Cloning and expression pattern analysis of high-affinity potassium transporter gene NtHAK5 in tobacco

【Objective】To clone high-affinity potassium transporter gene（NtHAK5）in tobacco，and analyze its expression pattern under different abiotic stresses，so as to provide a theoretical reference for further study on tobacco’ s function and mechanism of resistance to abiotic stresses，such as potassium stress.【Method】Coding region（CDS）sequence of NtHAK5 gene was amplified from Nicotiana tabacum K326，and then it was analyzed by bioinformatics software，and the pBWA（V） HS-NtHAK5-GLosgfp fusion expression vector was constructed.Tobacco epidermal cells were transfected，and fluorescent signals were observed to determine the subcellular localization of the protein.Quantitative real-time PCR（qRT-PCR）was used to detect the expression characteristics of NtHAK5 gene in different tissues and under different abiotic stress conditions，and plants treated with no stress（normal potassium supply of 2 mmol/L K⁺）were taken as control（CK）.【Result】 Full-length CDS sequence of tobacco NtHAK5 gene was 2418 bp，encoding a total of 805 amino acid residues.The molecular weight of the encoded protein was 89874.00 Da and the theoretical isoelectric point（pI）was 8.65，indicating that it was a stable hydrophobic protein containing transmembrane domain of typical HAK family proteins.In the secondary structure of NtHAK5 gene，the α-helix accounted for 40.12%，the extended chain accounted for 24.97%，the random coil accounted for 26.21%，and the β-sheet accounted for 8.70%.Amino acid similarity between NtHAK5 protein and tobacco NtHAK1 protein was 40.69%，while amino acid similarity between NtHAK5 protein and NrHAK1 tobacco and 40.44%.NtHAK1 protein had the highest similarity with Arabidopsis AtHAK5，58.26%.NtHAK5 gene promoter contained relevant cis-acting elements for anaerobic，low temperature，drought，abscisic acid，methyl jasmonate（MeJA）responses.NtHAK5 protein located on the cell membrane.Under low potassium stress treatment and normal potassium supply（CK）conditions，NtHAK5 gene was expressed in roots，stems，old leaves and new leaves，and the relative expression was the highest in old leaves.Relative expression of NtHAK5 gene under drought stress，chilling injury，salt stress and low potassium stress was significantly higher than that of CK（P<0.05），but relative expression under low nitrogen and low phosphorus conditions was not significantly different from that of CK（P>0.05）.【Conclusion】 NtHAK5 belongs to the HAK gene family with obvious tissue expression specificity，and participates in the response of tobacco plants to abiotic stresses such as drought stress，chilling stress，salt stress and low potassium stress.It is anticipated that K⁺ transporters are involved in the absorption，transport and reuse of K⁺ under abiotic stress.