The Research Of Potassium Nutrition Efficiency Response Of Transgenic AtCIPK23 Tobacco Plants And Molecular Cloning And Characterization Of Putative Potassium Transporter NtHAK1 In Tobacco

K⁺ palys a key role in plant development and is the most abundant cation ion in cytoplasm. Great difference exsits among orgnisms and genotypes in the same organism in K⁺ nutrition efficiency(KNE).In agricultural practice in China,potassium deficiency in soil is popular across the country due to less potassium fertilizer input and poor potassium deposit.Soil potassium deficiency is the most important factor that prevents crop output and quality from further improving.Therefore,the research of molecular mechanism of potassium absorption is the fundamental step to improve PNE by means of the combination of traditional breeding method and genetic engineering method.AtCIPK23,a protein kinase,interacting with CBL1/9,is crucial for the response to low-K⁺ stress in Arabidopsis.Low-K⁺ stress signal may activate CBL1/9,which then recruit AtCIPK23 to the PM,where AKT1 is phosphorylated by AtCIPK23.As the result,AKT1 is activated for K⁺ uptake under low-K⁺ conditions.In this study,AtCIPK23 was successfully transferred to tobacco by means of Agrobacterium mediated disc transformation.Molecular identification indicated that AtCIPK23 was expressed at transcriptional and translational level in transgenic tobacco plants. Phenotype screening under low-K⁺ stress under either MS plate or hydroponic condition showed that compared with WT,the growth of transgenic plants were less restrained,their main root longer,plants bigger and leaf color more normal.Under low-K⁺ stress,in contrast to WT,dry matter of transgenic plants increased by 30%-70%on LK MS medium,and by 26.3%,13.5%and 20.5%under hydroponic condition respectively;potassium concentration increased by 34.0%, 21.9%and 9.4%respectively on LK MS medium,and by 187.5%,179.2%and 237.5%(upper leaves)respectively under hydroponic condition.K⁺ uptake dynamics measurement showed that compared with WT,the K_M and C_minof transgenic plants decreased by 26.2%,48.5%and 52.3%; and 29.8%,49.6%and 70.2%respectively.As refers to Vmax,no obvious difference exists between material.To conclude,for the first time,we have acquired higher PNE transgenic AtCIPK23 tobacco plants by the transfomation of one protein kinase which regultes K⁺ channel activity.Meanwhile,a putative new potassium transporter gene NtHAK1 has been cloned in Nicotiana tabacum as one step to understand molecular mechnism of K⁺ absorption in tobacco. Bioinfomatics analysis showes that NtHAK1 may encode a protein of 648 amino acid,molecular weight 72 kDa.NtHAK1 may localize in membrane because it has 9 TMS and 4 helixes structure. Conserved domain analysis indicates that NtHAK1 contains K⁺ transporter conserved domain COG3158(kup)/pfam02705(K_trans).NtHAK1 shares higher similarity with AtKUP10, AtKUP11,AtKUP5,AtHAK5 and HvHAK2 in DNA sequence,and AtHAK5,AtKUP11,AtPOT5, AtPOT9,AtPOT10,AtPOT11,OstHAK2,OsHAK11,high affinity K⁺ transporter in reed and capsicum,and K⁺ transporter in broad bean and grapes in amino acid.Therefore,NtHAK1 may encode a membrane protein related to K⁺ uptake and transportation.Expression model analysis of NtHAK1 showed that it expressed in root,stem,leaf and flower,even though lower in root.Finally, NtHAK1 is not induced by low-K⁺ stress.To investigate the impacts of the transformation of K⁺ transpoter and channel genes on PNE of transgenic plants,genes from Arabidopsis such as AtAKT1,AtKC1,AtKUP1,AtHAK5,AtCBL1, AtCIPK23+AtAKT1 and AtCIPK23+AtCBL1 were transformed into tobacco.Plenty of T1 transgenic lines were acquired.AtKUP1 and AtHAK5 were selected for the functional study of their role in Arabidopsis K⁺ nutrition.The primary result showed that compared with CW,atkupl and athak5 grown normally on low-K⁺ MS medium.Potassium concentration measurement indicated that there was no difference between CW and atkup1 and/or athak5 grown normally on either MS or low-K⁺ MS medium.