Molecular cloning, expression and characterization of BmIDGF gene from Bombyx mori.
Imaginal disc growth factors (IDGF) play a key role in insect development, but their mechanism remains unclear. In this study, we cloned a novel IDGF gene in Bombyx mori and designated it as BmlDGF. We found that the BmlDGF gene contains eight exons and seven introns, encoding a peptide of 434 amino-acid residues. The protein was predicted to contain one conserved motif of the glycosyl hydrolases family 18 and fall into group V chitinases. Sequence alignment showed that BmIDGF shares extensive homology with other invertebrate IDGF. RT-PCR analysis showed that BmIDGF is expressed in all developmental stages of silkworm larvae and various larvae tissues, which was further confirmed by Western blot analysis. Subcellular localization analysis indicated that BmIDGF is located in the extracellular space. We also successfully expressed it in E. coli and further characterized it by SDS-PAGE and mass spectrometry. Taken together, our data suggests that BmIDGF is a chitinase-like extracellular protein, and provides an excellent platform for subsequent studies on its enzyme activity and role in B. mori development.
Pan
Ye
Y
Institute of Life Sciences, Jiangsu University, No. 301 Xuefu Road, Zhenjiang 212013, PR China.
Chen
Keping
K
Xia
Hengchuan
H
Yao
Qin
Q
Gao
Lu
L
Lü
Peng
P
Huojuan
He
Yuanqing
Y
Wang
Lin
L
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000235:genetics*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D006023:Glycoproteins / Q000235:genetics*; D019476:Insect Proteins / Q000235:genetics*; D007438:Introns; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D006023:Glycoproteins; D019476:Insect Proteins; D012313:RNA; D002688:Chitinases