唐古特白刺质膜Na^+/H^+逆向转运蛋白基因的克隆与表达分析

Isolation and expression analysis of a plasma membrane Na+/H+ antiporter from Nitraria tangutorum

土壤中过多的Na+会导致植物产生盐害,严重影响植物的生长和发育。质膜型Na+/H+逆向转运蛋白SOS1介导植物细胞内过量的Na+外排,在植物抵御盐胁迫过程中起着重要的作用。唐古特白刺隶属于蒺藜科白刺属,是中国特有的盐生植物,主要生长于西北地区的荒漠或盐渍化荒漠,具有极强的抗逆能力。应用简并RT-PCR及RACE技术克隆获得唐古特白刺Na+/H+逆向转运蛋白NtSOS1基因(GenBank登录号KC292267)。序列分析显示,该cDNA全长4 008bp,包含3 492bp的开放阅读框,编码分子量为127.59kDa的蛋白质。疏水性分析预测NtSOS1基因编码蛋白N端具有11个跨膜结构域,C端具有一个面向胞质的长亲水尾部,包含保守环核苷酸结合区域、自我抑制基序及磷酸化位点等多个活性调控结构域。NtSOS1氨基酸序列与葡萄、霸王、拟南芥等植物的SOS1序列同源性较高,分别可达71.77%,68.94%,62.65%。系统发育分析表明NtSOS1基因为质膜型Na+/H+逆向转运蛋白,与液泡型Na+/H+逆向转运蛋白属于不同分支。半定量RT-PCR结果显示冷、热、盐和干旱胁迫都可以诱导NtSOS1基因的表达,同时,NtSOS1基因随着盐处理浓度增加呈现出先上升(0～300 mmol/L)后下降(400mmol/L)的趋势。结合植物的生境特点和NtSOS1表达模式分析,该基因表达水平的升高可能在唐古特白刺适应恶劣生境的过程中发挥积极作用。本研究的开展为利用NtSOS1基因进行作物与牧草改良并深入研究唐古特白刺抗逆分子机制奠定了基础。

Excessive concentrations of the sodium ion in soil is a major constraint to plant ment. The plasma membrane Na＋/H＋ antiporter salt overly sensitive SOS1 is responsi from cells, and it plays a crucial role in plant salinity tole Nitratia genus in Zygophyllaceae, is a native and typical growth and develop ble for Na＋ effluxes rance. Nitraria tangutorum, a shrub belonging to the halophyte mainly growing in deserts or salinity deserts in the northwest of China. It is an excellent species noted for its superior tolerance ability to diverse abiotic stresses. A full length cDNA of NtSOS1 （GenBank accession number KC292267） was isolated from this spe cies by means of degenerate PCR and RACE. NtSOS1 cDNA was 4 008 bp long and comprised a 3 492 bp open reading frame that was predicted to encode a within its N terminal portion, and a long hy 127. droph 59 kDa protein with 11 hypothetical transmembrane domains cyclic nucleotide binding domain, auto-inhibitory mo cyt tifs oplasmic tail with some regulatory domains including and phospho The amino acid sequences of NtSOS1 show 71.77%, 68.94% and 62 rylati 65% on sites in its C-terminal portion. identity to homologues from Vitis vinifera, Zygophyllum xanthoxylum and Arabidopsis thaliana, respectively. Phylogenetic analysis showed that NtSOS1 appeared to be a plasma membrane Na＋/H＋ antiporter and clustered distantly with vacuolar Na＋/H1 antiporters. The level of NtSOS1 mRNA was clearly up-regulated in the presence of NaC1, PEG, cold and heat stress. Furthermore, NtSOS1 transcript abundance was positively associated with NaC1 concen- tration till 300 mmol/L, while it was reduced under 400 mmol/L increased expression of NtSOS1 may be involved in the adaptabili ment. Our work offers a solid foundation for genetic improvement NaC1 treatment. These results suggest that ty of of N. tangutorum to the hostile environ the crops and herbages with NtSOS1 gene and in-depth study of the molecular mechanisms of stress tolerance of N. tangutorum