罗布麻K^+通道编码基因AvAKT1的克隆与表达分析

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

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AvAKT 1,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,AvAKT 1主要在罗布麻根中表达,在茎和叶中表达量极低;AvAKT 1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。AvAKT 1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。

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 AvAKT 1,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 AvAKT 1 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 AvAKT 1 belonged to GroupⅠ(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 AvAKT 1 is expressed especially in roots.Furthermore,AvAKT 1 was induced strongly by supplying of 5 mmol·L^-1 K^+in the medium.The expression level of AvAKT 1 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 AvAKT 1 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.