关键词: 葡萄, Shaker类钾离子通道, SKOR, 基因克隆与表达, 膜片钳技术

Abstract:

【Objective】The aim of this study was to isolate and characterize potassium (K⁺) channel gene SKOR (stelar K⁺ outward rectifier) from grape genome, based on the analysis of the tissue-specific expression patterns of VviSKOR gene and response to K⁺ depletion, K⁺ excess, NaCl and ABA treatments, as well as function study by patch clamping electrophysiological technology.【Method】By carrying out homology-based cloning, a putative K⁺ channel gene VviSKOR was screened and isolated from grape genome. The details of VviSKOR gene were analyzed, and the protein encoded via utilizing bioinformatical analysis software. With the help of MEGA7.0 software, a phylogenetic tree was constructed by multiple alignments of SKOR proteins from grape, Arabidopsis, rice, maize, sorghum, slender false brome, soybean, tomato, cucumber, poplar, peach, pear, strawberry, apple, papaya, citrus, banana and pineapple. Using quantitative real-time PCR (qRT-PCR), the expression profiles of VviSKOR gene and its response to K⁺ depletion, NaCl and ABA treatments were analyzed. Preliminary analized the physiological function of VviSKOR gene by using patch clamping.【Result】VviSKOR was isolated and determined from grape genome. VviSKOR contained the functional domains of cyclic nucleotide-binding domain, ion channel transmembrane, ankyrin repeats and KHA domain, which belonged to the classic plant potassium channels. The amino acid sequences of SKOR protein from 18 plants shared an overall identity of 58.92%. Phylogenetic tree analysis showed that VviSKOR was closely clustered with homolog of CsaSKOR from cucumber. SKOR members from 4 grass family plants (maize, rice, slender false brome and sorghum) were prone to be clustered together, while SKOR members from Rosaceae, including strawberry, apple, pear and peach, were prone to be clustered together. VviSKOR was mainly localized in plasma membrane, which contained 6 transmembrane domains (TMs), and the theoretical isoelectric point (PI) was 6.24. Twelve kinds of cis-acting regulatory elements, including stress response, hormone response and cell cycle regulation, were found in the promoter region of VviSKOR gene. Database expression profile analysis showed that VviSKOR gene was expressed in different tissues or organs in grape, and the highest percentage was predicted in roots, followed by leaves and xylem. qRT-PCR analysis showed that VviSKOR gene was mainly expressed in roots of both 7-year-old ‘Marselan' and young seedlings. Moreover, VviSKOR gene was more sensitive to K⁺ depletion, ABA and NaCl treatments, whose expression were decreased under K⁺ depletion and ABA treatments, but induced under NaCl treatment in all tested tissues, including roots, and roots. Expression of VviSKOR gene had no response to NaCl treatment. Preliminary evidence of patch clamp analysis revealed that outward current was recorded in HEK293-T cells transfected with pTracer-CMV3-SKOR plasmid, and the amount of released K⁺ was reduced with elevated external-K⁺, indicating that VviSKOR was an outwardly rectifying K⁺ channel. In addition, the recorded current increased with the enhancement of voltage, indicating that VviSKOR was also a voltage dependent K⁺ channel.【Conclusion】VviSKOR gene was mainly expressed in grape roots (mature trees and seedlings). VviSKOR was closely clustered with homolog of CsaSKOR from cucumber in phylogenetic tree. Expression of VviSKOR gene was prone to be regulated by K⁺ depletion, ABA, and NaCl treatment. VviSKOR was an outwardly rectifying K⁺ channel that dominated the K⁺ release in grape roots.

Key words: grape, Shaker type potassium channel, SKOR, gene cloning and expression, patch clamping