关键词: 棉花, 过氧化物酶, 克隆, 鉴定

Abstract:

Peroxidases are iron haem-containing oxidoreductase (EC 1.11.1.7) that reduce peroxides, mainly hydrogen peroxide, to water and subsequently oxidize small molecules, often aromatic oxygen donors. The plant peroxidase superfamily can be divided into three classes. Of these classes, the class Ⅲ peroxidases have important function in plant metabolism processes such as cell wall organization, germination, hormonal, and stress responses etc. Cotton fibers are single-celled seed trichomes and fiber cells develop in four distinct but overlapping phases - initiation, cell elongation (primary wall synthesis), cell wall deposition and maturation. The objective of the study was to clone peroxidase genes, further put a foundation to illustrate these genes function in cotton fiber developmental stages. In this paper, two cDNA clones encoding class Ⅲ peroxidases were separated from developmentally different cotton fiber pools of elite material 7235 library. GhPOD1 was a 1 489 bp cDNA sequence obtained via 5’ RACE technique, its open reading frame was 816 bp, and encoded a polypeptide containing 271 amino acids. BLAST analysis indicated this gene had high homologies with the peroxidase reported in Arabidopsis thaliana; the insert fragment of GhPOD2 was 1 355 bp, its open reading frame was 999 bp, and encoded a polypeptide containing 332 amino acids. BLAST analysis indicated this gene had a 99% amino acid identity with the peroxidase（pod8, L08199）reported in Gossypium hirsutum. GhPOD1 was expressed constitutively in every tissue, while GhPOD2 did not express in root, but express in stem, leaf, ovule and fiber cells and its expression in fiber cells was decreased after 14 DPA in transcriptional level. The phylogenetic analysis suggested GhPOD1 was distinctly different from other peroxidases, indicating that it was a novel G. hirsutum peroxidase; GhPOD2 and pod8 were in the same branch group, but they were also distinctly different from the other class Ⅲ peroxidases. Southern blotting analyses showed that there all were two copies of these two genes in the genome of upland cotton, deducing that sub-genome A and sub-genome D contain each of them. The work of transferring GhPOD1 and GhPOD2 into G. hirsutum L. by Agrobacterium mediated transformation is ongoing.

Key words: Gossypium hirsutum L., Peroxidase, Cloning, Characterization