水稻OsZAT12基因响应非生物胁迫和植物激素的研究

OsZAT12 gene responses to abiotic stresses and phytohormones in rice(Oryza sativa)

关键词:  水稻, C2H2锌指蛋白, OsZAT12, 非生物胁迫, 脱落酸

Key words:  rice(Oryza sativa), C2H2 zinc finger protein, OsZAT12, abiotic stresses, ABA

摘要:

C2H2锌指蛋白是真核生物体内一类重要的转录因子,在植物生长发育和应对非生物胁迫方面具有重要作用。实验室前期克隆了水稻C2H2锌指蛋白OsZAT12,该基因在水稻根中特异表达,定位于细胞核,异源过表达OsZAT12的拟南芥植株矮小。为进一步研究OsZAT12在水稻中的功能,该文分析了OsZAT12的启动子元件和转录活性,并采用qRT-PCR技术分析OsZAT12在非生物胁迫和植物激素处理下的响应模式。结果表明:(1)OsZAT12含有2个典型的C2H2锌指结构域和1个EAR motif,具有转录抑制活性,该基因的启动子中含有与非生物胁迫和植物激素相关的元件。(2)对野生型水稻进行非生物胁迫和激素处理发现,低温胁迫(4 ℃)和激素脱落酸(ABA)处理显著下调OsZAT12的表达; 而渗透胁迫(20% PEG 6 000)、激素油菜素甾醇(BR)或吲哚-3-乙酸(IAA)处理则显著上调OsZAT12的表达,这说明OsZAT12介导了水稻应对多种非生物胁迫和激素的变化。(3)利用含35S启动子的过表达载体和CRISPR/Cas9基因编辑技术分别得到纯合的OsZAT12过表达植株和OsZAT12敲除植株。(4)对过表达OsZAT12的水稻表型观察发现,相比于野生型,OsZAT12过表达植株在分蘖期、抽穗期和成熟期这3个时期的株高均显著降低; OsZAT12敲除植株的株高与野生型虽无明显差异,但每株穗数和结实率均显著低于野生型,这说明OsZAT12影响了水稻株型、穗型及结实率等农艺性状的建成。(5)实验进一步表明,过表达OsZAT12降低了水稻对外源ABA的敏感性,而OsZAT12敲除植株则相反。因此推测,OsZAT12对植株生长发育的影响可能与该基因响应多种非生物胁迫和激素信号的调控有关,该研究结果为将来利用OsZAT12进行水稻耐逆稳产分子设计育种提供了依据。

Abstract:

C2H2 zinc finger proteins are an important category of transcription factors in eukaryotes, which play important roles in plant growth and development and in response to abiotic stresses. OsZAT12, a C2H2 zinc finger protein in rice, which cloned in our previous study, was only expressed in rice roots and was localized in the nucleus. Overexpressing OsZAT12 in Arabidopsis thaliana exhibited dwarf phenotype. To further investigate the function of OsZAT12 in rice, qRT-PCR was used to analyze the response patterns of OsZAT12 under abiotic stresses and phytohormones treatment. The results were as follows:(1)OsZAT12 contained two typical C2H2 zinc finger domains and one EAR motif, and has transcriptional repressive activity. The promoter of the OsZAT12 contained elements related to abiotic stresses and phytohormones.(2)The results of abiotic stresses and phytohormones treatment in rice also revealed that low temperature stress(4 ℃)and phytohormone abscisic acid(ABA)treatment significantly down-regulated OsZAT12 expression, while osmotic stress(20% PEG 6 000), phytohormone brassinosteroid(BR)or indole-3-acetic acid(IAA)treatment significantly up-regulated the expression of OsZAT12. These results showed that OsZAT12 involved in the changes in response to abiotic stresses and phytohormones in rice.(3)Homozygous OsZAT12 overexpression plants and OsZAT12 knockout plants were obtained using overexpression vector with 35S promoter and CRISPR/Cas9 gene editing technology, respectively.(4)Observation of the phenotype of OsZAT12 overexpression rice showed that compared with the wild type, the plant height of OsZAT12 overexpression plants was significantly shorter at tillering stage, heading stage and maturity stage. The plant height of OsZAT12 knockout plants did not change significantly compared with the wild type, while the panicle number and seed-setting rate of them were significantly lower than those of the wild type. These results indicated that OsZAT12 affected the establishment of agronomic traits such as rice plant type, panicle type and seed-setting rate.(5)The results in this study further showed that overexpression of OsZAT12 reduced the sensitivity of rice to exogenous ABA, while the opposite phenotype was observed in OsZAT12 knockout plants. Therefore, it is speculated that the effect of OsZAT12 on plant growth and development may be related to the regulation of this gene in response to abiotic stresses and hormonal signals, and this study provides an experimental basis of using OsZAT12 for molecular design breeding of stress-tolerant and stable yield in rice.