Cloning And Functional Analysis Of Four Potassium Transporter KUP Gene Of Tobacco

Potassium is one of the main mineral necessary elements in tobaceo growth and development,which is almost involved in all material and energy metabolism during the growth and development of tobacco.Adequate potassium supply not only can ensure the normal physiological metabolism and growth of tobacco plant,but also can improve the quality of tobacco leaves.Tobacco potassium ion uptake and transport mainly through two ways:potassium ion channel and potassium transporter.KUP/HAKjKT family is the largest family among four plant potassium transporter family(KUP/HAK/KT family,Trk/HKT family,KEA family,CHX family).Several KUP/HAK/KT potassium transporter genes have been cloned in Arabidopsis,rice,barley and maize plants,but these genes have been reported less in tobacco.In this study,four KUP/HAK/KT potassium transporter genes KUPI,KUP2,KUP3 and KUP4 were cloned from tobacco cultivar K326.The functions of these genes were studied by bioinformatics analysis,fluorescence quantitative PCR analysis and yeast functional complementation assay.The main results were as follows:(2)Four new potassium transporter genes were cloned by homology cloning,the full-length of genes were 2310bp,2532bp,2565bp and 2532bp,these genes were named NtKUP1,NtKUP2,NtKUP3 and NtKUP4.(2)KUP1,KUP2,KUP3 and KUP4 contained twelve transmembrane domains,hydrophobic structure and typical conserved domain of KUP/HAK/KT family.Further analysis showed that KUP1 belong to KUP/HAK/KT family cluster Ⅱmembers,KUP2,KUP3 and KUP4 belong to KUP/HAK/KT family cluster Ⅰmembers.(3)KUP1,KUP2,KUP3 and KUP4 were expressed in root,stem,leaf and flower of tobacco K326 by qRT-PCR analysis.These results were consistented with the expression characteristics of the KUP/HAK/KT family in plant tissues.(4)KUP1,KUP2,KUP3 and KUP4 were responsed to low potassium stress by qRT-PCR analysis.The expression level of KUP2 and KUP3 were decreased significantly,and the expression of KUP4 and KUP1 were increased significantly.(5)Yeast functional complementation test results showed the potassium deficient yeast strain R5421 with four KUP genes could grow on the low potassium medium,which indicated that they were high affinity potassium transporters.The yeast cells with four KUP genes were sensitive to Ca2+ during growth.(6)The tobacco potassium transporter genes(KUP1,KUP2)were successfully introduced into tobacco K326 by agrobacterium mediated method.Seven transgenic tobacco plants overexpression KUP1 were obtained,and eight transgenic tobacco plants overexpression KUP2 were obtained through PCR detection.