Molecular Properties And Biological Functions Of GFAT And GNA In Locusta Migratoria

Chitin was widely distributed in epidermis and peritrophic matrix of insects, many enzymes were involved in chitin biosynthesis pathway. The importance of chitin in insect development and the absence of chitin in vertebrates had led to a hypothesis of attacking insect chitin biosynthesis pathway as a novel target for developing safe and effective insecticides. However, the roles of these enzymes in insect development have not been well elucidated. In this thesis, glutamine:fructose-6-phosphate amidotransferase (GFAT) and Glucosamine-6-phosphate-N-acetyltransferase (GNA) of Locusta migratoria, two key enzymes in chitin biosynthesis pathway, were selected to study their sequences, mRNA expression pattern and biological function.A partial cDNA sequnence of GFAT was obtained from Locust Database, blast analysis showed this sequence had intact3’terminal sequence and miss5’end sequence.5’-RACE PCR was performed to get full length cDNA sequence of LmGFAT. The obtained complete cDNA sequence of LmGFAT was3202bp, contained ORF2292bp encoded764amino acid residues, predicted molecular weight was85.6kD. LmGFAT showed high indentity with other GFATs by animo acid sequence alignment. Howerve, LmGFAT presented additional74amino acid residues at N-terminal compared with other known insect GFATs. The catalytic cites were very conserved in GFATs from both prokaryotes and eukaryotes. Five potential PKA phosphorylation site S271, S326, S380, S567and S581were predicted, which could be involvoed in PKA or AMPK phosphorylation regulation. Phylogenetic tree indicated that GFATs were clustered based on the evolution relationship, in addition, GFAT1and GFAT2of mammal were separated to two different clusters.Tissue specific expression pattern showed that LmGFAT had the highest expression in integument and higher in fat body and wing pad in second day of the5th-instar nymph. The developmental expression pattern in each day of the5th instar nymphs showed that LmGFAT was highly expressed in first day of the5th instar nymphs, and then declined at the following days of the same stage. After LmGFAT was down regulated by RNA interference,93%insects were dead when molting to adult. In conclusion, LmGFAT should be involved in chitin biosynthesis, and regulated by feedback of UDP-GLcNAc and phosphorylation of PKA and AMPK.The cDNA sequence of LmGNA was searched out from Locust database by using bioinformatics methods. The complete cDNA sequence consists of1230bp nucleotides that contain a612bp ORF coding203amino acid residues. The amino acid sequences of GNAs from different species appeared high identity, the residues responsible for binding substrate GlcN6P are exactly same, which suggested GNAs are highly conserved among different species. Recombinant LmGNA protein was expressed in E. coli BL21(DE3) with pET-28a(+) vector containing LmGNA ORF, and then purified by Ni-NTA affinity resin, the enzyme activity was analyzed by spectrophotometer. The optimal enzyme activity was detected at37-50℃and optimum pH was between8.0-9.5. The Km value for D-GlcN6P was42.43μM when the concentration of Acetyl-CoA was200μM, in contrast, the Km value for Acetyl-CoA was133.60μM when the concentration of D-GlcN6P was200μM. Tissue specific expression pattern indicated that LmGNA was mainly expressed in epicuticle, fat body and muscle at the2nd day of the5th-instar nymph. The developmental expression pattern from the1st day to7th day of the5th-instar nymph stage showed that LmGNA was highly expressed after new molting, and then declined at the following days of the same stage. RNAi results indicated the mRNA of LmGNA was effectively silenced, however, ecdysis and development of Locusts didn’t be affected. These data suggested that N-acetylglucosamine kinase, a rescue pathway could be involved in producing N-acetylglucosamine-6-phosphate (GlcNAc6P) instead of GNA for chitin biosynthesis.In this thesis, cDNA cloning, mRNA expression and biological function of LmGFAT and LmGNA were studied. The results would be helpful to improve the understanding of the complexity of insect biosynthesis and explore the chitin metabolic mechanism, which should provide important data for developing new strategies for pest control.