蚕豆VfNHX1基因克隆及初步功能验证

Cloning and Preliminary Functional Verification of VfNHX1 in Vicia faba L.

【目的】探究蚕豆(Vicia faba L.)VfNHX1基因在响应盐胁迫过程中的作用。【方法】通过3′和5′RACE方法,从蚕豆中克隆了1个Na^(+)/H^(+)逆向转运蛋白编码基因VfNHX1,并对其进行了生物信息学分析、亚细胞定位、盐胁迫下的表达分析和初步功能验证。【结果】(1)该基因全长2255 bp,CDS编码区长1629 bp,编码542个氨基酸;(2)生物信息学分析显示,该蛋白有10个跨膜区,不具有信号肽,是一个结构稳定的膜蛋白,且包含1个NHX蛋白家族特有的Na-H Exchanger结构域;亚细胞定位分析显示VfNHX1定位在液泡膜上;(3)实时荧光定量PCR(qRT-PCR)分析显示,在NaCl处理后,叶片中VfNHX1表达量呈现先降低后升高,随即又下降的变化趋势,且在12 h时达最高值;根中VfNHX1表达量先降低后升高,在48 h时表达量显著升高(P<0.01);(4)酵母生长试验结果表明,VfNHX1可以提高盐敏感酵母突变体AXT4K对高盐的耐受能力。【结论】VfNHX1基因能够响应盐胁迫,是蚕豆潜在抗盐功能基因。

Objective Role of VfNHX1 of faba bean in response to salt stress was studied.Methods A Na^(+)/H^(+) reverse transporter protein-encoding gene,VfNHX1,was cloned from Vicia faba L.by 3'and 5'RACE for a bioinformatics analysis,subcellular localization determination,expression under salt stress,and a preliminary verification on the function.Results(1)VfNHX1 sequence was 2255 bp with a CDS coding region of 1629 bp encoding 542 amino acids.(2)As a stable transmembrane protein,VfNHX1 had 10 transmembrane regions without signal peptide and contained a typical Na-H exchanger conserved functional domain of NHX family.It was in the vacuolar membrane.(3)In the leaves,the expression of VfNHX1 under NaCl stress determined by qRT-PCR showed a trend of decreasing,then increasing,followed by decreasing with a peak reached in 12 h.In the roots,the expression declined initially and then rose significantly at 48 h(P<0.01).(4)The presence of VfNHX1 in a yeast growth experiment indeed heightened the NaCl tolerance of the salt-sensitive yeast mutant AXT4K.Conclusion VfNHX1 in V.faba L.was able to respond to salt stress and could be a functional gene to boost the salt resistance of the plant.