Expression and characterization of a-glucosidase III in the dwarf honeybee, Apis florea （Hymenoptera： Apoidea： Apidae）  

作　　者：CHANPEN CHANCHAO RUMPALAI PADOONGSUPALAI POLKIT SANGVANICH 

机构地区：[1]DepartmentofBiology [2]ProgramofBiotechnology [3]DepartmentofChemistry,FacultyofScience,ChulalongkornUniversity,Bangkok,Thailand

出　　处：《Insect Science》2007年第4期283-293,共11页昆虫科学（英文版）

摘　　要：Alpha-glucosidase is synthesized in the hypopharyngeal glands located in the head of worker bees including Apisflorea. To analyze the developmental stage-specific expression of the a-glucosidase gene inA.florea, total RNA was isolated from eggs, and the heads of nurse and forager bees. By reverse transcription polymerase chain reaction （RT- PCR）, it was shown that the highest expression levels of the a-glucosidase Ⅲ gene, in the three examined developmental stadia, were found in forager bees, with much lower expression levels in nurse bees and no detectable expression in eggs. A complete aglucosidase III eDNA was obtained by RT-PCR and sequenced. The 1 701 bp eDNA nucleotide sequence and the predicted 567 amino acids it encodes were assayed by BLASTn, BLASTp and BLASTx programs and revealed a 95% and 94% similarity to the A. mellifera aglucosidase III gene at the DNA and amino acid sequence levels, respectively. For purification of the active encoded enzyme, forager bee heads were homogenized in sodium phosphate buffer solution and the crude extract （0.30 U/mg） sequentially precipitated with 95% saturated ammonium sulfate （0.18 U/mg）, and purified by DEAE cellulose ion exchange chromatography （0.17 U/mg）, and gel filtration on Superdex 200 （0.52 U/mg）. After resolution through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single enzymically active band （73 kDa） was identified from renatured substrate gels. Excision of this band, elution of the protein and tryptic peptide digestives identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） revealed six matching masses to the A. mellifera （Q17958） and predicted A.florea α glucosidase Ⅲ protein with 12% coverage, supporting the probable purification of the same α-glucosidase III protein as that encoded by the cloned cDNA.Alpha-glucosidase is synthesized in the hypopharyngeal glands located in the head of worker bees including Apisflorea. To analyze the developmental stage-specific expression of the a-glucosidase gene inA.florea, total RNA was isolated from eggs, and the heads of nurse and forager bees. By reverse transcription polymerase chain reaction （RT- PCR）, it was shown that the highest expression levels of the a-glucosidase Ⅲ gene, in the three examined developmental stadia, were found in forager bees, with much lower expression levels in nurse bees and no detectable expression in eggs. A complete aglucosidase III eDNA was obtained by RT-PCR and sequenced. The 1 701 bp eDNA nucleotide sequence and the predicted 567 amino acids it encodes were assayed by BLASTn, BLASTp and BLASTx programs and revealed a 95% and 94% similarity to the A. mellifera aglucosidase III gene at the DNA and amino acid sequence levels, respectively. For purification of the active encoded enzyme, forager bee heads were homogenized in sodium phosphate buffer solution and the crude extract （0.30 U/mg） sequentially precipitated with 95% saturated ammonium sulfate （0.18 U/mg）, and purified by DEAE cellulose ion exchange chromatography （0.17 U/mg）, and gel filtration on Superdex 200 （0.52 U/mg）. After resolution through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single enzymically active band （73 kDa） was identified from renatured substrate gels. Excision of this band, elution of the protein and tryptic peptide digestives identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） revealed six matching masses to the A. mellifera （Q17958） and predicted A.florea α glucosidase Ⅲ protein with 12% coverage, supporting the probable purification of the same α-glucosidase III protein as that encoded by the cloned cDNA.

关 键 词：Α-GLUCOSIDASE Apisflorea cDNA EXPRESSION homology purification 

分 类 号：Q966[生物学—昆虫学]