Adenylate Kinase Gene Gmadk Cloning,Vector Construction And Genetic Transformation In Soybean

Adenylate metabolism is an important part of the primary metabolism, and the amount of the adenylate compounds is considered to be one of the main factors affecting the cellular energy metabolism. Adenylate kinase (ADK, EC.2.7.4.3) is a phosphotransferase enzyme that catalyzes the interconversion of adenine nucleotides, which is required for the biosynthesis of ADP and is essential for homeostasis of adenosine phosphates. Adenylate kinase is the key enzyme that regulates the adenosine pool, and its expression level directly affects the distribution of adenosine nucleotides. Studies have reported that the transgenic potatoes with antisense ADK gene expression vector have reduced ADK activity, leading to the increase of adenosine content in transgenic potato tubers and a significant increase of39%in tuber yield. Others studies have shown that the ADK may be involved in plant tolerance to salt, drought and other abiotic stresses. No research on soybean ADK has been reported.Soybean genetic transformation is an important tool for molecular breeding, germplasm enhancement and studying gene function. Agrobacterium-mediated transformation method has the several advantages over the others such as low copy-number, low cost, little gene silencing, and high genetic stability. But there are still many problems in this method such as the long process, low efficiency, and high chimera ratio. These problems remain unresolved.In this study, based on the location of a major QTL for soybean salt tolerance provided by National Center for Soybean Improvement and the genomic information, a key enzyme in the adenylate metabolic pathways-adenylate kinase (GmADK) gene is a good candidate for this QTL. We cloned the GmADK gene by its coding sequence, and characterized the GmADK gene structure and its protein using bioinformalic tools. We constructed its over-expression vector and RNA interference vector, and transfered them into soybean. Details as follows: We cloned the adenylate kinase gene (ADK) from the soybean cultivar "Nannong1138-2" by RT-PCR, and constructed a plant over-expression vector and a RNA interference vector by Gateway technology, and did bioinformatic analyses. The results showed that the GmADK coding sequence is801base pairs long and encodes a polypeptide of267amino acids. It was predicted to locate in the plastid, including a typical protein functional domain of adenylate kinase ATP-AMP (Ap5A) binding site and a AMP binding site, the three-dimensional model of the ADK protein show a typical three-dimensional structure. The constructed vectors were introduced into Agrobacterium tumefaciens strain EHA105, and transferred to different soybean varieties, which would be useful to study the gene function of GmADK.In order to optimize the Agrobacterium-mediated soybean cotyledonary node genetic transformation system, we used the cotyledonary node explants from different soybean genotypes, the bar and hyg as selection marker genes, gus as a reporter gene, to study the various factors that affect the efficiency of induction and regeneration in soybean transformation. The results showed that different genotypes has different optimum sterilization time and different sensitivity to the selection markers. The1/2MS more suitable for the germination medium. Explant cut in different ways lead to the different regeneration rate of buds. The induction rate of soybean cotyledonary node buds is the highest and the infection result is the best when the Agrobacterium concentration OD600value is0.6, infection time is30min, co-cultured pH5.4, and co-cultured for5d.