沙冬青AmFAD2-1基因的克隆及其功能分析

细胞膜脂的不饱和度主要由脂肪酸去饱和酶(fatty acid desaturase,FAD)决定,二者与植物抵抗低温和高盐等逆境胁迫密切相关。沙冬青(Ammopiptanthus mongolicus)是中亚荒漠区唯一的常绿旱生阔叶植物,具有很强的耐寒、耐旱和耐盐碱等特性。该研究利用RT-PCR方法克隆到沙冬青油酸去饱和酶基因AmFAD2-1的cDNA和gDNA,其中后者不含内含子。在AmFAD2-1蛋白(含382个氨基酸残基)序列中含有FAD家族必须的保守组氨酸簇和内质网定位信号及跨膜结构域。进化分析表明,AmFAD2-1与大豆(Glycine max)和柠条锦鸡儿(Caragana korshinskii)等豆科植物的FAD2距离较近,而与玉米(Zea mays)和拟南芥(Arabidopsis thaliana)等其他植物的FAD2距离较远。qRT-PCR分析表明,在室内培养的沙冬青幼苗中,AmFAD2-1的表达量在低温处理2～24h和干旱处理6～13d期间显著上调,在盐胁迫处理期间则呈现升高、降低、再升高的变化趋势;在野外生长的沙冬青成株嫩叶中,其表达量在秋季和冬季总体上明显高于春季和夏季。成功构建了植物表达载体p3300-35S-AmFAD2-1并通过根癌农杆菌介导法转化野生型拟南芥,获得16株转AmFAD2-1基因植株。耐逆性鉴定表明,转基因株系的耐冻性较野生型拟南芥显著提高,其耐旱性和耐盐性也比后者有较明显的改善。

Cloning and Functional Analysis of AmFAD2 1 Gene from Ammopiptanthus mongolicus

The unsaturation degree of cellular membrane lipids is mainly determined by fatty acid desaturases (FADs), and both the unsaturation degree and FADs are closely related to the tolerance of plants to harsh environments such as low temperature and salt stress. Ammopiptanthus mongolicus is the only evergreen xerophytic broadleaf plant that is endemic to the central Asian desert and has very strong tolerance to abiotic stresses such as cold, drought and high salinity. In this study, both the cDNA and gDNA fragments for an oleic acid desaturase, AmFAD2 1, in this species were cloned by RT PCR, and the gDNA region has no intron. Except for the conserved histidine clusters for FAD family, there also exist the signal peptide for endoplasmic reticulum localization and transmembrane domains in the AmFAD2 1 sequence (containing 382 amino acid residues). Phylogenetic relationship analysis showed that AmFAD2 1 is closely related to the FAD2s from leguminous plants such as Glycine max and Caragana korshinskii but is distantly related to the FAD2s from other plant species such as Zea mays and Arabidopsis thaliana. qRT PCR analysis showed that in laboratory cultured A. mongolicus seedlings, the expression level of AmFAD2 1 increased significantly between 2 to 24 h of the cold treatment and between 6 to 13 d of the drought treatment. Moreover, AmFAD2 1 showed an expression pattern of increasing, decreasing and increasing again during the period of salt treatment. In young leaves of the A. mongolicus plants naturally growing in the wild, the expression levels of AmFAD2 1 during the autumn and winter months were overall obviously higher than those in the spring and summer. The plant expression vector p3300 35S AmFAD2 1 was successfully constructed and wild type Arabidopsis was transformed using the Agrobacterium tumefaciens mediated method. In total, 16 AmFAD2 1 transgenic plants were obtained. Stress tolerance evaluation experiments revealed that the transgenic lines had significantly higher freezing tolerance compared with the wild type Arabidopsis; their drought and salt tolerances were also improved.