibis population.After that,we analyzed the correlation between the expression level of different ?-defensin genes and further analyzed the expression difference of the crested ibis of different gender and ages.Secondly,based on the data of paternity testing and gene expression level,the correlation between the expression level of ?-defensin genes of parents and offsprings was analyzed.Finally,the CpG island in promoter of AvBD2 gene of the crested ibis was predicted,and the methylation level of 37 CpG sites in the promoter and gene body regions of AvBD2 gene in 10 individuls were detected by bisulfite sequencing,and the methylation level of AvBD2 gene in crested ibis at different gene expression level was preliminarily analyzed.The following are the main results of this study:(1)Five pairs of specific primers for ?-defensin genes were obtained and a realtime fluorescence quantitative PCR optimization system was established to detect the relative expression level of ?-defensin genes in the crested ibis.The expression profile of ?-defensin genes in the blood of the crested ibis population in Zhejiang Province was successfully constructed based on real-time fluorescence quantitative PCR.(2)The expression characteristics of ?-defensin genes in the blood of the crested ibis population in Zhejiang Province were elucidated: AvBD1?,AvBD2,AvBD3,AvBD4 and AvBD7 were widely expressed in the blood of the crested ibis population,and the proportion of individuals expressing the genes was 89.2%,100.0%,100.0%,100.0% and 83.8%.AvBD5(V5),AvBD9 and AvBD14 were not expressed in the blood of most individuals,and the proportion of individuals who did not express the genes was 95.9%,73% and 62.2%.The expression of AvBD8,AvBD10 and AvBD13 was tissue-specific,not expressed in the blood of all individuals while widely expressed in other tissues of the crested ibis.(3)For the five ?-defensin genes widely expressed in the crested ibis population,the relative expression level from high to low is AvBD3 > AvBD2 > AvBD4 > AvBD1? > AvBD7.The relative expression level of AvBD3,AvBD2 and AvBD4 was significantly higher than AvBD1? and AvBD7 in the crested ibis population,indicating AvBD3,AvBD2 and AvBD4 genes may paly an important immune role in the captive population of the crested ibis.(4)The relative expression level of AvBD1?,AvBD2,AvBD3,AvBD4 and AvBD7 genes in the crested ibis population showed a significantly positive correlation among each other,which revealed that the coevolution of these five contiguous genes in the gene cluster might occur.(5)The expression level of ?-defensin genes in different individuals of crested ibis population was quite different,which showed that the immune capacity of different individuals was different.Among them,AvBD8,AvBD10 and AvBD13 were not expressed in crested ibis blood,but they were widely expressed in other tissues,which showed that their expression was tissue-specific.(6)The relative expression level of AvBD1?,AvBD2,AvBD3,AvBD4 and AvBD7 genes in the blood of birdlings of the crested ibis was higher than that of sub-adults and adults,and among which,the relative expression level of AvBD2 gene was significantly different in the crested ibis of different ages.These results showed that ?-defensins play an important role in the process of growth and development and environment adaption of crested ibis birdlings.(7)The expression of AvBD1?,AvBD2,AvBD3,AvBD4 and AvBD7 genes in the blood of crested ibis sub-adults was at the lowest level in the population,which indicates that sub-adults have a lower immune capacity and need more nutrition and attention.In addition,we found no significant difference in ?-defensin genes expression level between female and male crested ibis.(8)There was a moderate to high positive correlation between ?-defensin genes expression level of parents and offsprings individuals,and the correlation of ?-defensin genes expression level between some parents crested ibis and offsprings crested ibis reached a significant level.(9)The 2000 bp promoter region of AvBD2 gene of the crested ibis did not contain CpG island.Varying degrees of methylation occurred in 9 CpG sites in promoter region and 28 CpG sites in gene body region in AvBD2 gene of different individuals of the crested ibis.The average methylation level of CpG sites was 59.2% in promoter and 75.4% in gene body.(10)The high level of methylation at CpG8 site in the promoter region of AvBD2 gene in the crested ibis might down-regulate the gene expression in blood,while methylation at different CpG sites in the gene body region might have different regulatory effects on gene expression,suggesting the complexity of the regulation of DNA methylation on AvBD2 gene expression.This study provides theoretical reference and basis for further study on the expression and function of ?-defensins in the crested ibis,and has important guiding significance for the actual protection work in artificial breeding of the crested ibis.