Cloning And Functional Analysis Of Several Immune-related Genes Of Chinese Soft-shelled Turtle Pelodiscus Sinensis

Chinese soft-shell turtle Pelodiscus sinensis is an ancient ectothermic amniotes reptile with an evolutionary link between ectothermic anamniotic fishes and amphibians and endothermic amniotic birds and mammals. It is not only of great importance in life science research and protection of wildlife resources, but also of edible and medicinal values. It has become one of the major farmed species in China and southeastern Asian countries. However, deterioration of the breeding stock, severe infectious diseases and lack of basic research on its biology has greatly affected the sustainability of the turtle farming industry. This dissertation covers a series of experiments for cloning and functional studies of some immune-related genes in an attempt to explore the primary mechanisms of its innate immunity and responses to heat stress and pathogen associates molecular patterns (PAMP). The results could serve as good foundations for further research in immunobiological and phylogenetic aspects of turtles as well as in host-microbe interactions.1. Cloning and expression profiling of full-length tMyD88cDNA from Chinese soft-shelled turtle.Myeloid differentiation factor88(MyD88) is one of the key adaptor proteins to signal transduction that triggers downstream cascades involved in innate immunity. This study identified the MyD88gene from Chinese soft-shelled turtle (tMyD88), representing the first example from reptile species. The tMyD88has an894-bp ORF and encodes a polypeptide of297amino acids including a typical death domain (DD) at the N-terminus and a conservative Toll/IL-1R (TIR) domain at the C-terminus. Its mRNA expression in organs of turtles challenged with live cells of Aeromonas hydrophila were in the order from high to low levels as spleen, blood, lungs, liver, kidneys and intestines, as determined by real-time PCR. RAW264.7macrophage cells transfected with pcDNA-tMyD88showed higher NF-κB activity than the vector control (673.8vs410.7. P<0.05). Expression of proinflammatory cytokines IL-1(3and TNF-a was also significantly higher in RAW264.7cells expressing tMyD88than the cells containing control vector (P<0.01). These results indicate that tMyD88might possess an important role in defense against microbial infection in Chinese soft-shelled turtles similar to that in mammals.2. Identification of tHsc70, tGRP78and tHsp72in Chinese soft-shelled turtle and their expression in response to heat stressThe70kDa heat shock proteins (Hsp70s) are molecular chaperones that are not only involved in protein folding and transport, but also in initiating host immune responses. In this study, Hsp70s full-length cDNA molecules from P. sinensis were cloned and identified. The turtle Hsc70(heat shock cognate protein70. tHsc70) has a1941bp ORF encoding646amino acids and has identities to Hsc70of other vertebrates at80-87.7%(nucleotide) and95.5-99.5%(amino acid). Turtle GRP78(glucose regulated protein78, tGRP78) has an ORF of1980bp encoding660amino acids with nucleotide and amino acid identities at79.3-80.3%and92.3-94%respectively to that of other vertebrates. Turtle Hsp72(inducible heat shock protein70. tHsp72) has an ORF of1908bp coding for636amino acids and possess identities to Hscp72of other vertebrates at72-76.5%(nucleotide) and83.6-89.1%(amino acid). Two major domains for binding to ATPase (ABD) and to substrates (SBD), an ATP/GTP-binding motif (AEAYLGRKK), three signature regions characteristic of Hsp70s (IDLGTTYS, IFDLGGGTFDVSIL,1VLVGGSTRIPKIQ) and consensus plasmic(EEVD)/endoreticulum(KDEL) sequences are present in the deduced amino acids of these three molecules. The prokaryotic expression products of tHsc70and tHsp72reacted with mouse monoclonal (Hsc70) and polyclonal rabbit antibodies against human recombinant Hsp72. respectively, as shown by Western-blotting. Phylogenetically. these molecules fall into the same group to Alligator mississippiensis and Gallus gallus. There were differences of transcription of tHsc70, tGRP78and tHsp72mRNA of4tissues/organs from turtles stressed at40℃as compared to25℃. Heat stress for1to4hrs induced0.25to20.35-fold increase of tHsc70mRNA in live, hear, lung and