Computational protocol: Cloning, Characterization and Effect of TmPGRP-LE Gene Silencing on Survival of Tenebrio Molitor against Listeria monocytogenes Infection

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Protocol publication

[…] Total RNA from T. molitor larvae (n = 200) was isolated by TRIzol reagent (Molecular Research Centre, Inc., Cincinnati, OH, USA) after homogenization using TissueLyser (Qiagen, Valencia, CA, USA) and subsequently mRNA was purified using Absolutely mRNA Purification Kit (Stratagene, Santa Clara, CA, USA). The cDNA library was synthesized using Express cDNA Synthesis Kit (Stratagene, Santa Clara, CA, USA). The cDNAs of more than 500 bp in length were ligated into pBK-CMV vector (Agilent Technologies, Inc., Santa Clara, CA, USA) and packaged using the ZAP expression cDNA Gigapack® III Gold cloning kit (Stratagene, Santa Clara, CA, USA) according to manufacturer’s instructions. cDNA library clones (~2000) were cultured in Terrific broth (TB) medium containing kanamycin (50 mg/mL) at 37 °C for 15 h. Plasmid DNAs extracted from the selected colonies were sequenced by ABI 3730 XL capillary sequencer (Applied Biosystems, Foster City, CA, USA). Clones corresponding to the partial sequence of PGRP-LE (TmPGRP-LE) were identified, by conducting BLASTX with Swissprot analysis (EMBL-EBI, Hinxton, Cambridge, UK). By using the plasmid DNA as templates, full-length sequencing was performed by a clone-by-clone primer walking method using a Model 3730 XL sequence analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing reaction was performed using the BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequences were assembled using the Phrap program (University of Washington, Seattle, WA, USA) []. Based on the assembled sequences, primers corresponding to the terminal sequences were designed using Primer3 (version 0.4.0, Whitehead Institute, Cambridge, MA, USA) []. The primer walking procedure was repeated until poly (A) tail region for the sequence was identified. The full-length sequences were finished by trimming the vector-derived sequences from both ends using the cross match program. The consensus full-length cDNA sequence of TmPGRP-LE obtained from 200 T. molitor larvae has been registered with the European Nucleotide Archive-European Bioinformatics Institute (ENA-EBI, Hinxton, Cambridge, UK) with the accession number HF935084. […]

Pipeline specifications

Software tools BLASTX, Primer3
Databases ENA Full Length cDNA
Application qPCR
Organisms Listeria monocytogenes, Tenebrio molitor
Diseases Infection, Listeriosis
Chemicals Diaminopimelic Acid