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

Q3 Agricultural and Biological Sciences
Xiao-Rong WAN , Ai-Qiong MO , Xiao-Jian GUO , Miao-Xian YANG , Tu-Yuan YU , Jin-Ping CAO
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引用次数: 1

Abstract

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.

异位表达花生AhNCED1基因拟南芥对渗透胁迫耐受性的提高
AhNCED1基因在花生(arachhis hypogaea L.)植物对干旱胁迫的脱落酸(ABA)生物合成调控中起重要作用。构建了两个携带AhNCED1基因的二值载体p35S::ORF和pAtNCED3p::ORF,分别由来自pCAMBIA1301的CaMV 35S启动子和来自野生型拟南芥的AtNCED3基因启动子驱动。分别用含有p35S::ORF或pAtNCED3p::ORF载体的农杆菌对野生型和129B08/nced3突变体拟南芥植株进行转化,得到35S::ORF- wt和A3p::ORF- b08转基因植株。利用双反转录PCR (RT-PCR)证实了AhNCED1基因在拟南芥中的稳定表达。随后测试了野生型、129B08/nced3突变体和转基因拟南芥植株对外源ABA的敏感性和对渗透胁迫的耐受性。结果表明,129B08/nced3突变体对ABA的敏感性降低,而异位表达AhNCED1基因的拟南芥植株对ABA的敏感性升高。山梨醇胁迫下,129B08/nced3突变体种子的相对发芽率远低于野生型种子。而A3p::ORF-B08转基因种子的相对发芽率与野生型种子接近,显著高于129B08/nced3突变体种子。在300 mmol L−1山梨醇处理下,35S::ORF-WT转基因种子的相对发芽率高于野生型种子。300 mmol L−1山梨醇处理后,129B08/nced3突变体植株叶片高度褪绿,根系形成和幼苗生长受到严重抑制。而在此浓度山梨醇下,A3p::ORF-B08转基因幼苗的叶片仅发生轻微的褪绿,与野生型植物相似。山梨醇处理对35S::ORF-WT转基因幼苗的生长几乎没有影响。根据萌发试验和表型评价结果,129B08/nced3突变体对山梨醇诱导的非离子渗透胁迫敏感,花生AhNCED1基因的异位表达恢复了129B08/nced3突变体对山梨醇的敏感性,使转基因拟南芥对渗透胁迫的耐受性增强。
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来源期刊
CiteScore
1.50
自引率
0.00%
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审稿时长
30 weeks
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