Improvement of Tolerance to Osmotic Stress in Arabidopsis Plants Ectopically Expressing Peanut AhNCED1 Gene

Gene AhNCED1 plays a vital role in regulation of abscisic acid (ABA) biosynthesis in peanut (Arachis hypogaea L.) plants in response to drought stress. Two binary vectors harboring AhNCED1 gene, p35S::ORF and pAtNCED3p::ORF, were established, which were driven by the CaMV 35S promoter from pCAMBIA1301 and the AtNCED3 gene promoter from wild type Arabidopsis, respectively. Wild type and 129B08/nced3 mutant Arabidopsis plants were separately transformed with Agrobacterium harboring p35S::ORF or pAtNCED3p::ORF vector, resulting in 35S::ORF-WT and A3p::ORF-B08 transgenic plants, accordingly. The stable expression of AhNCED1 gene in Arabidopsis plants was confirmed with duplex reverse transcription PCR (RT-PCR). The wild type, 129B08/nced3 mutant, and the transgenic Arabidopsis plants were subsequently tested for sensitivity to exogenous ABA and tolerance to osmotic stress. The results showed that the ABA sensitivity was declined in the 129B08/nced3 mutant and increased in the Arabidopsis plants ectopically expressing AhNCED1 gene. Under sorbitol stress, the relative germination rate of 129B08/nced3 mutant seeds was far lower than that of the wild type seeds. However, the relative germination rate of A3p::ORF-B08 transgenic seeds was close to that of the wild type seeds, which was significantly higher than that of 129B08/nced3 mutant seeds. The relative germination rate of 35S::ORF-WT transgenic seeds was higher than that of the wild type seeds in the treatment of 300 mmol L−1 sorbitol. When treated with 300 mmol L−1 sorbitol, the leaves of 129B08/nced3 mutant plants were highly chlorotic, and root formation and seedling growth were severely inhibited. In contrast, at this concentration of sorbitol, the leaves of A3p::ORF-B08 transgenic seedlings were only slightly chlorotic, just similar to those of the wild type plants. There was almost no influence on the growth of 35S::ORF-WT transgenic seedlings under the sorbitol treatment. Based on the results of germination assay and phenotypic evaluation, the 129B08/nced3 mutant was hypersensitive to the nonionic osmotic stress induced by sorbitol, and ectopic expression of peanut AhNCED1 gene reverted the hypersensitivity of 129B08/nced3 mutant Arabidopsis to sorbitol and conferred enhanced osmotic stress tolerance in transgenic Arabidopsis plants.

摘 要AhNCED1是干旱胁迫下调控花生ABA生物合成的关键基因。以pCAMBIA1301为基本双元表达载体, 分别构建CaMV 35S启动子和拟南芥AtNCED3基因启动子(AtNCED3p)驱动花生AhNCED1基因的2个植物双元表达载体p35S::ORF和pAtNCED3p::ORF, 通过根癌农杆菌介导法将上述两个表达载体分别转化野生型和129B08/nced3突变体拟南芥, 经潮霉素筛选和PCR鉴定分别获得35S::ORF-WT和A3p::ORF-B08转基因植株, RT-PCR证实花生AhNCED1基因已在转基因植株中稳定表达, 并对野生型、129B08/nced3突变体和转基因拟南芥进行外源ABA敏感性和耐渗透胁迫能力分析。结果表明, 129B08/nced3突变体对外源ABA的敏感性下降, 而花生AhNCED1基因在拟南芥中的异位表达提高了对外源ABA的敏感性。在山梨醇胁迫下, 129B08/nced3突变体种子的相对萌发率明显低于野生型的, 而A3p::ORF-B08转基因拟南芥种子的相对萌发率与野生型的相当, 显著高于129B08/nced3突变体的, 且300 mmol L–1山梨醇胁迫下, 35S::ORF-WT转基因拟南芥种子的相对萌发率明显高于野生型的。在300 mmol L–1山梨醇胁迫下, 129B08/nced3突变体幼苗叶片高度黄化, 根的形成和幼苗生长受到严重抑制, 而A3p::ORF-B08转基因突变体与野生型相似, 叶片仅轻度黄化, 幼苗生长势良好; 35S::ORF-WT转基因植株幼苗生长未受明显影响。这些结果说明, 拟南芥129B08/nced3突变体对山梨醇诱导的非离子渗透胁迫有超敏性, 异位表达花生AhNCED1基因能恢复该突变体对山梨醇的超敏性, 提高拟南芥的耐渗透胁迫能力。