罗布麻K+通道编码基因AvAKT1的克隆与表达分析

罗布麻是一种典型的钾高效植物,具有很强的K⁺吸收和利用效率,通过增强对K⁺的选择性吸收维持体内高且稳定的K⁺含量和K⁺/Na⁺以抵御盐和干旱胁迫。为了探究其分子机制,采用RT-PCR和RACE技术从罗布麻根中克隆得到Shaker家族成员K⁺通道蛋白基因AvAKT1,其cDNA全长序列2906 bp,编码899个氨基酸。氨基酸序列分析显示AvAKT1包含Shaker同源蛋白家族典型的6个跨膜域、1个P环以及环核苷酸结合位点(cyclic nucleotide-binding domain, cNBD)、锚蛋白区(ankyrin-related domain, ANKY)和富含疏水酸性残基区(a domain rich in hydrophobic and acidic residues, KHA)保守结构域;系统进化树将Shaker家族分为5个亚族,AvAKT1隶属第Ⅰ亚族(AKT1亚族),与烟草进化关系较近。实时荧光定量PCR分析结果表明,AvAKT1主要在罗布麻根中表达,在茎和叶中表达量极低;AvAKT1受5.0 mmol·L^-1 K⁺的显著诱导,且在-0.2 MPa渗透胁迫和25 mmol·L^-1 NaCl处理6 h时,在根中的表达水平显著上升。AvAKT1可能参与调控罗布麻低亲和性K⁺吸收过程,并在其应答干旱和盐逆境胁迫中发挥有效作用。

Cloning and expression analysis of the K+ channel gene AvAKT1 in Apocynum venetum

Apocynum venetum is a typical K⁺-efficient species with notably high K⁺ uptake and utilization efficiency under K⁺-deficiency conditions. Furthermore, A. venetum can maintain high plant tissue K⁺ contents and K⁺/Na⁺ ratio through enhancement of the capacity for selective absorption and transport of K⁺ under salinity or drought conditions. In order to investigate the underlying molecular mechanisms, a potassium channel gene, designated AvAKT1, was isolated from the root of A. venetum using RT-PCR and RACE methodologies. The full cDNA sequence was 2906 bp in length, encoding 899 amino acid residues. The deduced amino acids of AvAKT1 exhibited all the structural features shared by other plant Shaker-like K⁺-channel family members, including six transmembrane helices, a K⁺-selective pore-forming domain, a cyclic nucleotide-binding domain (cNBD), an ankyrin-related domain (ANKY) and a domain rich in hydrophobic and acidic residues (KHA). The phylogenetic analysis showed that AvAKT1 belonged to Group Ⅰ (AKT1-subfamily) in the Shaker-like K⁺ channel family, and formed a clade with the closest relation to the dicotyledon AKT1 homologue NtAKT1 from Nicotiana tabacum. Real-time fluorescent quantitative PCR suggested that AvAKT1 is expressed especially in roots. Furthermore, AvAKT1 was induced strongly by supplying of 5 mmol·L^-1 K⁺ in the medium. The expression level of AvAKT1 was significantly increased within a short time (e.g. 6 h) under -0.2 MPa osmotic stress or 25 mmol·L^-1 NaCl treatment. These results indicate that AvAKT1 is probably involved in the process of low affinity K⁺ absorption, and might play an effective role in response to salinity or drought in A. venetum.