muscles with its expression in liver significantly higher (20.35-fold, P<0.01) with4-hrs stress than control treatment at25℃. Heat stress for4hours also showed1.86-fold increase (P<0.05) in tGRP78expression as compared to the control. tHsp72showed the highest expression in liver.4.66-fold higher than control (P<0.05) at hour2after treatment and declined thereafter. Western blot analysis indicated that tHsc70expression was significantly higher (P<0.05) than the control treatment in liver of turtles heat stressed for1,2and4hrs with1-h adaptive recovery. Heat stress for1h,2hrs and4hrs induced higher expression of tGRP78in liver than the controls (P<0.05). In summary, we have successfully cloned three Hsp70family proteins from Chinese soft-shelled turtles. They are relatively conserved molecules as compared with those of other vertebrates and functional to heat stress though the expression profiles varied with tissues of turtles examined with highest response in liver.3. Effects of representative pathogen associated molecular patterns on expression of Toll-like receptors and immune-related genes.Partial cDNA fragments of tlr2(1112bp), tlr3(565bp), tlr4(990bp) and il-1β (159bp) were PCR-amplified and sequenced. Sequence comparison indicates that turtle TLR2has nucleotide identity of60.8-74.4%and amino acid similarity of58.6-76%to that of other vertebrates such as zebrafish and chicken. Domain analysis reveals that the deduced amino acid sequence of turtle TLR2contains a leucine rich repeat C-terminal domain, LRRCT)(aal-47), a transmembrane domain (aa49-71) and a Toll-like/IL-1receptor domain, TIR)(aa101-244). Turtle TLR3is homologous to the counterpart of other vertebrates including zebrafish and chicken between58-81.2%at the nucleotide level and51.9-76.1%at the amino acid level. It contains a transmembrane domain (aa10-32) and a TIR domain (aa62-188). The homology between turtle TLR4and those from other vertebrates is between48.5-67.4%and38.2-63%at the nucleotide and amino acid levels respectively. Deduced amino acid sequence of turtle tlr4contains three LRR (aa23-46,47-70and71-94), an LRRCT (aa107-157), a transmembrane domain (aal71-193) and a TIR domain (aa206-330). The putative turtle IL-1β has a high identity to the chicken counterpart (aa66-118)(60%at the amino acid level) and is homologous to those of other vertebrates between49.7-61.4%and28.3-49.1%at the nucleotide and amino acid levels respectively. Representative PAMPs were used to examine the response of turtle whole blood cells to stimulation. Turtle tlr2was found to have higher expression in the blood cells exposed to4000ng/ml of zymosan for2hrs. Expression of il-1β increased with increasing concentration of zymosan treatment. The mRNA expression of tlr3exhibited a tendency of decrease with increasing levels of Poly(I:C) between40-8000ng/ml). In whole blood incubated at28℃, tHsp72mRNA expression increased at both lower (40ng/ml) and higher (4000ng/ml) concentrations. mRNA expression of tlr4, tMyD88and il-1β increased with increasing concentrations. Expression of tlr3mRNA increased by1.46-fold in Poly(1:C) treated whole blood cells incubated at37℃, as compared with the same treatment but incubated at28℃. These results provide preliminary evidence that the TLRs, tMyD88, tHsp70s and il-1β in Chinese soft-shelled turtle could respond to PAMP molecules to some degree, though with varying intensity.In summary, this study has generated for the first time the full-length cDNA sequence data of tMyD88, tHsc70, tGRP78and tHsp72as well as partial cDNA of tlr2, tlr3, tlr4and il-1β from Chinese soft-shelled turtle. These molecules are clustered with those from avian species and reptiles as the sister group, but fall into a different subgroup with those of mammals, amphibians and fish. Majority of these molecules responded to stimulation with PAMPs or heat stress and the degree and profile of the responses vary with stimuli or their concentration, indicating that they possess biological functions similar to those from other vertebrates. Findings in the present study may provide good foundation for further research into the structure-function relationship of these molecules, mechanisms of innate immunity in turtles and its role from the evolutionary perspective.