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Title: SEQUENCES OF CDNAS AND EXPRESSION OF GENES ENCODING CHITIN SYNTHASE AND CHITINASE IN THE MIDGUT OF SPODOPTERA FRUGIPERDA

Author
item BOLOGNESI, RENATA - KANSAS STATE UNIV
item ARAKANE, YASUYUKI - KANSAS STATE UNIV
item MUTHUKRISHNAN, SUBBARATNAM - KANSAS STATE UNIV
item KRAMER, KARL - 5430-05-30 RETIRED
item TERRA, WALTER - UNIV DE SAO PAULO, BRAZIL
item FERREIRA, CLELIA - UNIV DE SAO PAULO, BRAZIL
item Beeman, Richard

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/29/2005
Publication Date: 9/30/2005
Citation: Bolognesi, R., Arakane, Y., Muthukrishnan, S., Kramer, K.J., Terra, W.R., Ferreira, C., Beeman, R.W. 2005. Sequences of cDNAs and expression of genes encoding chitin synthase and chitinase in the midgut of Spodoptera frugiperda. Insect Biochemistry and Molecular Biology 35: 1249-1259.

Interpretive Summary: Together with scientists at Kansas State University and the University of Sao Paulo in Brazil, we describe the cloning, sequencing and gut expression patterns of genes of the fall armyworm involved in chitin metabolism in different developmental stages and investigate the physiological functions of two enzymes, chitin synthase and chitinase. It was discovered that the genes encoding these enzymes are tightly regulated during larval development, in mutually exclusive temporal patterns to precisely coordinate the synthesis of the chitinous gut lining during the feeding stage and its degradation during larval-larval and larval-pupal molts. This regulation is accomplished at the level of mRNA transcription. The studies provide new biological insights and will contribute to our knowledge about regulated chitin synthesis and degradation in the insect midgut, the understanding of which is very limited at present. Understanding this regulation is a significant step that will allow development of reagents for use in future research on the inhibition of chitin metabolism using chitin inhibitors (CIs). The results obtained provide new information about structure-activity-regulation relationships, and add to the knowledge base for metabolic enzymes and insect molecular science. The long-term goal of this research is to facilitate a more effective application of CIs for the control of insect pests.

Technical Abstract: The focus of this study was on the characterization and expression of genes encoding enzymes responsible for the synthesis and degradation of chitin, chitin synthase (SfCHSB) and chitinase (SfCHI), respectively, in the midgut of the fall armyworm, Spodoptera frugiperda,. Sequences of cDNAs for SfCHSB and SfCHI were determined by amplification of overlapping PCR fragments and the expression patterns of these two genes were analyzed during insect development by RT-PCR. SfCHSB encodes a protein of 1523 amino acids containing several transmembrane segments, whereas SfCHI encodes a protein of 555 amino acids composed of a catalytic domain, a linker region, and a chitin-binding domain. SfCHSB is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are expressed along the whole midgut. Chitin staining revealed that this polysaccharide is present in the peritrophic membrane (PM) only when SfCHSB is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHSB is responsible for PM chitin synthesis during the larval feeding stages and SfCHI carries out PM chitin degradation during larval-pupal molting, suggesting mutually exclusive temporal patterns of expression of these genes.