Expression Patterns Of β-defensins In Pelodiscus Sinensis

Aiming at exploring the expression pattern,location of β-defensin gene and their seasonal expresion in intestine in Pelodiscus sinensis,this study minined multiple gene sequences annotated as β-defensin from previous transcriptome sequencing results in the laboratory.Subseuqent bioinformatic anaysis,namely phylogenetic relathionship analysis,with β-defensin genes in some closely reated species and P.sinensis,at the protein level sugested that they are the homologus molecues of β-defensin in P.sinensis.In the following study,semi-quantitative PCR was used to analyze its expression patterns in selected 9 tissues.Moreover,the seasonal expression pattern of the selected intestinalβ-defensin gene in S5 and S8 segment,representing the small intestine and large intestine,respectively,were evaluated by q PCR methods.Finally,with in situ hybridization technology based on mRNA probe,the expression of Chinese soft-turtle β-defensin 1 gene(PsBD1)and β-defensin 2 gene(PsBD2)in S5 segment were located.The results of these studies are as the following:1.The results of phylogenetic tree constructionAfter using Multiple Sequence Alignment by CLUSTALW online tool,the predicted Chinese soft-shelled turtle β-defensin molecues were compared with the discovered crocodile and some Chinese soft-turtle homologes,and the phylogenetic tree was constructed using the neighbor-joining method in MEGA11.0.13 software.The results of revealed that thoes moleuces identified have a close evolutionary relationship with the crocodile and Chinese soft-shelled turtle β-defensins,which implied that they are highly likely to be β-defensin in Chinese soft-shelled turtle.2.Expression pattern of β-defensin genes in selected tissues with Semi-quantitative PCRThe results showed that β-defensin genes were widely expressed in lung,skin,plastron and carapace.No less than 10 β-defensin genes were found to express in each tissue.In spleen,heart,fat,brain and liver,less were found to express.A total of 21β-defensin genes were detected in the whole intestinal tissue,with an obviously distict expression pattern in different segments of the intestine.Surprisingly,the expression level of a large number of β-defensin genes in S9(posterior end of the large intestine)was very high.3.Seasonal expression of selected β-defensin gene in Chinese soft-turtle S5 and S8.The results showed that there were seasonal expression changes in some β-defensin genes in S5 and S8 of Chinese soft-turtle.Generally,more β-defensin genes were found to significantly increase the expression in the summer(July),compared with other selected sampling time points.In S5,with 18 S r RNA as the internal reference gene,nine β-defensin genes were found with various gene expression level in different sampling time points,among which six genes,namely PsBD15,PsBD17,PsBD19,PsBD26,PsBD27 and PsBD35,showed significant increases in July,while two genes,namely PsBD1 and PsBD11,showed significantly elevated expression levels during hibernation(December),and PsBD8 expression levels were significantly enhanced in May.In S8,with rpl13α as the internal reference gene,six β-defensin genes were found to express diffrently in most seasons,among which five genes,namely PsBD11,PsBD16,PsBD26,PsBD32,PsBD35,showed significant increases at the mRNA level in July,while PsBD1 showed a significant increase in the expression in May.Unlike in S5,there was no β-defensin gene significantly elevated during hibernation in S8.4.Expression location of PsBD1 and PsBD2 in the S5 segment of the Chinese soft-turtle intestineThe results in S5 with in situ hybridization based on mRNA probe showed that PsBD1 and PsBD2 were mainly expressed in villi epithelial cells and microvascular system.Conclusion:1.The expression pattern in selected tissues showed that a large number of Chinese soft-shelled turtle β-defensin genes were expressed on the surfece exposed to the envirments such as the intestine,skin,plastron and carapace,which indicated that Chinese soft-turtle β-defensin gene may act as antibacterial agent against the invasion of enviromental bacteria.2.Real-time quantitative results showed that the β-defensin genes in S5 and S8 were expressed at various levels in different seasons,which may be associated with dynamic changes in the intestinal microecological community of Chinese soft-shelled turtle.3.In situ hybridization results showed that PsBD1 and PsBD2 in S5 were mainly expressed in small intestinal villi cells.