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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。
In order to study the adaptive mechanism of citrus rootstocks to acid soils, the malate dehydrogenase gene CjMDH (GenBank accession No. ABI75147) was cloned from the cDNA library of Ziyang orange. The cDNA sequence of this gene is 1368 bp in length and has an open reading frame of 1239 bp. It is deduced that it encodes 412 amino acid residues. There is one NAD binding site and 8 malate binding sites of malate dehydrogenase in the encoded amino acid sequence, iPSORT analysis showed that the gene encoding amino acid sequence N-terminal chloroplast transfer peptide. Homology analysis showed that the MDH identity of CjMDH with Arabidopsis thaliana, tobacco, soybean, pea, rice, alfalfa and other plants was about 80% and the similarity was over 85%. The amino acid residues are highly conserved at the NAD and malate binding sites associated with malate dehydrogenase function, indicating that these two regions are important functional domains of MDH. The results of Northern blot and Real-time RT-PCR showed that the gene was predominantly expressed in roots and leaves of orange, and the highest expression was found in roots and lowest in stems. The CjMDH gene was constructed into a CaMV35S-driven vector and transformed into tobacco. Three CjMDH gene highly expressed lines were obtained from the transgenic tobacco lines. The transgenic lines with high expression level were tested for aluminum tolerance. The preliminary results showed that overexpression of CjMDH in tobacco could improve the tolerance of plants to aluminum toxicity.