Fruit-specific expression of papaya subtilase gene

Roohaida Othman, Azimi Nuraziyan

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 2316 bp encoding 772 amino acids with a calculated molecular mass of 82.6 kDa 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 NH2-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 (∼80 kDa) was expressed as recombinant pro-enzyme (∼97 kDa), 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 ∼70 kDa 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.

Original languageEnglish
Pages (from-to)131-137
Number of pages7
JournalJournal of Plant Physiology
Volume167
Issue number2
DOIs
Publication statusPublished - 15 Jan 2010

Fingerprint

Carica
papayas
Fruit
Subtilisin
subtilisin
nucleic acid hybridization
fruits
Amino Acids
ripening
amino acids
Complementary DNA
Genes
serine proteinases
genes
antiserum
Immune Sera
Amino Acid Sequence
Proteins
amino acid sequences
proteins

Keywords

  • cDNA clone
  • Expression
  • Genomic DNA
  • Serine protease
  • Subtilase

ASJC Scopus subject areas

  • Plant Science
  • Physiology
  • Agronomy and Crop Science

Cite this

Fruit-specific expression of papaya subtilase gene. / Othman, Roohaida; Nuraziyan, Azimi.

In: Journal of Plant Physiology, Vol. 167, No. 2, 15.01.2010, p. 131-137.

Research output: Contribution to journalArticle

Othman, Roohaida ; Nuraziyan, Azimi. / Fruit-specific expression of papaya subtilase gene. In: Journal of Plant Physiology. 2010 ; Vol. 167, No. 2. pp. 131-137.
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