【作者】 卢敏；

【导师】 王天宇；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2013， 博士

【摘要】 干旱、盐碱等非生物逆境胁迫严重影响植物的正常生长发育，而植物在长期进化过程中会形成一系列有效的作用机制，其中包括调控胁迫相关基因的表达以应对外界不良环境。逆境胁迫诱导表达基因可大致分为两大类：即功能蛋白编码基因和参与逆境胁迫信号转导过程的转录调控蛋白编码基因。NAC转录因子是具有多种生物学功能的植物特异转录调控因子，在植物生长发育、形态建成、激素调节和逆境胁迫应答过程中发挥着重要作用。目前在模式植物如拟南芥和水稻中的NAC基因与植物抗逆性相关研究方面已经取得了重要进展，然而在一些重要的C4禾本科作物如玉米和高粱中与逆境胁迫相关的NAC基因的研究报道较少。本研究克隆了两个NAC家族基因，玉米ZmSNAC1和高粱SbSNAC1基因,分别对其进行了基因表达模式分析和抗逆相关生物学功能验证。主要实验结果如下：1.玉米ZmSNAC1基因的克隆与功能分析首先，从玉米耐旱自交系“CN165”中分离得到一个玉米NAC家族基因ZmSNAC1，进化分析表明玉米ZmSNAC1与水稻SNAC1进化关系较近。诱导表达分析结果表明ZmSNAC1受非生物逆境胁迫如低温、高盐、干旱和植物激素脱落酸（ABA）的诱导表达上调，受水杨酸（SA）诱导表达下调；组织特异性表达分析结果证明ZmSNAC1在玉米根中和穗位叶的相对表达量较高。亚细胞定位实验结果表明ZmSNAC1定位在拟南芥原生质体细胞核中，转录激活分析证明ZmSNAC1蛋白具有转录激活活性。进一步通过过表达ZmSNAC1基因对其进行转基因拟南芥株系在不同生长发育时期的抗逆表型鉴定，在种子萌发阶段，ZmSNAC1过表达转基因株系能够显著提高其对ABA和渗透胁迫的敏感性；在苗期，转基因株系通过降低离体叶片失水速率来提高植株的耐脱水性；对生殖生长发育时期内ZmSNAC1过表达转基因株系的耐旱、耐盐性表型鉴定结果表明，在干旱胁迫条件下，转基因株系较野生型株系存活率提高了50-52%，转基因株系的相对电导率较野生型株系降低了17-21%，叶绿素含量较对照株系提高了36-47%，脯氨酸含量提高了17-23%；在300mM NaCl胁迫条件下，转基因株系的存活率提高了36-40%。抗逆功能研究结果证明过表达ZmSNAC1显著提高了转基因株系的耐旱和耐盐性。2.高粱SbSNAC1基因的克隆与功能分析从新疆耐旱高粱品种“XGL-1”中克隆得到高粱SbSNAC1基因，对其氨基酸序列分析结果发现SbSNAC1蛋白主要由位于N-末端的典型的NAC保守结构域和序列高度多样性的C-末端两部分组成。基因表达模式研究发现SbSNAC1基因受非生物逆境胁迫诱导表达上调，且响应植物激素ABA的诱导；组织特异性表达分析结果表明SbSNAC1在高粱根中表达量最高。转录激活实验证明SbSNAC1蛋白的C-末端为其转录激活区，而其N-末端没有转录激活活性，且215-235氨基酸区段为转录激活的必须区段。亚细胞定位结果证明SbSNAC1为细胞核蛋白，且其核定位信号主要存在于SbSNAC1的NAC保守结构域中（N-末端）。进一步通过遗传转化拟南芥的方式对其进行了抗逆功能鉴定，结果表明SbSNAC1过表达转基因株系较对照株系显著提高植株的耐旱性，且在干旱胁迫处理下，转基因拟南芥中一些胁迫应答基因（如RD29B、RAB18、ABI1等）的表达量较野生型显著上调。综合以上研究结果表明SbSNAC1作为应答逆境胁迫的转录因子在植物抗逆调控网络中起正向调控作用，为将来基因工程改良作物抗逆性提供了重要的候选基因。更多还原

【Abstract】 Abiotic stresses that cause adverse effects on the growth and development of plants. Thephysiologic response to these stresses arises out of changes in gene expression. The products of thesestress induced genes can be classified into two groups: functional genes and the regulatory genes thatinvolved in signal transduction in the stress response. NAC proteins are plant-specific transcriptionfactors that play essential roles in stress responses. However, only little information regardingstress-related NAC genes is available in grass family such as maize and sorghum. In this study, wecharacterized two NAC family members which were designed as ZmSNAC1and SbSNAC1. Weexamined their expression profiles and analyzed their abiotic stress-related functions, respectively.1. Isolation and functional analysis of maize ZmSNAC1A maize NAC gene, ZmSNAC1, was isolated from the maize inbred line “CN165” and functionallycharacterized. ZmSNAC1had more close relationship with rice SNAC1according to the phylogeneticanalysis. Expression analysis revealed that ZmSNAC1was strongly induced by low temperature, highsalinity, drought stress, and abscisic acid (ABA) treatment, but downregulated by salicylic acid (SA)treatment. Subcellular localization experiments in Arabidopsis protoplast cells indicated that ZmSNAC1was localized in the nucleus. Transactivation assays demonstrated that ZmSNAC1functioned as atranscriptional activator. Overexpression of ZmSNAC1in Arabidopsis led to hypersensitivity to ABAand osmotic stress at the germination stage, but enhanced tolerance to dehydration compared towild-type (WT) seedlings. Moreover, over-expression of ZmSNAC1in Arabidopsis conferred enhanceddrought and salt tolerance, which was supported not only by the improved survival percentage inZmSNAC1transgenic lines compared with the WT lines but also by physiological changes, such as thereduced electrolyte leakage and higher chlorophyll content in transgenic plants. These results indicatedthat ZmSNAC1may function as a positive regulator in multiple pathways of plants response to abioticstresses and is potentially useful in transgenic breeding to improve stress tolerance in crops.2. Isolation and functional analysis of sorghum SbSNAC1SbSNAC1is a member of the plant-specific NAC transcription factor superfamily that plays animportant role in the abiotic stress response in sorghum. The SbSNAC1protein consists of a typicalNAC conserved domain at its N terminus and a diverse C-terminal region. The expression of SbSNAC1was induced by various abiotic stresses, such as drought and salinity. SbSNAC1is also expressed at arelatively higher concentration in roots and responds to the phytohormone abscisic acid. Transactivationanalysis indicated that the transactivation activity of SbSNAC1is located in the C-terminal region,whereas no activity was detected in the conserved NAC-domain, localized in the N-terminus. The testedregion (215-235aa) of the C-terminus was considered to be necessary for its transactivation activity.Subcellular localization assays using constructs of different SbSNAC1fragments fused with greenfluorescent protein revealed that the SbSNAC1protein localized in the nucleus, and that the nuclearlocalization signal was present in the N-terminal section. Furthermore, transgenic plants overexpressingSbSNAC1had an improved drought stress tolerance compared with wild-type plants, but no obviousretardation was detected in plant growth and development. SbSNAC1-overexpressed transgenic plants showed increased transcription of the stress-responsive genes under drought treatment. These resultssuggest that SbSNAC1functions as a stress-responsive transcription factor in positive modulation ofabiotic stress tolerance, and may have applications in the engineering of drought-tolerant crops.更多还原