Molecular cloning, characterization and expression analysis of PtrHOS1, a novel gene of cold responses from trifoliate orange [Poncirus trifoliata (L.) Raf.

Abstract

High expression of osmotically responsive genes 1 (HOS1) encodes an ubiquitin E3 ligase that promotes the degradation of transcription factor Inducer of CBF Expression 1 (ICE1). Inactivation of ICE1 reduces CBF-induced activation of many cold-responsive genes, and thus, HOS1 act as a negative regulator of cold-responsive genes. In this paper, a novel HOS1 gene, designated PtrHOS1 (Genebank accession number FJ844367), was cloned by RT-PCR and RACE-PCR from trifoliate orange [Poncirus trifoliata (L.) Raf.]. The full length of PtrHOS1 is 3,434 bp with an open reading frame of 2,922 bp, encoding a protein of 974 amino acids with a molecular weight of 110.2 kDa and a theoretical isoelectric point of 5.55. Sequence alignment showed that PtrHOS1 protein had a conserved RING finger domain in its N-terminal region and shared high identity with other plant species HOS1-like proteins. Semi-quantitative RT-PCR analysis revealed that PtrHOS1 could be constitutively expressed at high levels in leaves, stems and roots. Interestingly, the PtrHOS1 expression had a declined period in leaves, stems and roots after cold and ABA treatments, which suggested that the PtrHOS1 expression was down regulated both by cold and ABA. Moreover, the decline was first occurred in leaves (30 min), followed with stems (2 h) and roots (4 h) after cold treatments. These results probably suggest that the leaves of trifoliate orange first sense the cold stress, followed with stems and roots. Oppositely, after ABA treatments, the significant decline of PtrHOS1 expression was first occurred in roots (15 min), followed with stems and leaves (30 min). Our results provide useful information for further studies about cold acclimation mechanism in citrus.