Sep 1, 1993

Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta

Insect Biochemistry and Molecular Biology
K J KramerSubbaratnam Muthukrishnan

Abstract

Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5-7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth i...Continue Reading

Mentioned in this Paper

Genes, Insect
N Syndrome
Genes, Reiterated
Transcription, Genetic
Endochitinase
Gene Expression
Antheraea
Poly(A) Tail
Tadpole
Gastrointestinal System

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