Isolation, molecular cloning and characterization of a cold-responsive gene, AmDUF1517, from Ammopiptanthus mongolicus

Abstract

Temperature is one of important factors that influence plant growth and development. Using cDNA-amplified fragment length polymorphism approach, we previously screened 344 temperature-related transcript-derived fragments (TDFs) from Ammopiptanthus mongolicus. In this study, we confirmed that 15 of these TDFs were upregulated in response to low- or high-temperature by using semi-quantitative RT-PCR. Based on the rapid amplification of cDNA ends, PCR and genome walking approaches, full-length cDNA and promoter sequence of AmDUF1517 was cloned and identified. The 906 bp open reading frame of the AmDUF1517 gene encoded for a protein of 301 amino acids residues. The corresponding genomic DNA sequence contains two exons and one intron. Bioinformatic analysis showed that a predicted cleavage site for chloroplast transit peptide, a DUF1517 domain, two transmembrane domains and two putative sumoylation sites were conserved between AmDUF1517 and its homolog from Arabidopsis thaliana (AtDUF1517). We further showed that GFP-tagged AmDUF1517 was indeed targeted to the chloroplast in Arabidopsis protoplast. The transcript levels of AmDUF1517 were increased specifically in leaves in response to cold stress. In addition, treatment of ethylene, salicylic acid, gibberellic acid or NaCl induced the transcription of AmDUF1517. Mutation of DUF1517 in Arabidopsis exhibited enhanced sensitivity to cold stress, which was coupled with increased electrolyte leakage, malondialdehyde content and decreased contents of soluble sugar, proline. Interestingly, heterologous expression of AmDUF1517 in Arabidopsisatduf1517 mutants significantly rescued their cold-sensitive phenotypes. Altogether, our data suggest the potential roles of both AmDUF1517 and AtDUF1517 in the regulation of cold stress tolerance.