Molecular Characteristics And Functional Analysis Of Chitin Deacetylase Genes In Oxya Chinensis (Orthoptera:Acrididae)

Chitin is a naturally occurring polysaccharide that is one of the main components in the cell walls of fungi and exoskeletons of insects and crustaceans. The extracellular matrix of insect exoskeleton is modified in different ways to make the cuticle either very rigid and thick or thin and flexible, or to generate specialized structures such as mandibles with sharp cutting edges. Other insect structures such as the highly branched tracheal network as well as the foregut originated from ectoderm are also lined with a chitinous cuticle. Chitin deacetylases (CDAs) are metalloproteins that belong to a family of extracellular chitin-modifying enzymes, which deacetylated chitin, the β-(1,4)-linked N-acetylglucosamine homopolymer, to form chitosan, a polymer of (3-1,4-linked N-acetyl-D-Glucosamine residues. This modification may contribute to an affinity with a variety of proteins distinct from those that bind specifically to chitin. In this study, we utilized Oxya chinensis as a model to explore the molecular characteristics and function of CDAs. The results will provide a reasonable basis for the effective management of O. chinensis. The contents are as follows:1. The cDNAs search of OcCDA genes and molecular characteristics analysisBy searching the transcriptome of O. chinensis, one fragment and two full-length cDNA sequences of CDA genes were obtained, and rapid-amplification of cDNA ends (RACE) was used to amplify 5 end sequence of the partial cDNA. Sequence analysis showed that all these three cDNA sequences had three conservative regions (ChBD, LDLa and CDA). Based on phylogentic analysis, the obtained sequences were named as OcCDA 1, OcCDA2a and OcCDA2b, respectively.2. Tissue-specific and developmental expression patterns of OcCDA genesReal-time quantitative PCR (qPCR) was applied to detect the relative expression levels of OcCDAs in different tissues of the 5th instar nymphs and at different developmental stages. The tissue expression results of OcCDAs indicated that OcCDAl, OcCDA2 and OcCDA2a were predominately expressed in foregut, lower expressed in integument, hindgut and fat body; OcCDA2b showed the highest expression in integument, higher expression in foregut. The developmental expression results of OcCDAs indicated that the expression level of OcCDA1, OcCDA2, OcCDA2a and OcCDA2b were the highest in the integument of the 1st day of the 5th instar nymphs, and then declined in the later developmental days.3. Chitin staining in foregut of O. chinensis and microscopic observationThe well-developed 5th instar nymphs of O. chinensis were selected for dissection, and the foregut was used to make frozen microsection followed by chitin staining. The results discoveried that there was a lot of chitin in foregut, and chitin was mainly distributed in internal membranes of the intestine, the outer wall also has a little. This result provides a direct evidence that the chitin was high expression of OcCDAs in the foregut of O. chinensis might contribute to the formation of cuticle and internal membranes in the foregut.4. Functional analysis of OcCDAThe double-stranded RNAs of OcCDA1, OcCDA2a, OcCDA2b and OcCDA2 were injected into the second day of 5th instar nymphs, respectively. The silencing efficiency was detected by qPCR at 24 h after injection, and the results showed that all genes were specifically silenced. The nymphs injected with dsOcCDAl, dsOcCDA2 and dsOcCDA2a were slow development and unable to molt the old cuticle, then finally died, and the mortalities were up to 92.5%,94.4% and 87.5%. However, the nymphs ccould successfully molt to adults after dsOcCDA2b injection.