Silencing of two alternative splicing-derived mRNA variants of chitin synthase 1 gene by RNAi is lethal to the oriental migratory locust, Locusta migratoria manilensis (Meyen)

Abstract

Chitin synthases are crucial enzymes responsible for chitin biosynthesis in fungi, nematodes and arthropods. We characterized two alternative splicing-derived variants of chitin synthase 1 gene (LmCHS1) from the oriental migratory locust, Locusta migratoria manilensis (Meyen). Each cDNA of the two variants (LmCHS1A and LmCHS1B) consists of 5116 nucleotides that include a 4728-nucleotide open reading frame (ORF) encoding 1576 amino acid residues, and 67- and 321-bp non-coding regions at the 5'- and 3'-ends of the cDNA, respectively. The two variants differ only in one exon consisting of 177 nucleotides that encode 59 amino acid residues. The amino acid sequences within this alternative splicing region are 75% identical between the two variants. Both variants were expressed in all the developmental stages. However, LmCHS1A was predominately expressed in the integument whereas LmCHS1B was mainly expressed in the trachea. Our RNAi-based gene silencing study resulted in a dramatic reduction in the levels of the corresponding mRNA in the locust nymphs injected with dsRNA of LmCHS1, or either of its two variants, LmCHS1A and LmCHS1B. Consequentially, 95, 88 and 51% of mortalities were observed in the locusts injected with the LmCHS1, LmCHS1A and LmCHS1B dsRNA, respectively. The phenotypes resulted from the injection of LmCHS1A dsRNA were similar to those from the injection of LmCHS1 dsRNA, whereas the locusts injected with LmCHS1B dsRNA exhibited crimpled cuticle phenotype. Our results suggest that both variants of chitin synthase 1 are essential for insect growth and development.