关键词: 山新杨, Shaker类钾离子通道, SKOR, 基因克隆, 基因表达, 膜片钳

Abstract:

Objective: Plant SKOR(Stelar K⁺ outward rectifier) is a typical outwardly rectifying Shaker type potassium(K⁺) channel, mediated long-distance K⁺ transport from roots to shoots. The objectives of this study were to isolate a K⁺ channel gene PdbSKOR from the hybrid of Populus davidiana × P.bolleana, to characterize the tissue-specific expression patterns of PdbSKOR gene and responses to K⁺ depletion, K⁺ excess, drought and low temperature treatments, and to determine electrophysiological function. Method: By carrying out homology-based cloning, a putative K⁺ channel gene PdbSKOR was characterized and cloned from P.davidiana × P.bolleana. The details of PdbSKOR gene and encoded protein were analyzed. MEGA7.0 software was used to construct a phylogenetic tree by multiple alignment of 14 SKOR proteins from different genera and families, including the hybrid of P.davidiana×P.bolleana. Expression profiles of PdbSKOR and responses to K⁺ depletion, K⁺ excess(60 mmol·L^-1 KCl), drought(15% PEG6000) and low temperature(4℃) treatments, especially in the roots, were analyzed using quantitative real-time RT-PCR(qRT-PCR). The electrophysiological function of PdbSKOR was preliminarily determined using patch clamping. Result: The K⁺ channel gene PdbSKOR(GenBank No. MT335814) was cloned from hybrid of P.davidiana × P.bolleana. PdbSKOR possessed the functional domains of 6 ion trans-membrane domains(S1-S6), cyclic nucleotide binding domain, ankyrin domain and KHA dimerisation domain, which belonging to the classic plant potassium channels. The amino acid sequences of SKOR proteins from 14 woody plants shared an overall identity of 81.09%, and the highest identity(96%) was observed in the S6 transmembrane region. The phylogenetic tree showed that SKOR homologs from different families and genera are quite different during evolution, while those from the same Family and Genus are relatively close in genetic distance. Particularly, P.davidiana ×P.bolleana and Salix purpurea belong to the same family of Salicaceae, PdbSKOR was closely clustered with SpuSKOR in the phylogenetic tree. Eighteen cis-acting regulatory elements, including development regulation, hormone response and stress response, were observed in the promoter region of PdbSKOR gene. qRT-PCR verification showed that PdbSKOR was dominately expressed in roots of 3-year-old hybrid of P.davidiana ×P.bolleana, followed by full bloom flower and inflorescence, and weakly expressed in stems, leaves and fruit flocs. Moreover, the relative expression of PdbSKOR was the highest in roots of tissue-culture plantlets, and was more sensitive to K⁺ depletion, drought and low temperature treatments, whose expression was decreased under K⁺ depletion and drought but induced under low temperature treatment. Expression of PdbSKOR had little response to K⁺ excess treatment. Patch clamp analysis demonstrated that the activity of PdbSKOR channel was activated when the cell membrane voltage reached at +20 mV, and the channel activity was increased correspondingly with the enhancement of positive membrane voltage, typical outward currents were recorded and enhanced alongside with the decrease of external K⁺ concentration. All these findings showed that PdbSKOR was a typical voltage dependent outwardly rectifying K⁺ channel. Conclusion: PdbSKOR gene was cloned and characterized from hybrid of P. davidiana × P. bolleana. PdbSKOR was closely clustered with homolog of SpuSKOR from S.purpurea in the phylogenetic tree. PdbSKOR was mainly expressed in roots of both mature trees and tissue-culture plantlets, and was prone to be regulated by K⁺ depletion, drought, and low temperature treatment. PdbSKOR is a voltage dependent outwardly rectifying K⁺ channel that dominates the K⁺ release in roots of P.davidiana × P.bolleana.

Key words: Populus davidiana×P. bolleana, Shaker type potassium channel, SKOR, gene cloning, gene expression, patch clamping