Construction Of Blood Expression Profile And Effects Of Stocking Density On Expression Levels Of β-defensin Genes In Chinese Alligator

Chinese alligator(Alligator sinensis)as a kind of crocodilian endemic to China,has been threaten by over-hunting and habitat reduction in the past one hundred years and is still in an extremely endangered state.In the late1970 s,Chinese government established reserves and carried out captive breeding projects to protect Chinese alligator from extinction.After forty years of protection,the number of captive populations of Chinese alligator has been increasing,which provides abundant resources for the reconstruction and rejuvenation of wild populations.The key to success of reintroduction project is to ensure the adaptation potential of captive individuals to the wild environment.However,the study found that long-term captive has reduced adaptability of captive animals to wild environment;and high stocking density will adversely affect animal behavior,physiology and immune function.Therefore,it is necessary to carry out research on the physiology and immunity of captive populations of Chinese alligator,which can provide more information for protection and reintroduction of Chinese alligator.In this study,we took seven-year-old Chinese alligator population in Changxing Chinese Alligator Nature Reserve as research object,and studied the defensin,an important component of immune system.First,reverse transcription quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of 9 β-defensin genes in blood samples of 41 seven-year-old Chinese alligators,and the blood expression profile of β-defensin genes was obtained.After that,to explored the effect of stocking density on immune function of Chinese alligator captive populations,the difference of defensin expression levels between populations under different sexes and stocking densities were analyzed.Finally,to explore the regulatory mechanism of defensin genes at the epigenetic level,AsBD8 gene with great expression differences between individuals was chose for promoter DNA methylation analysis.The methylation level of CpG sites in AsBD8 promoter of two groups which great difference existed among individuals in defensin expression level were detected by bisulfite sequencing PCR(BSP)and its correlation with expression variations among individuals were analyzed.The main results are as follows:(1)Nine pairs of qPCR primers for β-defensin genes were screened,and blood expression profiles of β-defensin gene family of Chinese alligator were obtained.(2)The β-defensin genes AsBD5 and AsBD8 were expressed in blood of Chinese alligator population,and there was a significant positive correlation between the expression patterns.(3)There were significant differences between two populations under different stocking densities.The defensin expression level of Chinese alligator population with higher stocking density was significantly higher,indicate that there are some effects of stocking density on the immune status of Chinese alligator captive population.(4)There was no significant difference of methylation level of every detected CpG sites in AsBD8 promoter between two groups,indicate that the regulation of AsBD8 expression levels among different individuals has no relationship with its promoter DNA methylation.This study provides the theory foundation for go further research on functions and expression regulation mechanisms of β-defensin gene in Chinese alligator,and provides new clues for the protection and population management of Chinese alligator.