Cloning and Expression Analysis of Two Cotton (Gossypium Hirsutum L.) Genes Encoding Cell Wall Proline-rich Proteins

Abstract

Two cotton (Gossypium hirsutum L.) genes, ghprpl and ghprpl, encoding cell wall proline-rich proteins (PRPs) have been cloned and characterized. The ghprpl gene has an open reading frame (ORF) that encodes a PRP of 299 amino acids (aa), whereas the ghprp2 gene contains an ORF that codes for a 310-aa PRP. The GhPRPl has an 80% identity in aa sequence with that of GhPRP2. Like other plant cell wall PRPs, both cotton PRPs have a hydrophobic signal peptide at their N-termini, followed by repeating peptide units. Northern blot analyses showed that the ghprp1 gene is predominantly expressed in the fiber during the elongation stage of fiber development. Reverse transcription (RT)-PCR analysis showed that ghprp1 is expressed in both fiber and root tissues, whereas ghprpl is in roots only. The ghprp1 gene was shown to be present in the A₁, A², D¹ and D₅ genomes of Gossypium by PCR amplification, whereas the ghprpl gene is only present in the A₁ and A₂ genomes. The ghprp1 gene was over-expressed in the yeast Pichia pastoris, and the expressed GhPRP1 protein was used as an antigen to raise polyclonal antibodies (anti-GhPRP1). Western analysis using the anti-GhPRP1 probe detected a major protein band (50 kDa) in 5-31-day postanthesis (DPA) fibers. However, the 50 kDa protein was absent in other cotton tissues.

Key Words:

• Cotton fiber
• Differential expression
• Polymerase chain reaction
• Repeating peptide units
• Reverse transcription
• Signal peptide