| 40%of the arable land on earth is the acidic soil. Al toxicity is considered to bethe main point that limits plant growth and crop yield on acid soil. For soybean, Alinduced root secretion of citric acid is the most important mechanism to resist Altoxicity.We found that GmNADP-ME gene can up-regulation in resistance toaluminum stress according to the preliminary transcriptome experiments.in thisstudy,in order to analysis function of GmNADP-ME, we combined withbioinformatics analysis and focus on the root secretion citric acid induced Al toxicityin the soybean. The main methods and results are as follows: choosing resistanceof Altoxicity soybean variety jiyu70as experimental materials,using reverse transcription(reverse-transcription,RT) aluminum treated soybean roots cDNA astemplate,amplified the open reading frame of NADP-ME(open reading frame,ORF).the full length was1769bp, By sequence alignment and phylogenetic tree analysis,wefound that GmNADP-ME is similar to Phaseolus vulgaris L.and,ArabidopsisNADP-ME,subcellular localization showed that GmNADP-ME protein is located inthe cytoplasm,chloroplast and other organs, in accordance with the characteristics ofNADP-ME protein. We found the expression of the transcription of GmNADP-MEgene began to increase rapidly after4h aluminum treatment,and the expressionreached the highest value,then the expression gradually decreased with the prolongingof treatment. Using GmNADP-ME was constructed into the plants expression vectorpCAMBIA3301by using In-Fusion cloning technique,and obtained the fusionoverexpression plants, Soybean was infected by Agrobacterium rhizogenes mediatedof K599,using the luminometer to detect luciferase infusion GmNADP-ME plant, andgained the positive plants with GmNADP-ME gene.positive soybean roots containingGmNADP-ME gene. Staining observation of Al toxicity degree with hematoxylinassay,and determinate the rate of secretion of citric acid under the stress of Al by enzyme assay.Compared with the wild type and load vector,showed that overexpression of GmNADP-ME can reduce the amount of aluminum in soybean roottips,and can produce more citric acid. According to the above study,indicating thatGmNADP-ME is an early gene in response to Al treatment,combined with itsbiological function,suggesting that GmNADP-ME may provide carbon skeleton forthree tricarboxylic acid cycle,so as to promote the accumulation of citric acid in thecells,thereby enhancing the secretion of citrate under aluminum stress,and ultimatelyimprove the aluminum tolerance in Soybean. |
