杜梨NHX基因家族的鉴定及其在非生物胁迫下的表达分析

Identification of NHX gene family in Pyrus betulaefolia and its expression under abiotic stress

[目的]NHX基因亚家族为钠氢逆转运体,具有Na^+/H^+ exchange(PF00999)蛋白结构域,广泛存在于多种物种中,主要参与植物响应盐胁迫过程离子平衡的重建。分析鉴定杜梨中NHX基因及其耐盐机制有助于实际生产中更为有效地利用这一砧木资源。[方法]结合已公布的梨基因组数据以及拟南芥相关数据,通过生物信息学手段,鉴定杜梨NHX基因家族成员;通过MEGA7.0软件进行序列比对以及进化分析;通过在线工具Pfam、SMART以及GSDS进行基因结构分析;通过定量PCR技术分析非生物胁迫下基因在不同组织中的表达特征;应用火焰石墨炉原子吸收光谱仪测定NaCl胁迫下杜梨不同组织中Na^+与K^+含量。[结果]成功鉴定出16个候选基因,其中有4个基因属于NHX亚家族,4个基因属于CHX亚家族,8个基因属于KEA亚家族。预测的候选基因均含有Na^+/H^+ exchange功能域;NaCl胁迫下,预测的16个基因在叶片中的表达差异大,但在8h以后均为负调控;在茎中,PEG6000处理下,PbNHX1的表达量显著增高,且高于同一时期NaCl胁迫下的表达水平,仅在48h时低于同一时期NaCl胁迫下的表达水平;在根中,基因PbN-HX12、PbNHX13、PbNHX14和PbNHX15在NaCl胁迫和PEG6000处理下在任何时间段均为负调控。[结论]在盐胁迫过程中,与拟南芥NHX基因进化关系最近的PbNHX1和PbNHX11呈现出负调控模式,与拟南芥KEA基因进化关系相近的PbNHX10和PbNHX7可能参与K^+的运输。

【Objective】NHXs belong to sodium hydrogen reverse transporter, which is widely distributed in many species, with Na^+/H^+ exchange (PF00999) protein domain. NHXs is mainly involved in the process of keeping ion balance in response to salt stress in plants. Identification and expression analysis under stress of NHXs in Pyrus betulaefolia would be useful to understand the reveal salt tolerant. Resistant mechanism of Pyrus betulaefolia to the salinity and be helpful to the better use of this rootstock resources.【Methods】Identification of NHX gene family members of Pyrus betulaefolia were carried out with bioinformatics, combining published pear genome and Arabidopsis related data. MEGA7.0 software was employed to sequence alignment and phylogenetic analysis;Pfam, SMART and GSDS software were used to analyze gene structure. The plantlets of Pyru betulaefolia was cultured in Hoagland inoculum and were treated with 250 mmol ·L^-1 NaCl and 10% PEG6000 for 0, 1, 8, 16, 24, 48, 72 and 84 h, respectively. Plant materials in Hoagland inoculum without NaCl and PEG were used as control.The total RNA was extracted from roots, stems and leaves of the plantlets with different treatments, and the expression level of the gene family under abiotic stress was analyzed systematically. qRT-PCR was further used to analyse the expression of PbNHXs under PEG and NaCl stress. Na+ content and K+ content in different tissues of NaCl stress were determined by flame graphite furnace atomic absorption spectrometry.【Results】Sixteen candidate genes were successfully identified, of which four genes belonged to NHX gene family, four genes belonged to CHX gene family and eight genes belonged to KEA gene family. The longest length NHX protein (PbNHX13) encoded 857 amino acids, the shortest protein (PbNHX12) encoded 401proteins, theoretical PI range from 5.14 (PbNHX10) to 9.92 (PbNHX12). All proteins except PbNHX7 and PbNHX10, contained 8-13 transmembrane structures. In the PbNHX1-PbNHX16 group of genes, the number of introns spaned a little bit, PbNHX12 had only one intron, and PbNHX3 had 25 introns. The predicted candidate genes contained Na+/H+ exchange domain, PbNHX7 and PbNHX10 contain TrKA_N domain, also. Under NaCl stress, the predicted expression trend of 16 genes in leaves was fluctuating, but all of them were negatively regulated after 8 hour. Eleven genes (PbNHX1-4, PbNHX6, PbNHX7, PbNHX11-15) appeared to peak at 8 h and 48 h in the stem, the expression of PbNHX1, 2, 3, 6, 11 was the highest at 48 h and the lowest at 1 h;similar expression patterns of PbNHX2, PbNHX3, PbNHX4, PbNHX7, PbNHX10, PbNHX11, PbNHX14, PbNHX15 in leaves, reached the highest expression level at 1 h, decrease to minimum expression at 1-8 h. The expression patterns of PbNHX5 and PbNHX12 are very similar, the expression level decreased gradually at 0-16 h, it tended to level off after 16 hours. PbNHX1-PbNHX16 in the roots, it was more vulnerable to the positive regulation of PEG6000 stress compared with the NaCl stress. The trend expression of PbNHX7 and PbNHX10 in stem was very similar, taking "M" shape, and a low peak at 16 hours. The expression of PbNHX5, PbNHX8 and PbNHX9 genes in NaCl treatment was higher than that in PEG treatment at the same time, only in 8 hours. Under the treatment of PEG6000, the expression of PbNHX1 in stem increased significantly, and was higher than that under NaCl stress in the same period, and was lower than that under NaCl stress in the same period only at 48 h. In leaves, the expression patterns of PbNHX2-5, PbNHX7, PbNHX9-11 and PbNHX13-15 genes were very similar. There were two peaks of PbNHX2 and PbNHX13 at 8 hour and 72 hour. Two peaks of high expression of the other eight genes occurred at 1 h and 72 h. In roots, PbNHX12、PbNHX13、PbNHX14 and PbNHX15 were negatively regulated at any time after NaCl stress and PEG6000 treatment. Determination of Na+ content and K^+ content in three tissues of Pyrus betulaefolia under Salt stress (0-84 h) showed that K^+ content in leaves increased slowly. K^+ content in stem showed a trend of "W" change and slight downward trend in the root at 24 h. Na^+ content in leaves had a inflection point at 16 hour, Na^+ content rised linearly after 16 hours. Na^+ content in stem was also increasing, but the increase was more moderate after 48 hours. Na^+ content in the root increased slowly with a wave-like trend, but it showed a downward trend at the 72-84 h.【Conclusion】PbNHX1 and PbNHX11, which was closely related to the evolution of NHX gene in Arabidopsis thaliana, showed a negative regulatory pattern during salt stress. PbNHX10 and PbNHX7, which were closely related to the evolution of Arabidopsis KEA gene, might be involved in K^+ transport during salt stressin Pyrus betulaefolia.