Identification and immune-related analysis of SNPs in Litopenaeus vannamei Toll3 receptor

Abstract

Tolls and Toll-like receptors (TLRs), as innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens, play a critical role in antimicrobial immune responses. Here, we report on single nucleotide polymorphisms (SNPs) of Litopenaeus vannamei Toll3 (LvToll3). Multiple sequence alignment of the L. vannamei Toll3 Leucine rich repeat C-terminal domain (LvToll3-LRR-CT) with other L. vannamei Tolls LRR-CT domains showed 39.23% - 43.96% homology at the nucleic acid level and 20.31% - 30.00% identity at the amino acid level. Analysis of different shrimp tissues by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) revealed that LvToll3-LRR-CT had genetic polymorphisms at both the genomic deoxyribonucleic acid (gDNA) and complementary deoxyribonucleic acid (cDNA) levels. Further, high-throughput sequencing analysis confirmed the presence of 8 non-synonymous SNP (nsSNP) and 1 nsSNPs with frequency greater than 1% at the gDNA level, while 13 nsSNPs and 2 nsSNPs with frequency greater than 1% at the cDNA level. In silico analysis revealed that the α-helix secondary structure and tertiary structure of LvToll3 changed when 3 SNPs (C2039T, T2041C, T2228C) were mutated. Interestingly, 2 novel bands on PCR-DGGE, which were identified as 2 nsSNPs (C2140A, T2186A) were observed following challenge with Streptococcus iniae but not with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). Moreover, the secondary and tertiary structures of LvToll3 changed when the nsSNP T2186A was mutated. The present findings therefore provide novel insight into the molecular basis of shrimp innate immune response to pathogens through the generation of specific SNPs.