In silico prediction of commonly used reference genes in qPCR for Lissachatina fulica

Lissachatina fulica is the largest invasive agricultural pest with the highest prevalence in Asian countries. At the same time, L. fulica is one of the few animal species that can regenerate organs, including the nervous system and eyes. However, its genome is poorly studied, which restricts further molecular and genetic studies of this organism. At the same time, one of the most accessible methods to study genomes is qPCR, the accuracy of the results of which depend on the selected reference genes. Therefore, the aim of this study was to predict the sequences of some genes in L. fulica using genes that are most often used as reference genes for qPCR.

In searching for homologous genes in L. fulica, actin (Act), β-tubulin (Tubb), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were selected as commonly used reference genes in qPCR for molluscs. The corresponding amino acid sequences of their proteins were used for more accurate prediction. The amino acid sequences of ACT, TUBB, and GAPDH of the gastropod molluscs Aplysia californica, Pomacea canaliculata, and Biomphalaria glabrata as the closest related species to L. fulica were taken from the NCBI Protein database. Multiple sequence alignment was performed using Clustal Omega followed by refining the results by MUSCLE in Unipro UGENE (v. 45.0). HMM3 profiles were also constructed in Unipro UGENE and then used to search for homologous sequences in L. fulica (GigaDB repository) using HMMER (v. 3.3.2).

As a result, we found in L. fulica homologous sequences for GAPDH (Afu019240), TUBB (Afu012850), and ACT (Afu006397) and the corresponding mRNA of genes encoding these proteins. Thus, the first attempt was carried out to annotate the L. fulica genome in order to identify reference genes, which is a necessary element of any molecular and genetic study.

This research was supported by the IKBFU grant 434-К-23 “Priority 2030”.