Identification, Expression And Single Nucleotide Polymorphisms Of SpToll Gene From Green Mud Crab, Scylla Paramamosa

Toll-like receptors (TLRs) is one of the most important pattern recognition receptors (PRRs),playing a crucial role in innate immune responses that eradicate invading pathogens and bridging innate and adaptive immunity. Nowadays, totally 9 kinds of Toll protein have been found in Drosophila pseudoobscura and 13 kinds of TLRs have been found in mammalian. Nevertheless, whether the TLRs existed in green mud crab is still unknown. TLRs from Scylla paramamosa (SpToll) has been deeply studied using molecular biotechnologies and immunolgic technologies in this research. Details of the result were listed unther.1. Cloning and bioinformation analysises of SpToll.SpToll gene was found in Scylla paramamosa using general primers PCR and rapid ampilification of cDNA ends (RACE) technique. The full-length cDNA of SpToll is 3843 bp with an open reading frame of 3018 bp, which encodes a protein of 1006 amino acids. SpToll is a typical single-pass transmembrane protein containing characteristic domain architecture of Toll and Toll-like receptors. SpToll gene showed high similarity to MjToll gene, LvToll gene and DmToll gene with 43%, 39% and 32% identity respectively and all of them were found in crustacean. It consists of an extracellular domain, which is composed of several leucine-rich repeats (LRRs), followed by a transmembrane segment, and a cytoplasmic Toll/Interleukin-1R (TIR) domain. 14 potential N-linked glycosylation sites were identi?ed in the SpToll that may in?uence the surface pattern recognition of the receptor. In this study, we confirm that SpToll existes in Scylla paramamosa, which could help to understand the immunity mechanism of crustacean more deeply.2. Expression of SpToll and Studies of PAMPs recognized by SpToll.We found that SpToll was widely expressed in many tissues including heart, gill, hepatopancreas, stomach, intestine, muscle, eyes and hemocyte. Real-time PCR analysis demonstrated that expression patterns of SpToll were distinctly modulated after bacterial or LPS stimulation, with significant enhancement after 48 hours post-V.Parahemolyticus challenge but reduced levels markedly and immediately after LPS stimulation. However, there was no significant change after Poly I: C stimulation. These results suggested that SpToll may be involved in innate host defense, especially against bacterial invadsion, but not viral invdsion.3. Single nucleotide polymorphisms of LRRs in SpToll.Futher more, many SNP sites from LRRs region of SpToll were indentified in this study. The results showed that each crab have average 55 SNP sites in DNA level and 61 in cDNA level. The frequency of these SNPs was relatively low, approximately 1% to 4% (based on sequenced clones counting for a DNA/cDNA library), except one site 1847 of the SpToll cDNA sequence, with the mutant frequency of 50% puls. The c.1847A>G mutation results in a lysine to glycine substitution at amino acid position 450 (Lys450Glu). It anchored the SpToll insertion in LRR13, residues between 438 and 451, leading changed from positive charge to negative charge at residues 450 and then leading secondary structure change of polypeptide, which may finally influence the ability of SpToll binding PAMPs.One step further, PCR product sequencing technique was used to study the distribution of each gene types of c.1847A>G in a crab population. Three gene types were found: A/A, A/G and G/G, with there frequencies of 55%, 33% and 12%, respectively. Allele frequencies of A/G were 0.7143/0.2857.The chi-square test showed this population was at Hardy-Weinberg disequilibration (P<0.05). The genetic diversity indexes of H/PIC were 0.5918/0.3249 in the green mud crab population indicating that the locus was moderate polymorphic. It suggested that the 1847 SNP sites could be used as a new molecular genetic marker for crabs'anti-disease breeding.In this study,we mainly found that there existed in Scylla paramamosain a SpToll gene ,a Toll receptor-homologous gene,which possibly participated in immune responses by primarily recognizing and resisting invaders,like pathogenicbacteria.These results were helpful to illuminate the defense mechanism in Scylla paramamosain and the coevolution relationship between the low frequency mutations in PRRs and the high frequency mutations in PAMPs ,which thus played a significant role for their potential theoretical and practical use in the immune control of Scylla paramamosain as well as the selection of excellent disease-resistant Scylla paramamosain variety.