ADNA Cloning Of Il6 From Chinese Soft-shelled Turtle And Its Expression Partern In Acute Cold Stress And Bacterial Immune Response

In this paper we divided the Chinese soft-shelled turtle's intestine at first.The nine segments of turtle were studied by morphological and histological methods.Then nine genes which had been reported in the intestine of mammals and fish were used to distinguish the segments of the Chinese soft-shelled turtle.Secondly,in order to study the immune function of the intestine,we focused on the interleukin 6.After the full-length c DNA of the turtle il6 gene were cloned,the bioinformatics analysis was carried out: promoter analysis,gene structure,gene synteny,similarity and identity analysis,phylogenetic tree and protein structure prediction.The distribution of il6 in tissues was analyzed by q PCR.Finally,in order to investigate the effect of cold stress on il6 function.We carried the experiment in two temperatures(25°C,15°C)and two kinds of bacteria were used for stimulation.The expression of il6 in spleen and intestine were detected at 5 time points(6h,12 h,24h,72 h and 7d)after bacterial infection.We used stimulants(LPS,Con A and Poly I:C)to stimulate the primary cells of the spleen,to further detect the expression of il6 in vitro.The results shown that:1.Combining traditional observation with the molecular method to divide the Chinese soft-shelled turtle intestineBy using several maker genes,we would suggest a more precise segmentation of turtle intestine,which is slight different from the traditional studies.The Region covering from S1 to S3(29.9% of the total length from the beginning of the intestine,29.9%)is equivalent to mammalian duodenum.And those covering S4(40.2%)and S5(65.4%),S8(92.7%)and S9 are equivalent to mammalian ileum and large intestine,respectively.As for S6(81.3%)and S7(87.3%),its functional maybe variable and depends on the used marker genes.They may be either larger intestine or the transition part from small intestine to larger intestine.2.Cloning and tissues specific expression analysis of il6 in Chinese soft-shelled turtlePromoter prediction showed that the promoter of turtle il6 contained conserved transcription factor binding sites.The full length of il6 c DNA of Chinese soft-shelled turtle is 2069 bp with a 663 bp ORF.And the length of il6 ns is 2194 bp with a 615 bp ORF.The difference between the two genes is in the signal peptide.There is a conserved gene location relationship between turtle and birds or mammals.The similarity and identity is high to that of birds.As to the gene structure,the turtle il6 is similar to mammals and fish,while il6 ns to the birds.Secondary structure consists of alpha-helix,extension strand,random coil and beta-turn.The result of tertiary structure analysis showed that the protein contains the IL-6 family signature(C–X(9)–C–X(6)–G–L–X(2)–Y–X(3)–L).The sequence contains the polyadenylation sequence in the end.Phylogenetic analysis demonstrated that the reptile il6 clustered firstly with birds,secondly with mammals,and finally with fish.Meantime,we use the human IL6 as a model to construct the 3D structure of turtle IL6.The result of tissues distribution analysis showed that the two il6 genes expression in vivo is different.3.Effects of cold stress on intestinal immune factor-interleukin-6 in Chinese soft-shelled turtleAfter A.hydrophila or S.aureus infection,the expression of il6/il6 ns increased at first and then decreased during the infection course,and the expression patterns of these genes showed the "delayed response" and "enhanced expression" in the cold stressed groups.In addition,we found that the response of il6 was stronger than il6 ns after the infection.In vitro,we isolated the primary cells from the spleen,the expression of il6/il6 ns in primary cells was significantly up-regulated(P<0.01)at 6h under the stimulation of Poly I:C,and decreased at 24 h and 48 h.But under the stimulation of Con A or LPS,the expression of il6/il6 ns was not significantly changed.Conclusions:1.By using several biomarker genes homologous to mammalian and zebrafish.We suggested a more precise segmentation of turtle intestine.2.We cloned two il6 c DNAs from Chinese soft-shelled turtle.Bioinformatics analysis was carried out and tissues specific expression of the two transcripts was observed.3.Cold stress affected the immune response of turtle il6 to the bacteria,especially in the intestine.We found the two il6 transcripts have a different expression pattern,indicating that they may have different functions.