The Expression And Regulation Of A High Affinity K~+Transporter Gene From Salicornia Europaea

Potassium is an essential nutrient for development and enables plants to resist abiotic strsee. The high-affinity K+transporters paly an important role in K+uptake when plants encounter potassium deficiency. So far, the researchs of HAK/KUP/KT family genes expression patterns are limited to K+starvation and salinity. How phytohormone and NH4+regulate their expression is rarely known, and other abiotic stresses which HAK/KUP/KT may response to are not reported. We have isolated AlHAK1and SeHAKl encoding high K+teansporters from Aelluropus littoralis and Salicornia Europaea respectively. AlHAK1could be induced by phytohormone and abiotic stresses. The result of semiquantitative PCR indicated that SeHAK1was induced by K+starvation. Other factors regulating SeHAK1are known little. For clarifying the regulatory pattern at molecular level, real-time PCR was used to study how SeHAK1was regulated by phytohormone and abiotic as well as when and where SeHAKl expressed. Gene transcription or not and transcription strength are determined by promoters. SeHAK1promoter was isolated with anchor PCR in order to explore its roles in mechanism of regulation.The real-time PCR assay for studying expression of SeHAK1was established. The primers were designed according to kouse keeping genes actin and eEF which were isolated from Salicornia Europaea. Analysis of melting curves indicated that the PCR amplifications of eEF and SeHAK1were specific except for actin. So eEF served as internal control. Standard curve showed that PCR efficiency of eEF and SeHAK1was almost the same.SeHAK1could be detected in both root and shoot, and its expression in root was as4.2times high as that in shoot. Compared to expression in one-month-old plants, SeHAKl was expressed2.9times higher than that in one-week-old plants. Interesting, the expression pattern of SeHAK1responsing to salinity was significantly different. SeHAKl was induced in one-month-old plants but inhibited notablely by salinity in one-week-old plants. The Phytohormone such as ABA and GA3upregulated expression of SeHAK1. It was induced markedly by abiotic including K+starvation, drought, cold and PH but inhibited significantly by NH4+. All of these results revealed that SeHAK1might response to abiotic and phytohormone.In order to study the roles of SeHAKl promoter in gene expression, a fragment of1858bp upstream of SeHAKl was isolated with anchored PCR. Analysis this promoter with on-line software (PLACE and plantCARE) indicated that there were various cis-elements such as HSE (response to heat), ARE (response to anaerobic), MBS (response to drought), ABRE (response to abscisic acid) and GARE-motif (response to gibberellin). The result implied that why SeHAK1could response tophytohormone and abiotic may attribute to these cis-elements.SeHAK1promoter fragments of5’ series delections were inserted into modified binary vector for Agrobacterium-mediated tobacco plants transformation. Histochemical staining of plant tissues and organs revealed that all of the four series SeHAK1promoter franments drived GUS expression in leaves, stems and roots.