Studies On Potassium Transporter Genes Oshaka And Oskb And Their Relationship With Salt Tolerance In Rice

Potassium(K+) is an important macronutrient and the most abundant cation in higher plants which plays a key role in various cellular processes and involved in osmoregulation of stress resistance in plants. Its accumulation from soil and its distribution throughout diverse plant tissues is mediated by transporter proteins. According to the protein transmembrane structure, it can be fell into five major branches:Shaker channel, KCO channels, KUP/HAK/KT transporters, TrK/HKT transporter and K/H antiporter. In this paper, we research OsHAKa of HAK transporters and OsKβ of Shaker channel and its role in salt tolerance in rice.By studying physiological indicators of OsHAKα and OsKβ mutant and the corresponding wild-type in salt stress including phenotype, potassium and sodium ion content, mortality, and fresh and dry weight, to prove their mutant is sensitive to salt in salt injury. It was found that the OsHAKa mutant in200mmol/L NaCl concentration, seed germination time delay, the germination rate was70%lower. OsKβ mutants in certain salt concentration range(0-250mmol/L), the seed germination rate was not significantly change. Salt concentration in the0-300mmol/L, two mutant gene mutant root under low concentration of salt solution has the certain variable length. High salt treatment inhibited the growth of the root length, the length is about50%of the control seedlings. Two gene mutant are subject to inhibition salt solution, height of about50%of the control seedlings.120mmol/L NaCl concentration of processing rice seedlings8d, two gene mutant around1.25times higher than wild type corresponding mortality, dry weight decreased about60%, fresh weight decreased by about75%.120mmol/L NaCl processing0d to8d, Na+content of OsHAKα mutants with the increase the number of days is elevated, K+content is reduced, in8d, Na+content is1.3times that of wild type corresponding, K+content is75%. The content of Na+and K+of OsKβ mutants did not change significantly with the number of days, but The mutants exhibit high mortality rates than the corresponding wild-type. The results show that OsHAKα and OsKβ may be positive regulation in rice salt resistance. The results provides the basis for the further study of gene function and mechanism of action.In order to further verify the role of the two genes in salt tolerance, we use the method of RNA interference. First, we select two different length specific fragments with a different length cDNA fragments from3’end of OsHAKα, in the same way, we select one specific fragment of OsKβ, and another specific fragment from intermediate transmembrane functional areas. Use of rice protoplast for material first, by double-stranded RNA instantaneous interference system which can be used to study gene function rapidly, to put the vitro-synthesized dsRNA from the four specific fragments were ligated to the T7promoter into rice protoplasts, results of semi-quantitative RT-PCR showed that the expression level of OsHAKα and OsKβ were obviously declined. The interference rate of OsHAKα can reach65%, the interference rate of OsKfβ can reach60%. The four pieces into the protoplast can obviously lower the expression of genes.The specific fragment we chose from OsHAKα and OsKβ is ultimately in order to turn into rice seedlings can be inherited stably. The two positive and antisense gene fragments were inserted into the both ends of the functional intron PDK joined in expression vector pSuper1300. We constructed the expression vector ihpRNA containing the intron hairpin structure. By agrobacterium-mediated, the vectors were transfected rice callus. After resistance screening and PCR detection, we get positive transgenic plants. Semi-quantitative RT-PCR results showed that the expression level of OsHAKα and OsKβ were obviously declined in the transgenic rice plants. It showed that the interfering carrier can effectively restrain the expression of genes of OsHAKα and OsKβ. The ihpRNA interference vector was successfully constructed, it will laid the groundwork for further identification of the gene’s function.