香橙苹果酸脱氢酶CjMDH基因表达特性及转基因烟草的耐铝性分析

Malate, ubiquitously existing in plan,t plays a crucial role in variousmetabolic pathways, including respiration, nitrogen fixation, phosphorus acquisition, and aluminum tolerance. Malate dehydrogenase(MDH) catalyzes the interconversion ofoxaloacetate andmalate. Previous results suggested that overexpressing MDHgene transgenic plants elevated tolerance to aluminum toxicity.Citrus junos, one ofmain rootstocks of Citrus fruits, iswidely used in the southern ofChina. Asmostof soils are acid in the south, to investigate themechanism of tolerance to aluminum inCitrusplants, a clone referredCMj DH(GenBank accession No. ABI75147) was isolated from the cDNA library of root fromCitrus junos‘Ziyang’.CMj DHhas an open reading frame (ORF) of1 239 bp encoding a putative proteinwith 412 amino acid residues, which showed high identity to otherMDHs in plants. The deduced protein contains aNAD binding site and eightmalate binding sites. Chloroplast transitpepitewas found inN-terminal ofCMj DHthrough iPSORT software. BlastP analysis result reveals over 80% identity and 85% similarity withArabidopsis thaliana, Nicotiana tabacum, Glycine max, Medicago sativa. Amino acids comparison between CMj DH and otherplantsMDH revealshighly identity inNAD andmalate binding site. Southern blotsuggested thatCMj DHis a single copy gene inCitrus junosgenome. Northern blot and Real-time RT-PCR data indicated thatCMj DHexpressed strongly in root and lea.f Subsequently, theCMj DHwas constructed into over-expression vector driven by constitutive CaMV35S promoter. The constructionwas introduced into tobaccomediated byAgrobacterium tumerfaciens, the overexpression transgenic tobacco lineswere obtained throughNorthern blo.t Transgenic lines increasedAl-toleranc in hydroponic culture, providing evidence thatMDHis a potentialAl-tolerance gene for plant in acid soils.