铁皮石斛DoLEA2基因的克隆、表达及功能分析

Cloning, expression and function analysis of DOLEA2 gene from Dendrobium officinale

晚期胚胎富集蛋白(late embryogenesis abundant proteins,LEA)是一种亲水、热稳定性蛋白,与植物抗逆性密切相关,研究LEA家族基因对铁皮石斛抗逆性研究具有重要意义。本研究以铁皮石斛(Dendrobium officinale)种子为材料,采用RACE技术获得全长cDNA,命名为DoLEA2(Gen Bank登录号:KY626329)。该基因全长cDNA为1 224 bp,编码313个氨基酸,具有晚期胚胎富集蛋白(LEA_2)结构域和水分胁迫高敏感响应结构域,所编码蛋白与其他物种LEA2蛋白具有较高相似性,在进化上与单子叶植物聚为一支,且与蝴蝶兰(Phalaenopsis aphrodite)亲缘关系最近。铁皮石斛DoLEA2在叶的表达量最高,根和茎次之。该基因在Escherichia coli BL21(DE3)中能够高效表达,其优化表达条件为:37℃添加终浓度0.5 mmol·L^(-1)的异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导4 h。盐胁迫条件下与对照组相比,重组pET28a-DoLEA2菌株表现出较好的耐盐性。本研究首次获得了铁皮石斛LEA2全长cDNA序列,验证了其耐盐胁迫功能,为进一步研究铁皮石斛抗逆性分子机制提供科学依据。

LEA（late embryogenesis abundant） proteins that are highly hydrophilic and thermally stable play a role in plant defense. The full-length c DNA of DoLEA2 was cloned by rapid amplification of cDNA ends（RACE） from Dendrobium officinale（Gen Bank number： KY626329）. The cDNA is 1 224 bp and encodes 313 amino acids. The deduced DoLEA2 protein contained LEA_2 and WHy domains. Multiple sequence alignment revealed that DoLEA2 shared a high homology with other species. Phylogenetic tree showed that DoLEA2 belonged to the monocotyledon and its closest relative was P. aphrodite. DoLEA2 was differentially expressed in the different organ. The expression was most abundant in the leaves, followed by that of the roots and stem. Do LEA2 could express in Escherichia coli BL21（DE3）, and the best induction conditions were 0.5 mmol·L^（-1） IPTG at 37 ℃ for 4 h. The growth curves of E. coli BL21（DE3） showed that the recombinant DoLEA2 protein improved tolerate against salt stress over the control. This study represents the first time of cloning and identification of the function of LEA2 in D. officinale. The result sets up an important foundation for the molecular mechanism of stress resistance in Dendrobium officinale.