Effects Mutation Of OsHAK16 And Genetic Variation Of OsHAK1 And OsHAK5 On Potassium Use Efficiency And Salt Tolerance In Rice

Potassium(K)accounts for 2-10%of plant dry weight,far higher than other inorganic cations.In the process of plant growth and development,K can maintain the balance of cation and anion,regulate cellular osmotic and cell membrane potential,promote photosynthesis efficiency,regulate enzyme activity,etc.The low available K content in many soils,especially in the Southern part of China,is generally insufficient and becomes one of the limiting factors of sustainable development of agriculture.Understanding the molecular mechanism of plant K absorption and transport will help to breed K efficient cultivars.HAK/KUP/KT transporter proteins are widely contained in microorganisms and plants.The members of HAK/KUP/KT family in the plant are divided into four or five sub-groups,and studies of the physiological function of the sub-group-I show that they are involved in the high affinity transport of K.OsHAK1 and OsHAK5 belonging to the subfamily are located in plasma membrane and are involved in root K uptake and allocation of K from root to shoot and salt tolerance.Therefore,in-depth study of the physiological function of HAK/KUP/KT transporter is expected to provide a theoretical basis and help for breeding K-efficient cultivars.In this dissertation,the physiological function of osHAK1 6 gene,another transporter located in subgroup-I of HAK/KUP/KT family,was analyzed.Meanwhile,the representative materials of OsHAK1 and OsHAK5 genes with genetic variation were selected from the deep sequenced rice core germplasms,which were used to evaluate their differences in tolerance of low K and high salt supplies.The main results obtained are as follows:1.Subcellular localization analysis shows that OsHAK16 is expressed mainly on the cytoplasm membrane,indicating that the transporter may be involved in root K uptake and distribution between cells.The staining of GUS report driven by OsHAK16-promoter indicated that OsHAK1 6 is expressed at the root,root-shoot junction,stems,leaves,glumes.Limiting K supply enhanced expression of OsHAK16 in rice roots.2.In comparison to WT,OsHAK16 knockout mutant showed significant lower root K uptake rate,lower biomass at both low(0.1 mM)and normal(1 mM)K supply conditions.The mutation of OsHAK16 also reduced K transport from root to shoot.When K in the culture solution was equally replaced with 0.2 mM or 1 mM(Rb),the mutation of OsHAK16 also significantly reduced the root uptake and allocation rate of Rb between root and shoot,further confirming the function of OsHAK16 in rice K nutrition.3.In the yeast heterogeneous system,external expression of OsHAK16 was able to increase K accumulation in a salt-sensitive yeast cells G19 cultured at a high salt(NaCl)medium while maintaining the Na content basically unchanged.As indicated in biomass increasement,OsHAK16 expressing resulted in significant higher K/Na ratio and improvement of the yeast tolerance to salt stress.4.Compared to WT,rice OsHAK16 mutants were more sensitive to the salt stress,with significantly lower dry weight and K concentration at both root and shoot,while the Na concentration was significantly increased,resulting in larger decrease of K/Na rato,indicating that OsHAK16 functions in enhancing the salt resistance of rice.5.By analyzing the natural genetic variation of OsHAK1 gene sequences,18 representative materials belonging to different OsHAK1 haplotypes were selected.A comparative study of the absorption and utilization of K and salt-resistant stress in the seedling period of these 18 materials found that the four materials numbered 1-3,1-6,1-14 and 1-18 showed higher physiological utilization efficiency of K under low K supply(0.1mM).The five materials,numbered 1-2,1-3,1-5,1-8,and 1-18,not only showed higher biomass accumulation,but also highly tolerance to salt stress.Adding additional 50 mM NaCl to the nutrient solution for two weeks did not significantly affect the increase of biomass accumulation of the four materials,especially for 1-18 that maintained exceptional high K/Na ratio.In addition,grown in low K paddy field,the three materials 1-8,1-13,1-18 displayed similar effective tillers,plant height,grain yield,and grain/straw ratio with a local high yield cultivar,Wuyunjin 27,of which the material 1-18 show the excellent characteristics of tolerance to low K and high salt with high yield potential.6.By analyzing the natural genetic variation of the OsHAK5 gene sequences,20 representative materials belonging to different OsHAK5 haplotypes were selected.A comparative study of the 20 materials in K absorption and utilization and salt-stress tolerance was conducted in the culture solution.It was found that the growth of the 8 materials numbered 5-1,5-2,5-11,5-13,5-17 and 5-20 was much less affected by limited K supply(0.1mM),showing higher physiological use efficiency of K.Moreover,addition of 50 mM NaCl to the nutrient solution for two weeks did not significantly affect the bioaccumulation of the four materials,5-3,5-14,5-15,5-17.The amount of K and K/Na ration in these four materials was significantly higher than that of other materials.In low available K field conditions,the three materials5-1,5-14 and 5-17 showed the similar effective tillers,plant height,grain yield with slightly lower grain to straw ratio with Wuyunjin 27.In summary,this study revealed the preliminary function of OsHAK16 participating in the root K absorption and internal transportation,isolated several K efficient elite materials from the representative OsHAK1 and OsHAK5 haplotypes.These materials can be considered to be used for molecular marker-assisted breeding of high K efficiency,salt tolerance with high yield potential.