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

Characterization of Malate Dehydrogenase Gene from Citrus junos and Its Transgenic Tobacco's Tolerance to Aluminium Toxicity

为研究柑橘砧木香橙对酸性土壤的适应机理,从‘资阳香橙’根cDNA文库中随机测序克隆了苹果酸脱氢酶基因CjMDH(GenBank accession No.ABI75147)。该基因cDNA序列长1368bp,其开放阅读框为1239bp,推测其编码412个氨基酸残基,在编码氨基酸序列中存在1个苹果酸脱氢酶的NAD结合位点和8个苹果酸结合位点,iPSORT分析表明该基因编码氨基酸序列N端存在叶绿体转移肽。同源性分析显示CjMDH与拟南芥、烟草、大豆、豌豆、水稻、苜蓿等植物的MDH一致性约为80%,相似性在85%以上。在与苹果酸脱氢酶功能有关的NAD和苹果酸结合位点处氨基酸残基高度保守,表明这两个区域是MDH重要的功能区。Northern blot和Real-time RT-PCR分析结果表明该基因在香橙根和叶中优势表达,在根中表达量最高,茎中表达最低。将CjMDH基因构建到组成性启动子CaMV35S驱动的载体中,对烟草进行转化,从转基因烟草株系中筛选获得了3个CjMDH基因高表达的株系。将表达量高的转基因株系进行耐铝试验,初步结果表明在烟草中超量表达CjMDH可以提高植株对铝毒的耐受能力。

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 ‘ Ziyang＇. CjMDH has an open reading frame （ORF） of 1 239 bp encoding a putative protein with 412 amino acid residues, which showed high identity to other MDHs in plants. The deduced protein contains a NAD binding site and eight malate binding sites. Chloroplast transit pepite was 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. 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.