Purification, cDNA cloning and northern blot analysis of trehalase of pupal midgut of the silkworm, Bombyx mori.
Trehalase (alpha-glucoside-1-glucohydrolase, EC 3.2.1.28) was purified from silkworm pupal midgut to homogeneity by DEAE-Sepharose CL-6B and hydroxyapatite chromatography, and native gel electrophoresis. The enzyme had a molecular mass of 70 kDa. The N-terminal amino-acid sequence of the intact trehalase and its three fragments by V8 proteinase digestion was determined. Based on the amino-acid sequence, degenerate oligonucleotides were synthesized and used as primers in a polymerase chain reaction (PCR). Using a 0.8 kb PCR product as a hybridization probe, trehalase clones were isolated from the pupal midgut cDNA library. Sequence analysis revealed that the isolated trehalase cDNA contains 3103 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis clearly showed a 3.0 kb transcript in midgut, and Malpighian tubule, but not in fat body, silk gland, ovary, trachea, brain and suboesophageal ganglion.
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Laboratory of Sericultural Science, School of Agriculture, Nagoya University, Japan.
Sato
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Yamashita
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D012831:Bombyx / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D011679:Pupa / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014198:Trehalase / Q000235:genetics* / Q000302:isolation & purification
D014198:Trehalase