关键词: 香橙, 苹果酸脱氢酶基因, 表达特性, 转基因烟草, 耐铝分析

Abstract:

Malate, ubiquitously existing in plant, plays a crucial role in various metabolic pathways, including respiration, nitrogen fixation, phosphorus acquisition, and aluminum tolerance. Malate dehydrogenase（MDH） catalyzes the interconversion of oxaloacetate and malate. Previous results suggested that overexpressing MDH gene transgenic plants elevated tolerance to aluminum toxicity. Citrus junos, one of main rootstocks of citrus fruits, is widely used in the Southern of China. As most of soils are acid in the south, to investigate the mechanism of tolerance to aluminum in citrus plants, a clone referred CjMDH (Genbank Accession No. ABI75147) was isolated from the cDNA library of root from Citrus junos cv.Ziyang. CjMDH has an open reading frame (ORF) of 1 269 bp encoding a putative protein with 412 amino acid residues, which shows high identity to other MDHs in plants. The deduced protein contains a NAD binding site and eight malate binding sites. Chloroplast transit pepite is found in N-terminal of CjMDH through iPSORT software. BlastP analysis result reveals over 80% identity and 85% similarity with Arabidopsis thaliana, Nicotiana tabacum, Glycine max, Medicago sativa. Amino acids comparison between CjMDH and other plants MDH reveals highly identity in NAD and malate binding site. Southern blot suggested that CjMDH is a single copy gene in Citrus junos genome. Northern blot and Real time RT-PCR data indicated that CjMDH expressed strongly in root and leaf, especially highest in root than leaf and stem. Subsequently, the CjMDH was constructed into over-expression vector driven by constitutive CaMV35S promoter. The construction was introduced into tobacco mediated by Agrobacterium tumerfaciens, the overexpression transgenic tobacco lines were obtained through Northern blot. Transgenic lines increased Al-tolerance in hydroponic culture, providing evidence that MDH is a potential Al-tolerance gene for plant in acid soils.

Key words: Citrus junos, MDH, expression characterization, transgenic tobacco, Al-tolerance