CDNA Cloning, Expression Patterns and Functional Analysis of β-N-acetylglucosaminidase in Locusta Migratoria
|Keywords||Locusta migratoria chitin degradation β-N-acetylglucosaminidase RNAinterference|
β-N-acetylglucosaminidases are widespread in bacteria, fungi and arthropods. There are often multiple genes that encoding P-N-acetylglucosaminidases in various insect species, which involve in different physiological processes including chitin degradation, glycoconjugates degradation and N-glycan modification. Because chitin is an indispensable structural component for insects but absent in higher plants and animals, the β-N-acetylglucosaminidases which is involved in chitin degradation could be an ideal potential target for pest control. The main contents of this thesis are as follows:1. Cloning, sequencing and phylogenetic analysis of β-N-acetylglucosaminidase in Locusta migratoria.17β-N-acetylglucosaminidase gene fragments from L. migratoria were identified by searching the sequences in the EST database. Fifth-instar nymphs were used for template to obtain full-length sequence of putative LmNAGl. The full-length cDNA consists of2667nucleotides, including an open reading frame (ORF) of1845nucleotides that encode614amino acid residues and233-and589-bp non-coding regions at the5’-and3’-end of the cDNA, respectively. Phylogenetic analysis of the NAGs from L. migratoria and several other insect species showed that LmNAGl was grouped with the enzymatically characterized NAGs in group I.2. Developmental stage and tissue-specific expression patterns of LmNAGl. RT-PCR and Real-time PCR were carried out to analyze the expression patterns of LmNAGl. Six selected tissues were dissected from day5,6and7of fifth-instar nymphs for tissue-specific expression analysis. The whole insects from the third day of each stage and integument dissected from each day during4th and5th-instar nymph stages of L. migratoria were used for developmental expression analysis. The results showed that LmNAGl was expressed in all the developmental stages and increased along with insect growth. The qPCR analyses showed that LmNAGl was predominantly expressed in foregut and hindgut, relatively low in the integument, midgut and wing pad, but hardly detectable in gastric caeca. The expression of LmNAGl in integument could be detected in whole4th-and5th-instar nymphs. The expression level of LmNAGl was significant high in the last two days of each instar, which is accordant with the ecdysone hormone (20-E) titer peak in both4th-and5th-instar nymphs. 3. Biofunctional analysis of LmNAGl gene. RNAi experiment was carried out to determine the biological function of LmNAGl. The results showed target gene could be down-regulated effectively in all selected tissues after dsLmNAGl injection, and the silence time could be kept more than72hours. The abnormal nymphs showed an "incomplete" ecdysis phenotype after the dsRNA injection and most of them eventually died from failed molting.4. β-N-acetylglucosaminidase total activity assay. The experiment determined the β-N-acetylglucosaminidase total activity of six selected tissues from day5,6and7of L. migratoria5th-instar nymphs. The results showed that the distribution of the enzyme activity obviously change in the six tissues of the last three days of5th-instar nymphs and the patterns correspond with the process of molting and other crucial physiological functions of insect. The total activity of LmNAG starts to significantly decrease between24h and48h after LmNAGl dsRNA injection.To summarize, the obtained LmNAGl gene plays a significant role in the developmental process of L. migratoria. Its biological function is closely related with the important process of molting. The knock-down of the gene could cause the mortality of the insect because of incomplete ecdysis and also supress suppress the total enzyme activity of β-N-acetylglucosaminidase in L. migratoria. The results suggest LmNAGl is a potential and promising target for RNAi-mediated biological pest control.