Cloning And Functional Verification Of Two Aluminum Resistant Genes GmNramp5Like And OsMGT1

Aluminum is not an essential mineral nutrition for plants, while it is widespread in soils. As much as 50% of the world’s potentially arable lands are acidic (pH values below 5.0). In acidic soils, Al is solubilized as highly phytotoxic Al3+, which inhibits root elongation at micromolar concentrations, and results in reduced uptake of water and nutrients. It can also rapidly trigger other stress responses such as elongation of root hair, mineral nutrient uptake, in vitro seedlings regeneration and the transmission between the cells and so on. Al toxicity constitutes a food security threat exceeded only by drought with regard to abiotic limitations on crop production.Our understanding of the physiology of Al3+resistance in important crop plants has increased greatly over the past 20 years, largely due to the application of genetics and molecular biology. It is of great significance that to increase the amount of crops and to improve the utilization rate of acidic soil, through transgenic technology to enhance plant resistance to aluminum toxicity on acidic soil. In this study, two aluminium resistance related genes were cloned and functional analyzed via plant growth system or yeast growth system. The main results of this study are listed below.1) The rice magnesium transporter gene OsMGTl, which was cloned in our previous study, was re-cloned with high fidelity Polymerase Chain Reaction (PCR) enzyme to add recognition sites for two specific restriction endonuclease enzymes. A plant expression vector pCambia1301-35S::OsMGT1 was further constructed using conventional molecular biology technologies. Construction of the plant expression vector was confirmed by PCR analysis and double restriction enzyme digestion.2) Using floral-dip method, the pCambia1301-35S:.OsMGT1 was transformed into Arabidopsis thaliana plants both wild type (Col-0) and ALMT/MATE double mutant. Antibiotics screening and PCR tests confirmed that OsMGT1 gene had been successfully integrated into the Arabidopsis thaliana genome. Aluminum resistance related test and analysis will be proceed once the transgenic plants were homozygous in higher generation.3) Soybean GmNramp5Like gene, a homologous gene to rice OsNratl which encoding an Aluminium transporter gene in rice, was cloned by homology-based cloning method. GmNramp5Like gene consists of 13 exons and 12 introns with the genomic sequence of 4043 base pairs. The length of its CDS is 1635 base pairs. It encodes a peptide of 544 amino acids. Phylogenetic tree analysis shows that GmNrampSLike gene is very similar to OsNratl gene, AtNrampl gene and AtNramp6 gene. Twelve transmembrane domains were predicted by TMHMM online software in GmNramp5Like encoded protein.4) GmNramp5Like gene was successfully cloned into yeast expression vector pYES2. Both blank vector control pYES2 and new constructed pYES2-GmNramp5Like were transformed into yeast strain By4741. Further results of yeast plant aluminum tolerance experiment showed that compared with control pYES2, the ability of aluminum resistance was significantly improved for the yeast with recombinant vector pYES2-GmNramp5Like. Thus, GmNramp5Like gene may be involved in the great improvement of aluminum resistance of yeast, which strongly suggesting that over expression of GmNramp5Like gene in plant might show us the same function.