Cloning and Functional Analysis of Calcineurin Subunits A and B in Development and Fecundity of Nilaparvata lugens (Stål)

Serine/threonine phosphatase calcineurin (CN) is a unique but confounding calcium/calmodulin- mediated enzyme, which is composed of a catalytic subunit A (CNA) and a regulatory subunit B (CNB). We cloned six transcripts for CNA named from NlCNA-X1 to NlCNA-X6, one CNB named NlCNB1 and one CNB homologous gene NlCNBH1 from Nilaparvata lugens. All of them are constitutively transcripted in various tissues and developmental stages. The primary structure of the six isoforms showed obvious differences in the length and composition of the amino acid sequence between the two binding domains of Ca2+/calmodulin (CaM) and CNB. Ca2+-binding EF-hand motifs were found in NlCNB1 and NlCNBH1. The specific gene silencing of NlCNA, NlCNB1 and NlCNBH1 respectively by RNAi resulted in drastical reduction in survival rate, female weight, eclosion rate and fecundity of N. lugens. These results showed that NlCNA, NlCNB1 and NlCNBH1 were required for N. lugens growth and reproduction. The negative effects of NlCNB1 silence on nymph mortality (97%), molting malformation (90%) and female sterile (50%) were more serious than those of NlCNA or NlCNBH1. qRT-PCR and enzyme-linked immunosorbent assay (ELISA) analyses indicated that the nymphs with silenced NlCNA, NlCNB1 or NlCNBH1 showed impaired hormone and energy metabolism. In nymphs, the contents of 20-hydroxyecdysone (20E) after NlCNB1 RNAi and phenoloxidase after NlCNA RNAi were particularly decreased. These results suggested that NlCNA is involved in immunity of N. lugens by regulation of phenoloxidase, while NlCNB1 may control the growth and development of N. lugens by 20E signaling pathway in addition to interact with CNA. Injection of 70ng/μL dsNlCNB1 resulted in 77.0% down regulation of NlCNB1, and the nymph mortality was up to 57.9% at 10 d after injection. Therefore, NlCNB1 could be a potential candidate target used for strategy design in control of N. lugens. Our results revealed the importance of CN in the regulation of the growth and development of N. lugens, which provided a basis for further study of the molecular mechanism of CN.