Cloning and postharvest expression of serine proteinase transcripts in the cultivated mushroom Agaricus bisporus

Increases in both the levels and the activity of serine proteinase have been previously described in the senescing mushroom Agaricus bisporus. cDNA encoding serine proteinase was amplified by reverse transcriptase-polymerase chain reaction using a degenerate primer based on the N-terminal sequence of a previously isolated A. bisporus serine proteinase and then cloned. The cDNA was sequenced and shown to be homologous to those of other fungal serine proteinases. Northern analysis showed that this serine proteinase gene (Spr1) was not expressed in freshly harvested sporophores but was strongly up-regulated postharvest and found almost entirely in the stipe of the sporophore (approximately 0.08% of mRNAs 2 days after harvest). Low-level expression was detectable in the flesh (pileus trama) and gill (lamellae) tissues of the cap, but none was detected in the skin (pilei pellis). In three of the cloned cDNAs, sequence analysis showed that the poly(A) tail starts at different positions. Expression of Spr1 in Escherichia coli caused restricted colony growth.