Isolation of Genes Involved in Soybean Response to Al Toxicity under Low pH Condition

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Central Research Institute for Food Crops
Al toxicity in plants is one of the major limitation of crop production on acid soils. Because of complex interaction between Al and a plant, it is very likely that there are 3 number of different mechanisms used by plants to confer Al resistance. Genetic studies of Al resistance have shown it to be a dominant, multigenic traits controlled by one or a few major genes and several minor genes. The aim of our investigations was to study such gene activities involved in molecular mechanisms of plant stress resistance. Therefore our research started with application of Differential Display RT PCR (DD) approach to find some differences in mRNA population between roots and suspensions cultured cells of sensitive (Malabar and Lumut) and tolerant (Tambora and Wills) soybean (G/ycrne max L. Merril) lines under stress and non-stress conditions. After set of DD experiments 12 clones specific to mRNA of stress treated tolerant lines were obtained and sequenced. Marathon cDNA amplification was used to prolong the DD fragments. Eight prolonged PCR fragments were obtained and enhanced expression of corresponding mRNA under stress conditions was verified by Reverse Northern Blotting. In order to isolate complete protein coding cDNA sequence, selected fragments were used to screen ^-ZAP cDNA library produced from root tip and suspension cultured cell cDNA from tolerant soybean lines. After putative search, clones were obtained. Their differential expression under conditions of aluminum stress was confirmed by Reverse Northern or Northern Blotting experiment. The clone II-58 1b2 is coding for protein of 168 amino acids. It has 80% homology to translation ally controlled tumor protein from plants. The clone II-63 2a3 is coding for protein of 319 amino acids having 77% identity with inosine-5'-mo no phosphate dehydrogenase from Arabidopsis thaliana. The clone 1-31 bb is coding for protein of 539 amino acids having 35-42% identity with ATP-dependent transporter family. In addition, several genes of probable interest in Al resistance mechanisms were tested for their A I-responsiveness by Reverse Northern Blotting. Eight genes, for example G-protein, a-subunrt, glutathione-S-transferase, calreticulin, ma I ate dehydrogenase, metallothionein MT1, either gave equal signal or did not give any signal at all. For soybean malate synthase, Walii and Wah4 (wheat aluminum induced) genes as well as for PEP carboxylase turned out to be possible to prove that their expression was enhanced under conditions of Al stress. In order to study influence of obtained genes on plant resistance to Al stress It is planned to over express selected genes in soybean protoplas and Nicotians plumbaginifolia as a model organism.
Al-toxicity, low pH, Gtycine max, genes