CLONING OF A CDNA-ENCODING A PUTATIVE CYSTEINE PROTEASE FROM GERMINATING MAIZE SEEDS

We constructed a cDNA expression library with mRNAs from the endosperm of germinating maize (Zea mays L.) seeds to isolate sequences encodin g proteases that degrade zein polypeptides. No immune-positive plaques were detected when the library was screened with polyclonal antibodie s reacting with proteases A,-A(4) a group of zein degrading cysteine p roteases isolated from germinating maize seeds. The library was subseq uently screened with a cDNA (pHVEP-4) encoding the cysteine endopeptid ase (EP-B) from the aleurone of germinating barley seeds. Five clones were isolated, and the longest cDNA (pMCP1OA) showing homology to the barley gene was characterized. This cDNA encodes a putative cysteine p rotease of 25 kDa, with an incomplete prosequence of 62 amino acids. T here is around 70% similarity between the primary sequences of pMCPIOA and pHVEP-4, with approximately 68% identity between the amino acid s equences deduced from both clones. Genes corresponding to pMCP1OA are represented no more than five times in the maize genome. Transcripts h ybridizing to pMCP1OA are absent in the dry seed, but are present betw een 2 and 6 days after germination.