Progress 07/01/05 to 06/30/07
Outputs
OUTPUTS: By screening the newly completed red flour beetle genome database we have identified 16 chitinase or chitinase-related genes and two peritrophin genes. The exon-intron organization of all of these genes has been completed. We have also cloned the protein coding regions of most of these genes. Three of these proteins (TcCht5, TcIDGF2 and TcIDGF4 have been expressed in an insect cell expression system using recombinant baculoviruses containing the corresponding cDNAs. These putative chitinase proteins were purified by ion-exchange and affinity column chromatography and their enzymatic activities have been investigated. The proteins, TcIDGF2 and TcIDGF4, have no chitinase activity against any of the substrates that we have tested. But these proteins bind strongly to chitin. On the other hand, the protein TcCht5 has good activity against both low molecular weight and high molecular weight substrates. We have determined the kinetic constants of this enzyme and its ability to bind
to chitin. We have determined the patterns of expression of the chitinase and peritrophin genes using reverse transcriptase-polymerase chain reactions (RT-PCR). There are differences in the expression patterns of the 16 different chitinase genes and the two peritrophin genes. We have carried out RNA interference studies for many of these chitinase and peritrophin genes using dsRNA for gene-specific regions. The dsRNA's were injected into young larvae, old larvae, prepupae or adults. After injection, the levels of the transcripts for these genes were monitored by RT-PCR. In all cases, we found substantial or complete loss of transcripts for the corresponding gene, but not for the other genes. We have also followed the effects of dsRNA injections on the development of the insects. These experiments have revealed that some chitinase genes affect specific molts while some affect all molts. We have analyzed the expression of the two peritrophin genes by in situ hybridization and RT-PCR.
These genes are expressed in the posterior part of the midgut only and there is partial overlap of the regions of the posterior midgut.
PARTICIPANTS: Individuals: S. Muthukrishnan PI - Designed project and analyzed data and edited publications; R. Beeman, PI - Designed project and analyzed data and edited publications; Q. Zhu - Performed the experiments, analyzed data and wrote the publications; Y. Arakane - Performed the experiments, analyzed data and wrote the publications; D. Banerjee - Performed the experiments, analyzed data; G. Osman - Performed the experiments, analyzed data. Partner Organizations: USDA- GMPRC, Manhattan, KS; Advance Genetic Engineering Research Institute, Giza, Egypt
TARGET AUDIENCES: Farmers, agricultural chemical industry, graduate students, visiting scientists.
Impacts
Knowledge of the full assortment of chitinase and related proteins and their biological role in insects will help identify suitable targets for insecticides. It may also be possible to generate transgenic plants that will produce dsRNA corresponding to some chitin binding proteins which might be useful in controlling targeted pests.
Publications
- Zhu, Q., Arakane, Y., Beeman, R. W., Kramer, K. J., & Muthukrishnan, S. M. (2007) Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum. Insect Biochem. Molec. Biol. Doi.10:1016/j.ibmb.2007.06.011
- Zhu, Q., Arakane, Y., Banerjee, D., Beeman, R. W., Kramer, K. J., & Muthukrishnan, S. M. (2007) Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects. Insect Biochem. Molec. Biol. Doi.10:1016/j.ibmb.2007.06.010.
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Progress 01/01/06 to 12/31/06
Outputs
By screening the newly completed red flour beetle genome database we have identified 16 chitinase or chitinase-related genes and two peritrophin genes. The exon-intron organization of all of these genes has been completed. We have also cloned the protein coding regions of most of these genes. Three of these proteins (TcCht5, TcIDGF2 and TcIDGF4 have been expressed in an insect cell expression system using recombinant baculoviruses containing the corresponding cDNAs. These putative chitinase proteins were purified by ion-exchange and affinity column chromatography and their enzymatic activities have been investigated. The proteins, TcIDGF2 and TcIDGF4, have no chitinase activity against any of the substrates that we have tested. But these proteins bind strongly to chitin. On the other hand, the protein TcCht5 has good activity against both low molecular weight and high molecular weight substrates. We have determined the kinetic constants of his enzyme and its ability
to bind to chitin. We have determined the pattern of expression of the chitinase and peritrophin genes using reverse transcriptase-polymerase chain reactions (RT-PCR). There are differences in the expression patterns of the 16 different chitinase genes and the two peritrophin genes. We have carried out RNA interference studies for many of these chitinase and peritrophin genes using dsRNA for gene-specific regions. The dsRNA were injected into young larvae, old larvae, prepupae or adults. After injection, the levels of the transcripts for these genes have been followed by RT-PCR. In all cases, we found substantial or complete loss of transcripts for the corresponding gene, but not for the other genes. We have also followed the effect of dsRNA injections on the development of the insects. These experiments have revealed that some chitinase and peritrophin genes affect specific molts while some affect all molts.
Impacts
Knowledge of the full assortment of chitinase and related proteins and their biological role in insects will help identify suitable targets for insecticides.
Publications
- Muthukrishnan, S., Arakane, Y., Zhu, Q., Hogenkamp, D., Specht, C.A., Beeman, R.A., Kanost, M. R., and Kramer, K. J. 2006. An integrated study of several chitin metabolism genes in Tribolium castaneum. Proceedings of the Fifth International Symposium on Molecular Insect Science at Tucson, AZ, May 20-24. p.49
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Progress 01/01/05 to 12/31/05
Outputs
By screening the newly completed red flour beetle genome database we have identified 13 chitinase or chitinase related genes and one peritrophin gene. The exon-intron organization of some of these genes has been completed and others are in progress. We have identified the protein coding regions in all of these genes. Full length cDNA clones for some of these genes have been obtained. One of these cDNAs has been expressed in an insect cell expression system using a recombinant baculovirus containing this cDNA. This putative chitinase protein was purified by ion-exchange and affinity column chromatography and its enzymatic activity has been investigated. We have determined the kinetic constants of this enzyme and its ability to bind to chitin.
Impacts
Knowledge of the full assortment of chitinase and related proteins and their biological role in insects will help identify suitable targets for insecticides.
Publications
- Zhu, Q. 2005. Characterization of Families of Chitinase Genes and Proteins from Tribolium castaneum, Drosophila melanogaster and Anopheles gambiae. Doctoral thesis, pp. 1-174.
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