There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
Subtilisin-like serine proteases (EC 3.4.21) consist of a widespread family of enzymes
that is involved in various processes including in plants. The full-length cDNA (CpSUB1)
and the corresponding genomic DNA for papaya subtilase have been obtained using rapid
amplification of cDNA ends (RACEs) and PCR primer walking techniques, respectively.
The cDNA clone contains an open reading frame of 2316bp encoding 772 amino acids with
a calculated molecular mass of 82.6kDa and an isoelectric point (pI) of 8.97. The
CpSUB1 gene is composed of nine exons and eight introns. The amino acid sequence encoded
by CpSUB1 shared high identity (>60%) with the amino acid sequence of other plant
subtilisin-like proteases. Sequence analysis of CpSUB1 revealed the presence of a
possible signal peptide (25 amino acid residues) and an NH(2)-terminal prosequence
(88 amino acid residues). In addition, papaya subtilase possesses the characteristic
subtilisin catalytic triad amino acids namely Asp, His and Ser, together with the
substrate-binding site, Asn. DNA hybridization analysis showed that subtilase gene
exists as a single copy in the papaya genome. RNA hybridization analyses showed that
expression of the subtilase transcripts was only detected in mesocarp but not in non-fruit
tissues. Gene expression in fruit tissues reached the highest level during the ripening
stage at which the fruits undergo dramatic softening process. Subsequently, pro-subtilase
( approximately 80kDa) was expressed as recombinant pro-enzyme ( approximately 97kDa),
which was used to generate antiserum against papaya subtilase, anti-sub. Protein gel
blot analysis using anti-sub towards total protein extracted from all ripening stages
revealed that a protein with a molecular mass of approximately 70kDa reacted with
the antiserum. Hence both RNA hybridization and protein gel blot analyses confirmed
the presence of subtilase during papaya fruit ripening, pointing to its possible involvement
in this important process.