Identification Of Low Potassium Tolerance Varieties And Function Analysis Of Regulated Genes On Potassium Stress In Soybean

Potassium is one of the most important inorganic cations required for plant growth and metabolism, the potassium of absorption and utilization by plant was mainly in the form of available K in the soil, and widely distributed in various tissues of the plant, in natural and agricultural production, Potassium deficiency frequently limits plants growth because of the low amount of soluble K that can be directly absorbed by plants, therefore, in order to adapt to the lack of potassium, maintain the potassium balance of the plant, plants have formed a number of adaptive mechanisms to reduce or respond to potassium deficiency in the long-term evolutionary process, including changes of root morphology in the structure, improvement of the rhizosphere soil chemical properties, induction of high-affinity potassium transporter protein and low-affinity potassium ion channel protein, regulation expression of response genes.Absorption and transportion of available potassium by plants from the external environment is mainly done through a variety affinity potassium transporter and channel protein which expressed in plant root cell membrance, so far, the study of potassium ion channel proteins and transport proteins were deeply investigated in the model plant: Arabidopsis.Homology-based cloning has allowed the isolation of a large number of genes encoding K+transporters and channels which control the plant potassium nutrition related traits. Potassium is essential element for the soybean, but it’s little known about the regulation mechanism of the soybean potassium transporter and channel proteins function and expression. In order to clarity the soybean potassium transporter and channel protein expression patterns in the low potassium stress conditions and the stress gene response mechanism, principal component analysis and membership functions had been used to evaluate the low K-resistant traits of ten soybean genotypes in this study, according to the relevant indicators of phenotypic properties and root characteristics, we screened the low K tolerance variety and intolerance variety. Through low K treatment for these two varieties, we used the Solexa/Ⅲumina sequcencing to understand the difference of response gene between the two varieties. Real-Time PCR was used to study the expression patterns of different potassium transporter and channel proteins, we selected the relative potassium transport genes to further research, a green fluorescent (GFP)-tagged gene was fused the GmKUP or GmKEA genes to analysis their subcellular localization. Meanwhile, verify the functionality of these genes by transformation of Arabidopsis thaliana and soybean, and hopes to get the high KUE transgenic plants, the results are as follows:1We used the principal components analysis and evaluate low potassium tolerance of soybean genotypes. The tolerance genetypes were as follows:You06-71, Xudou8, Xudou13and Zhongdou33, The intolerance genetypes were as follows:Edou4, Xiangchun10, Hengchun04-11, Zhonghuangl3, according to the relevant indicators of phenotypic properties and root characteristics, we screened the low K tolerance variety (You06-71) and intolerance variety (Hengchun04-11).2. Root and leaf tissues of soybean seedling were used to examine the expression pattern of six potassium transport and channel protein under low K stress (0h,0.5h,2h,6h,12h,3d,6d,9d,12d), including GmKAT1, GmKC1, GmKUP, GmKEA, GmKAB and GmKAT2. The result showed that the expression of GmKCl and GmKEA were significantly increased in the two soybean varieties, indicated GmKC1and GmKEA were intensity induced by K deficiency, the expression level of GmKUP and GmKATl in tolerance variety were more than in intolerance variety, supposeded the four genes had relationship with the regulation of potassium tolerance. In addition, the expression level of GmAKT2and GmKAB1were no significant difference, indicated GmAKT2and GmKABl were slightly induced by K deficiency.3. The TBpred prediction server was used for searchs that yielded unambiguous results with positive scores for the integral membrane protein. To verify the subcellular locations of GmKUP and GmKEA, a green fluorescent protein (GFP)-tagged gene was fused to the GmKUP and GmKEA genes. Transformed into onion epidermal cells by agrobacterium infection. A clear GFP signal was observed at the periphery of onion epidermal cells bombarded with the GmKUP/GFP or GmKEA/GFP construction, whereas the signal was seen throughout cells expressing free GFP. Localization of the GmKUP/GFP and GmKEA/GFP fusion proteins to the periphery of the cells indicated that the two proteins are targeted to the plasma membrane.4. The GmKUP and GmKEA gene were overexpressed in Arabidopsis to study their function under low K stress. The results showed that as the low K treatment time increased, the expression level of GmKEA gene was increased, but the K content of GmKEA over-expressed material was decreased and do not have the resistant phenotype for low K stress. Meanwhile, compared with the wild type, the GmKUP over-expressed material was superior on the index of target gene expression level, K content, K absorption rate, and phenotype under low K stress, and make up for the damage which caused by the low K stress. But compared with the normal MS medium treatment, the root of GmKUP over-expressed material was still slow growth, indicated the number and activity of transporter protein increased may still not fully meet the needs of potassium for plant.5. In order to verify these potassium-related genes, we had a preliminary study on the soybean genetic transformation and optimize the steps involved in the genetic transformation process, including the choice of the transformation receptor, the content of AS, the time and concentration of Agrobacterium infection,and obtained part of GM plant. These would establish the foundation for gene associated high KUE of genetic transformation in future.