Functional Studies Of Tobacco Shaker Potassium Channels NsylNKT6 And NtomSKOR1 In Potassium Absorption And Transport

Tobacco is a typical potassium-loving crop.Potassium not only participates in the growth and development of tobacco from many aspects,but also serves as an important quality element,regulates the physiological and biochemical reactions of tobacco,and affects the quality,combustion and safety of tobacco products.Tobacco absorption of potassium ions is mainly mediated by potassium channels and potassium transporters.The Shaker potassium channel is the earliest and most extensively studied type of potassium channels.It is of great significance to excavate the key Shaker potassium channel gene of tobacco and analyze its function and regulation mechanism in the process of potassium nutrition for genetic improvement of high potassium in tobacco leaves.In this study,we took the Shaker channel of Nicotiana sylvestris NsylNKT6 and the Shaker channel of N.tomentosiformis NtomSKOR1 as the research objects,through bioinformatics analysis,histochemical staining,phenotype identification of overexpressed materials,etc.,the function of the two genes in potassium absorption and transport was initially investigated.To clarify the tissue expression pattern of NsylNKT6,a cis-acting element prediction of the gene promoter sequence was performed.The results show that the NsylNKT6 promoter contains light-responsive elements,plant hormone-responsive elements(abscisic acid,ethylene,gibberellin,etc.),stress response elements(anaerobic,drought,disease resistance,wounds,etc.)and meristematic expression elements,etc.It is speculated that the expression of this gene may be regulated by various environmental signals such as light,hormones and stress.The transgenic material for the GUS reporter gene driven by the NsylNKT6 promoter was further obtained.GUS histochemical staining results showed that under normal culture conditions,GUS activity was detected in guard cells of the leaf at the seedling stage and in vascular tissues of the leaf veins,glandular hairs,and guard cells of the leaf during the rosette stage,and it was speculated that the gene was fuction in the above tissues.Using the yeast heterologous expression system,the potassium uptake activity of two different clone-encoded proteins(Nsyl NKT6-1and NsylNKT6-2)obtained in the previous period was detected.The results show that NsylNKT6-1 has no potassium absorption function,and NsylNKT6-2 can well supplement the growth of potassium absorption-deficient yeast on low potassium medium,and has a K~+inward transport capacity similar to the Arabidopsis Shaker potassium channel AKT1.Further bioinformatics analysis showed that the functional difference between the two proteins of NsylNKT6 may be caused by the difference in the properties of amino acids 187 and 398.The homozygous material for NsylNKT6 overexpression in Arabidopsis thaliana was obtained through transgenic,selfing and resistance screening,and its K~+accumulation level under different potassium supply levels was determined by hydroponic experiment.The results show that under low potassium(0.1 mM K~+)and normal potassium supply conditions(1.00mM K~+),the growth and the potassium content in the roots of the Arabidopsis overexpression material were significantly higher than those in the wild type,indicating that NsylNKT6 promoted K~+absorption and growth in Arabidopsis.Based on the above research,it is speculated that NsylNKT6 may be an important inward Shaker potassium channel,which may participate in the regulation of stomatal movement or mediate K~+absorption and transport of vascular tissues and the development of glandular hairs by regulating K~+absorption.The 2439 bp NtomSKOR1 promoter was cloned from N.tomentosiformis.Predictive analysis of cis-acting elements indicates that the promoter contains light-responsive elements,plant hormone-responsive elements(abscisic acid,auxin,salicylic acid,jasmonic acid,etc.),stress response elements(anaerobic,drought,low temperature,disease resistance and Wounds,etc.)and endosperm expression elements.A2484 bp NtomSKOR1 coding region was cloned from N.tomentosiformis.Bioinformatics analysis showed that the NtomSKOR1 protein belongs to the Shaker family outward rectified potassium ion channel subfamily Group V,which is predicted to have 5 transmembrane regions,contains Ion trans,cNMP binding,Anky,KHA conserved domain and Shaker potassium channel highly conserved motif TxxTxGYGD.The subcellular localization vector of NtomSKOR1 was constructed,and the subcellular localization of NtomSKOR1 was studied by Agrobacterium transient transformation experiment.The results show that the NtomSKOR1 protein is mainly localized in the cell membrane,which may be involved in the potassium efflux of the cell as a membrane-located Shaker potassium channel.At the same time,the ProNtomSKOR1::GUS transgenic tobacco material and tobacco materials over-expressing NtomSKOR1 were obtained through the leaf disc method,which laid a material foundation for subsequent research.