Radopholus similis is an important parasitic nematode of plants. Serine carboxypeptidases (SCPs) are peptidases that hydrolyse peptides and proteins and play critical roles in the development, invasion, and pathogenesis of certain parasitic nematodes and other animal pathogens. In this study, we obtained the full-length sequence of the SCP gene from R. similis (Rs-scp-1), which is 1665 bp long and includes a 1461-bp open reading frames encoding 486 amino acids with an 18-aa signal peptide. This gene is a double-copy gene in R. similis. Rs-scp-1 was expressed in the procorpus, esophageal glands and intestines of females and in the esophageal glands and intestines of juveniles. Rs-scp-1 expression levels were highest in females, followed by juveniles and males, and lowest in eggs. Rs-scp-1 expression levels were significantly suppressed after R. similis was soaked in Rs-scp-1 dsRNA for 12 h. Nematodes were then inoculated into Anthurium andraeanum after RNAi treatment. Compared with water treatment, R. similis treated with RNAi were reduced in number and pathogenicity. In summary, we obtained the first SCP gene from a plant parasitic nematode and confirmed its role in the parasitic process.
Six candidate reference genes were chosen from the transcriptome database of Radopholus similis using the bioinformatics method, including four conventional reference genes (actin, Eukaryotic translation initiation factor 5A (eIF5A), Tubulin alpha (a-tubulin), ubiquitin (UBI)) and two new candidate reference genes (Ribosomal protein S21 (Rps21) and Serine/threonine protein phosphatase PP1-β catalytic subunit (β-PP1)). In addition, a traditional reference gene 18S ribosomal RNA (18S rRNA) obtained from NCBI databases was also added to the analysis. Real-time PCR was used to detect the expression of seven candidate reference genes in six populations of R. similis and four developmental stages (female, male, larva and egg) of a population. The stability of the expression of candidate genes was evaluated by three software programs, BestKeeper, geNorm and NormFinder. The results showed that eIF5A is the most suitable reference gene for gene functional research of different populations, while both Rps21 and eIF5A are the most suitable reference genes for different developmental stages of a population. Therefore, eIF5A is the best reference gene for studying R. similis. However, one defect of this study is that only seven candidate reference genes were analyzed; ideally, more genes should be tested.
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