Cloning And Functional Study Of A Soybean E3Ubiquitin-Ligase Gene GmARI

Soybean is the major source crop of plant oil and protein. In the world, acid soil constitutes40%of the arable soil, and70%of the unarable soil. In China, the acid soils comprise approximately21%of the total land area. Aluminum(Al) is one of the most important factors limiting plant growth in acid soils. The molecular basis and mechanisms of the soybean tolerance to Al are not clear, which is important for the soybean breeding program. Previous research has discovered soybean genotypes and QTLs for Al tolerance, which laid a good foudation for the soybean breeding to enhance Al tolerance and the discovery of Al tolerance genes.With the development of biotechnology, researchers pay more attention to use genetic engineering to breed new cultivars with high yield, fine quality and resistance to diseases, pests and abiotic stresses. Soybean transformation is important for functional studies of genes and molecular breeding, but many difficulties still exist. Establishing a stable transformation system is critical in soybean research.Based on the previous studies on the location of an Al tolerance QTL, single nucleotide polymorphisms and the predicted gene function, we cloned two Al tolerance candidate genes and analyzed their functions. At the same time, based on previous studies, we chose seven soybean genotypes with good regeneration rates to compare their sprouting rate, and made some modifications for the soybean transformation protocol. The main results were as follows:1. According to the QTL mapping results and the predicted functions of the candidate genes, two E3ligase genes, named as GmARI(Glycine max ARIADNE)1and GmARI2gene, were cloned.The length of their ORF is1758bp and the putative corresponding proteins are composed of586amino acids. They both have15extons and14introns. The proteins belong to the RBR superfamily with a typical IBR(C6HC) domain and two RING domains.Both GmARI proteins were localized in the nucleus of arabiposis protoplasts cells. The transcripts of GmARI1and GmARI2were expressed in root, stem, leaf, apex, flower and pod with similar amount, and were up-regulated by Al3+, Na+, JA, Man, IAA, ACC, SA and GA3. The trend of induction was more obvious in roots than that in leaves. It is consistent with the fact that the Al toxicity is mainly acting on the roots. Therefore we proposed GmARI1gene might participates in the signaling pathway of Al tolerance. We also transferred the genes into arabidopsis, and treated the transgenetic plants with15μM Al3+(pH4.3). Under Al treatment, the roots of transgenetic plants were significant longer than the wild type. Therefore, the overexpression of GmARIl gene can improve the Al tolerance of plants.2. The comparison of the regeneration rates among soybean genotypes showed Dalihuang and Williams had higher regeneration rates. Using Dalihuang for the soybean transformation, we obtained16transgenic plants with kanamycin resistance. However, the transgenic plants later were found having no target genes, which might be due to the insensitivity of soybean to kanamycin. Later, we modified our soybean transformation protocol on the aspects of seed germination and explant treatments. In the new method, the explants were infected with Agrobaeterium immediately after wounding, which reduced the time period when explants exposured to the air. In addition, we put the GUS reporter gene in the vector to assess the transformation rate.