Transcriptome Analysis Of Taenia Saginata And The Research On Acetylcholinesterase Gene

Taenia saginata is an important zoonotic tapeworm. It is an intestinal parasite in humans causing taeniasis and cysticercosis in cattle. According to statistic, there are approximately 50 million humans infected with this parasite worldwide, it is relatively common in Africa, Middle East, Asia. Europe. Adult T. saginata and cysticercus T. saginata cause significant health problems to their hosts and huge socioeconomic losses. However, in the absence of complete genomic data on this species, new drugs development and scientific research work are limited. Here, we performed the first investigation of the transcriptome of the larval cysticercus form of this parasite using next-generation sequencing(NGS) technology. Meanwhile, a lot of related study based on transcriptome database were done.1. De novo transcriptome assembly by Illumina sequencing was performed, generating 52,829,606 high-quality reads that were assembled into 91,487 unigenes with an average sequence length of 2,122 base pairs. Based on a sequence similarity search against the seven public databases(Nr, Swiss-prot, GO, COG, KEGG) with a cut-off E-value of 10-5 using BLASTX, a total of 59,262(~64.77%) unigenes were annotated with gene descriptions, gene ontology terms, conserved protein domains, or metabolic pathways. In addition, a larger number of molecular markers were identified, including 12,902 single-nucleotide polymorphisms(SNPs) and 10,017 simple sequence repeats(SSRs).2. A comparative analysis was performed to investigate the characteristics of codon bias and influencing factors of the codon usage patterns among six tapeworm species(Taenia solium, Taenia saginata,Taenia asiatica, Hymenolepis microstoma, Echinococcus multilocularis and Echinococcus granulosus). The results indicated that the five species(excluding H.microstoma) had similar codon usage patterns, showing a strong bias towards a high representation of NNG and NNC codons. In contrast to this, H. microstoma tended to use A or T in the third position. The results of Neutrality analysis, Parity Rule 2(PR2) plot analysis and ENc-plot analysis suggested that both mutational and selection forces contributed to codon usage bias in six tapeworm species. Meanwhile, other factors such as protein length, gene expression, GC content of genes, the hydropathicity of each protein also influence codon usage. We also identified the optimal codons in every tapeworm.3. Two ACh E genes were cloned and a recombinant Pichia pastoris strain expression Taenia saginata ACh E were constructed. Bioinformatics analysis showed that Taenia saginata ACh Es shared more than 80% identity with Taenia solium ACh E, however, they only showed 37% identity with Human ACh E. The results of real-time quantitative PCR revealed that the expression level of ACh E1 and ACh E2 in the adult were higher than that in the larval stage. The results of immunochemistry demonstrated that ACh E1 and ACh E2 were predominantly located in the ovarian of gravid proglottids of Taenia saginata. Analysis of enzyme activity suggested that the activity of ACh E1 and ACh E2 were highest in solution at p H 7.4 and 37°C.4. Virtual screening method was used to find Taenia saginata ACh E inhibitors. The homology model of ACh E1 and ACh E2 were first built based on the crystal structures of the templates 3ZV7. Then, the model ACh E1 and ACh E2 were validated and used in docking-based virtual screening studies against Specs commercial compounds database. Finally, 15 molecules(for each ACh E) with potential ACh E inhibitory activity were obtained. Hydrogen-bonding and hydrophobic interactions were observed in the binding mode between these compounds and ACh E.