互花米草NHX2基因的克隆与功能鉴定

Cloning and Function Identification of NHX2 Gene from Spartina alterniflora

NHX2属于CPA1基因家族,编码Na^+/H^+逆向转运蛋白,控制液泡膜中活性K^+的摄取,同时调节气孔的关闭。该研究以耐盐植物互花米草为材料,采用PCR技术克隆NHX2基因,并将其转入拟南芥进行相关功能鉴定。结果显示:(1)成功克隆获得互花米草NHX2基因CDS序列(1 602 bp),命名为SaNHX2,该基因编码533个氨基酸,SaNHX2蛋白的分子量约为58.65 kD,定位于细胞核和细胞膜,表明SaNHX2基因可能发挥转录调控的功能。(2) qRT-PCR结果显示,在ABA、NaCl和干旱胁迫处理下,互花米草叶和根中SaNHX2基因的表达量均上调。(3)为进一步鉴定其功能,成功构建植物表达载体,将SaNHX2基因转入拟南芥;经RT-PCR检测结果显示,SaNHX2基因在转基因植株中过表达;高盐胁迫处理后,转SaNHX2基因拟南芥的主根长度、叶绿素总量和相关胁迫应答基因表达量均高于转空载拟南芥,表明转SaNHX2基因拟南芥的耐盐能力显著增强。研究表明,SaNHX2基因可能在盐胁迫调节机制中发挥调控作用,可作为改良农作物耐盐的重要候选基因。

NHX2 belongs to the CPA1 gene family and encodes a Na^+/H^+ reverse transporter which controls the uptake of active K^+ in vacuolar membrane and regulates the closure of stomatal. In this study, the NHX2 gene was cloned by PCR from the salt-tolerant plant Spartina alterniflora and transferred into Arabidopsis thaliana for function identification. The results showed that:(1) the full-length sequence of NHX2(1 602 bp), encodes 533 amino acids, which was named SaNHX2 gene. The molecular weight of SaNHX2 protein was about 58.65 kD and located in the nucleus and cell membrane. These results indicate that SaNHX2 gene may play a role in transcriptional regulation.(2) The qRT-PCR analysis results indicated that the expression of SaNHX2 gene was up-regulated in leaves and roots of S. alterniflora under ABA, NaCl and drought stresses.(3) In order to identify its function, plant expression vector was constructed and SaNHX2 gene was transformed into A. thaliana. After high salt stress treatment, the length of root, the total chlorophyll content and the expression of related stress response genes of transgenic A. thaliana with SaNHX2 gene were all higher than those of transgenic A. thaliana without SaNHX2 gene. The results indicated that SaNHX2 gene may play a regulatory role in salt stress regulation and may be an important candidate gene for improving crop salt tolerance.