海蓬子中高亲和钾离子转运体SbHKT1基因的克隆、表达及生物信息学分析

Cloning, Expression Pattern and Bioinformatic Analysis of High-Affinity Potassium Transporter Gene SbHKT1 from Halophyte Salicornia bigelovii

本研究利用RACE技术从真盐生植物海蓬子中获得了高亲和钾离子转运体SbHKT1基因1647bp完整的ORF框。序列分析结果表明,该基因编码548个氨基酸,分子量为62.10kD,理论等电点为9.33;氨基酸序列中第1个～第35个属信号肽序列,第197个～第537个属离子转运体(TrkH)家族特征序列;该基因编码的蛋白具有10个跨膜结构,N端跨膜区及中部膜上呈现明显的疏水性,C端及中部多个跨膜区呈现强疏水性,符合载体类运输蛋白的特点,因此推测SbHKT1蛋白为跨膜运输蛋白。Blast分析显示该蛋白与碱蓬SsHKT1氨基酸同源性高达77%,与冰叶日中花、赤桉和小麦HKT类蛋白的同源性分别为63%、52%和46%。SbHkt1基因表达存在组织特异性:正常生长条件在根、茎中表达较低,在叶片中几乎看不到表达;在高盐低钾的环境下,各组织表达明显升高,高盐低钾胁迫处理8h,其根部表达处于高峰期;经100μmol/L脱落酸处理4h,根部表达达到最高;干旱胁迫(20%PEG6000)处理2h,根部表达量明显上升。由此推断,该基因参与了植物在高盐低钾、渗透、干旱等非生物胁迫下的生理调控。由于目前已克隆的HKT类蛋白基因多来自非盐生植物,对盐生植物内源HKT基因的研究相对较少,因此,海蓬子内源HKT1基因的全长的获得有助于我们进一步研究该基因在高盐钾饥饿环境下运输钾离子,调节植物体内Na+/K+平衡的功能,对于揭示真盐生植物的耐盐机制,将其运用于非盐生植物,培育新的耐盐品种具有一定的意义。

A putative high-affinity potassium transporter gene SbHKT1 was isolated from halophyte Salicornia bigelovii by RACE cloning.Sequence anlysis showed that the gene was 1 723 bp in whole length,has an open reading frame （ORF） of 1 647 bp encoding a 62.10 kD protein with 548 amino acids and theoretical pI of 9.33.Amino acid sequence from 1 to 35 is signal peptide sequence,and from 197 to 537 is TrKH characteristic sequence.The protein encoded by SbHKT1 gene contains 10 transmembrane domains.Then the tranmembrane area near N-end and the outer membrane area in middle of the peptide show obvious hydrophobicity some transmembrane areas near C-end and in middle of peptide show high hydrophobicity.Since these characters are similar to the features of cation transporter,and we predicted SbHKT1 was a transmembrane peptide.The Blast result showed the amino acid sequence had 77% homology with Suaeda salsa SsHKT1 and shared homology 63% with Mesembryanthemum crystallinum McHKT1,52% with Eucalyptus camaldulensis EcHKT1,46% with Triticum aestivum TaHKT1.The tissue-specific expression pattern of the SbHKT1 showed that under normal living conditions,its transcripts were detected weakly in roots and stems,and nearly no expression in leaves.However,under high salt and low potassium environment,the expression of SbHKT1 was increased obviously.The expression of SbHKT1 reached at peak in roots after being treated with high salt and potassium starvation for 8 h,100 μmol/L ABA for 4 h,and 20% PEG6000 for 2 h.All of these results demonstrate SbHKT1 takes part in plant physiologic regulations under some abiotic stress such high salt /low potassium environment,osmotic stress and drought stress.Recently,most of the genes of HKT-like protein were cloned from non-halophyte.There is rare research about the HKT gene of halophyte.So the full length SbHKT1 gene cloning from Salicornia bigelovii helps people focus on the potassium transporting and Na＋/K＋ balancing function of this gene under high salt with potassium starvation environment.It is significant to uncover the salt tolerance mechanism of halophyte,to apply this mechanism to non-halophyte and to breed salt tolerant varieties ultimately.