Molecular Characteristics And Functional Analysis Of Trehalase Genes In Locusta Migratoria

Trehalose is the main blood sugar of insects, which is involved in energy metabolism. Trehalase is the only enzyme to hydrolyze trehalose. Because higher animals lack this metabolic process, it could be served as a potential target for developing safe and effective insecticides. In this paper, the molecular characteristics and function of trehalase genes were explored in Locusta migratoria. The results will provide a reasonable basis for the effective management of L. migratoria. The contents are as follows:1. The cDNAs search of trehalase genes from L. migratoria and molecular characteristics analysisBy searching the transcriptome of L. migratoria, three full-length cDNA sequences of trehalase genes and one fragment were obtained, and based on partial cDNAfragment, its full cDNA sequence was got by further searching the Locusta genome. Blast analysis showed that all these four trehalases had two conservative regions (PGGRFrEyYyWDtY and qWDyPNaWpP), and a glycine enrichment region (GGGGEY).TMHMM Server v.2.0 found that one trehalase had a transmembrane domain, named LmTreM. The remains were soluble trehalases, named LmTreSl, LmTreS2, LmTreS3, respectively. SignalP 4.1 Server software’s analysis indicated that only LmTreSS had a signal peptide.2.Tissue-specific and developmental expression patterns of trehalase genesEF-1α and β-actin are chose for the most suitable reference genes in different tissues and developmental stages of fifth-instar nymph of L. migratoriaby qPCR. The mRNA expression of trehalase geneswas analyzed with qPCR. The results indicated that trehalase genes showed different expression characteristics. LmTreM was exclusively expressed in muscle, LmTreSl was mainly expressed in Malpighian tubes, midgut and hindgut, and LmTreS2 were highest expressed in integument, while LmTreSS was expressed in all tissues. Then the highly expressed tissues were chosen to detect the developmental expression. qPCR results showed LmTreM was highly expressed in muscle of the middle days of the 5th instar nymphs. During the development of Malpighian tubes, the mRNA expression of LmTreSl was increased accordingly, and also LmTreSl was highly expressed in midgut and hindgut of middle days of the 5th instar nymphs.The expression of LmTreS2 in integument decreased with developmental day increased, and LmTreS3 was constantly expressed throughout the developmental days.3. Determination of the trehalase activity of L. migratoriaThe enzyme activity was determined by using the hexokinase-HK method. We selected muscle of the 5th instar nymphs to detect the activity of typical membrane-bound trehalase LmTreM, and dissected Malpighian tubes, midgut and hindgut to detect the activity of typical soluble trehalase. It was found that LmTreM showed high enzyme activity in the 1st day ofthe 5th instar nymphs, and relatively lower in other 6 days, the enzyme activity of soluble trehalase gradually increased during the developmental days in Malpighian tubes, and constantly determined in midgut and hindgut.4. Functional study on trehalase genes of I. migratoriaTo further explore biological functions of trehalase genes during L. migratoria development, RNAi was performed in the 2nd day of the 5th instar nymphs.The whole body was pooled together to extract total RNA. The silencing efficiency was detected by qPCR after injecting dsRNA of LmTreM, LmTreSl, LmTreS2and LmTreS3, respectively, the results showed that all genes were specifically silenced, and did not find cross silencing phenomena. However, the 5th instar nymphs can successfully molt to adults after injected dsRNA, which suggested that silencing of trehalase genes can not affect the molting process of L. migratoria.