Five aminopeptidase-N genes （EpAPN1-5） of the tortricid moth Epiphyas postvittana have been isolated from a midgut expressed sequence tag （EST） library. Relative RNA expression of these genes was measured by quant...Five aminopeptidase-N genes （EpAPN1-5） of the tortricid moth Epiphyas postvittana have been isolated from a midgut expressed sequence tag （EST） library. Relative RNA expression of these genes was measured by quantitative reverse transcription polymerase chain reaction using actin as a reference gene. Measurements were made on various tissues of fifth instar larvae, over all stages of the life cycle and under differing dietary conditions： different protein sources and levels, and in presence of trypsin and metalloprotease inhibitors. Gene expression for all five genes was the greatest in midgut tissue, but was also detected in the hindgut, fat body and Malpighian tubules. EpAPN4 was consistently the highest expressed, with EpAPN3 at about half that level; EpAPN5 was the least expressed. During larval stages expression was high, generally increasing over the instars, after an early peak as neonates or first instars. Expression in other life stages was much lower. Males and females showed differing expression in the pupal and adult stages： female expression was higher in the pupa, this reversed in the adult. Gene expression levels and ratios both changed with diet. A natural apple leaf diet depressed levels. Diets with the most impaired amino acid balance induced the greatest change; generally EpAPN1 increased by the greatest proportion. The addition of proteinase inhibitor also increased gene expression, and it was noteworthy that the trypsin inhibitor addition, which has been shown to double aminopeptidase activity, also doubled levels of gene expression.展开更多
文摘Five aminopeptidase-N genes （EpAPN1-5） of the tortricid moth Epiphyas postvittana have been isolated from a midgut expressed sequence tag （EST） library. Relative RNA expression of these genes was measured by quantitative reverse transcription polymerase chain reaction using actin as a reference gene. Measurements were made on various tissues of fifth instar larvae, over all stages of the life cycle and under differing dietary conditions： different protein sources and levels, and in presence of trypsin and metalloprotease inhibitors. Gene expression for all five genes was the greatest in midgut tissue, but was also detected in the hindgut, fat body and Malpighian tubules. EpAPN4 was consistently the highest expressed, with EpAPN3 at about half that level; EpAPN5 was the least expressed. During larval stages expression was high, generally increasing over the instars, after an early peak as neonates or first instars. Expression in other life stages was much lower. Males and females showed differing expression in the pupal and adult stages： female expression was higher in the pupa, this reversed in the adult. Gene expression levels and ratios both changed with diet. A natural apple leaf diet depressed levels. Diets with the most impaired amino acid balance induced the greatest change; generally EpAPN1 increased by the greatest proportion. The addition of proteinase inhibitor also increased gene expression, and it was noteworthy that the trypsin inhibitor addition, which has been shown to double aminopeptidase activity, also doubled levels of gene expression.
