西藏野生垂穗披碱草EnPLA1基因克隆与表达分析

为探究α-亚麻酸调控植物低温胁迫应答的分子机制，本研究从西藏野生垂穗披碱草（Elymus nutans Griseb.）中克隆了其合成酶磷脂酶基因（EnPLA1），对其进行生物信息学、基因表达模式和亚细胞定位分析，并采用外源添加的方法分析α-亚麻酸在西藏野生垂穗披碱草低温适应中的作用。结果表明：EnPLA1基因的编码序列（Coding sequence，CDS）全长为1 305 bp，编码434个氨基酸；分子量为47.44 KD，等电点为6.05；EnPLA1蛋白为亲水性蛋白且偏酸性，含有1个高度保守的Lipase（class 3）结构域和4个跨膜结构域；二级结构以α-螺旋和无规则卷曲为主；EnPLA1蛋白与粗山羊草（Aegilops tauschii subsp. Tauschii），圆锥小麦（Triticum turgidum subsp.Durum），大麦（Hordeum vulgare subsp.Vulgare）的氨基酸序列相似性达80.76%，且该蛋白主要定位在细胞质。EnPLA1基因在垂穗披碱草的根、茎、叶中均有表达，其中叶中表达高于茎和根；低温诱导叶和茎中EnPLA1基因表达（P<0.05），对根中表达影响较小；25 μM α-亚麻酸处理显著地提高植物的抗寒性。本研究为深入地阐明EnPLA1基因调控垂穗披碱草低温适应机制奠定基础，并为利用优良野生种质资源改良牧草及作物提供思路。

Cloning and Expression Analysis of EnPLA1 Gene in Tibetan wild Elymus nutans Griseb.

To determine the molecular mechanism of α-linolenic acid regulating cold tolerance in plants, the full-length CDS sequence of EnPLA1 gene, which codes a enzyme of α-linolenic acid biosynthesis, was cloned from Tibetan wild Elymus nutans Griseb.. The results showed that the CDS of EnPLA1 gene was 1 305 bp, encoding 434 amino acids. The molecular weight of 47.44 KD and the isoelectric point was 6.05. The EnPLA1 protein was hydrophilic and partial acidic, containing 1 highly conservative Lipase (class 3) domains and 4 transmembrane domains. The secondary structure was mainly composed of α-helix and random coil. The EnPLA1 protein was highly similar to the amino acid sequence of Aegilops tauschii subsp. Tauschii, Triticum turgidum subsp. Durum and Hordeum vulgare subsp. Vulgare, and similarity was up to 80.76%. The EnPLA1 protein was localized predominantly in cytoplasm. The EnPLA1 gene was expressed in the roots, stems and leaves of E. nutans, which was higher than that in stems and roots. The expression of EnPLA1 gene was significantly regulated in leaves and stems and less effect on root expression when it was exposed to cold stress (P <0.05). The 25 μM of α-LA significantly improved the cold resistance of the plants. This study laid the foundation for the adaptation mechanism of EnPLA1 gene and provided ideas for genetic improvement of forage and crops with excellent wild germplasm resources.