含异位表达花生AhNCED1基因的拟南芥提高耐渗透胁迫能力

doi:10.3724/SP.J.1006.2010.01440

摘要/Abstract

摘要： AhNCED1是干旱胁迫下调控花生ABA生物合成的关键基因。以pCABIA1301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基因的两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。

关键词: AhNCED1基因, 异位表达, 转基因拟南芥, 渗透胁迫, ABA敏感性

Abstract: The phytohormone abscisic acid (ABA) is a key regulator of seed development, root growth, stomatal aperture in higher plants and it is also involved in adaptation of plants to various stresses. The oxidative cleavage of cis-epoxycarotenoids catalyzed by nine-cis-epoxycarotenoid dioxygenase (NCED) is considered to be the rate-limiting step in ABA biosynthesis in higher plants. The AhNCED1 gene plays a vital role in the regulation of ABA biosynthesis in peanut plants in response to drought stress. Two binary vectors, p35S::ORF and pAtNCED3p::ORF, were established which harbored the AhNCED1 gene, respectively, driven by the CaMV 35S promoter originated from pCAMBIA1301 and the AtNCED3 gene promoter from wild type Arabidopsis. Wild type and 129B08/nced3 mutant Arabidopsis plants were separately transformed with Agrobacterium harboring p35S::ORF or pAtNCED3p::ORF vectors, generating 35S::ORF-WT and A3p::ORF-B08 transgenic plants, respectively, after hygromycin screening and PCR detection. The stable expression of AhNCED1 gene in Arabidopsis plants was confirmed by duplex RT-PCR performance. Wild type, 129B08/nced3 mutant and transgenic Arabidopsis plants were subsequently tested for sensitivity to exogenous ABA and tolerance to osmotic stress. The results showed that the ABA sensitivity of 129B08/nced3 mutant declined, and that of Arabidopsis plants ectopically expressing the AhNCED1 gene increased. Under sorbitol stress, the relative germination rate of 129B08/nced3 mutant seeds was far lower than that of wild type seeds; however, the relative germination rate of A3p::ORF-B08 transgenic seeds was close to that of wild type seeds, significantly higher than that of 129B08/nced3 mutant seeds. The relative germination rate of 35S::ORF-WT transgenic seeds was higher than that of wild type seeds at the treatment of 300 mmol L^–1 sorbitol. Under 300 mmol L^–1 sorbitol stress, leaf of 129B08/nced3 mutant plants was highly chlorotic, and root formation and seedling growth were severely inhibited. In contrast, at this concentration of sorbitol, leaf of A3p::ORF-B08 transgenic seedlings was only slightly chlorotic, just similar to those of wild-type plants; the growth of 35S::ORF-WT transgenic seedlings was nearly not affected. Germination assay and phenotypic evaluation revealed that 129B08/nced3 mutant was hypersensitive to the nonionic osmotic stress induced by sorbitol, and that ectopic expression of peanut AhNCED1 gene reverted the hypersensitivity of 129B08/nced3 mutant Arabidopsis to sorbitol and conferred enhanced osmotic stress tolerance of transgenic Arabidopsis plants. These observations and an improved understanding of the roles of AhNCED1 gene in stresses adaptation could provide the basis for engineering greater stress tolerance in crops.

Key words: AhNCED1 gene, Ectopic expression, Transgenic Arabidopsis, Osmotic stress, ABA sensitivity

万小荣,莫爱琼,郭小建,杨妙贤,余土元,曹锦萍. 含异位表达花生AhNCED1基因的拟南芥提高耐渗透胁迫能力[J]. 作物学报, 2010, 36(09): 1440-1449.

WAN Xiao-Rong, MO Ai-Qiong, GUO Xiao-Jian, YANG Miao-Xian, YU Shi-Yuan, CAO Jin-Ping. Osmotic Stress Tolerance Improvement of Arabidopsis Plants Ectopically Expressing Peanut AhNCED1 Gene[J]. Acta Agron Sin, 2010, 36(09): 1440-1449.

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