【作者】 张欣；

【导师】 万建民；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 低温胁迫引起作物大幅度减产是世界范围内普遍存在的问题。植物在长期的进化过程中形成了多种适应机制,其中包括胁迫条件下一些基因被诱导表达,并通过直接或间接的方式调控下游靶基因的表达,从而在提高植物抗逆性中发挥重要作用。为深入了解冷敏感植物如番茄、水稻低温逆境胁迫反应的分子机理和发现新的耐冷调控基因,我们在前期利用番茄cDNA芯片(约含8700个基因)分析番茄低温转录谱基础上,发现一些受低温高度诱导的新型转录因子,它们均不受番茄CBF转录因子调控。根据这些基因在低温逆境胁迫下的表达水平及表达模式,筛选出2个受低温诱导高表达的基因,番茄SlCZFP1(Solanum lycopersicum Cold Zinc Finger Protein 1)和SlCMYB1(Solanum lycopersicum Cold MYB 1)。本文通过对其序列、表达及生物学功能的研究,探索了SlCZFP1和SlCMYB1与植物抗逆性之间的关系,以期阐明利用低温诱导转录调控因子提高番茄、水稻等冷敏感型作物抗低温胁迫的分子机理和可能的调控路径,为转录调控因子在农作物抗冷基因工程的应用提供理论依据及基因资源。1.本研究从番茄中克隆了1个编码C2H2型锌指蛋白基因SlCZFP1。该基因受低温、高盐和干旱胁迫诱导表达,但不受ABA诱导,说明SlCZFP1在应答低温、高盐、干旱胁迫时的表达可能是不依赖于ABA途径的。洋葱表皮亚细胞定位研究表明SlCZFP1为核定位基因。将SlCZFP1基因分别在拟南芥和水稻中过表达,可显著提高转基因拟南芥及水稻植株的抗冻、抗冷性,但不能提高植株的抗旱性和耐盐性,此外,过表达SlCZFP1转基因植株中低温等非生物胁迫应答基因如COR15a,COR6.6,COR47,OsGOLS3,OsCOR413等基因的表达水平明显增加,推测SlCZFP1可能是植物抗低温胁迫的正调节因子,可能调控与CBF途径平行的低温信号转导过程。这些研究结果为通过遗传工程的方法操控低温信号通路改良冷敏感植物抗冷性提供了理论依据。2.本文还克隆了1个属于MYB转录因子家族R2R3-MYB亚类的番茄SlCMYB1基因。该基因不仅受低温和高盐诱导高表达,而且还受干旱和ABA胁迫诱导表达,说明SlCMYB1在应答低温、高盐、干旱胁迫时的表达可能是依赖于ABA途径的。洋葱表皮亚细胞定位研究表明SlCMYB1为核定位基因。为了深入了解所克隆转录因子的生理功能及与非生物逆境应答的关系,构建了SlCMYB1基因过表达载体并成功转化了番茄和水稻,筛选到12个转基因番茄和16个转基因水稻株系。对番茄转基因纯系的筛选和耐逆性鉴定正在进行中。对过表达SlCMYB1水稻转基因植株初步功能分析结果表明,转基因水稻植株的苗期抗冷性显著高于对照,一些低温胁迫应答基因(脯氨酸合成的关键酶基因OsP5CS1,膜转运蛋白基因OsWCOR413)的表达水平明显增加,说明SlCMYB1可能和OsP5CS1,OsWCOR413基因在同一低温调控信号转导路径上,关于SlCMYB1基因对干旱、高盐等其他逆境胁迫的作用机理正在进一步研究中。更多还原

【Abstract】 Low temperature stresses cause major losses in crop productivity worldwide. Plants vary greatly in their abilities to survive freezing temperatures. At one extreme are plants from tropical and subtropical regions including tomato and rice, which suffer injury when exposed to chilling temperature. In sharp contrast, plants from temperate regions are not only chilling-tolerant, but many of them, such as Arabidopsis and wheat, can survive freezing after exposure to low nonfreezing temperatures. Understanding what accounts for the differences in low temperature tolerance between those plant species and the molecular basis of cold acclimation is a fundamental biology question and has the potential to provide new approaches to improve cold tolerance in crop plants. We identified two cDNAs by analyzing the previous microarray data of the low temperature transcriptome of tomato. In this study, we present isolation and functional characterization of these two genes, designated SlCZFP1 (for Solanum lycopersicum Cold Zinc Finger Protein 1) and SlCMYB1 (for Solanum lycopersicum Cold MYB1). We provide evidence that two genes play important roles in cold stress tolerance in plants. The information will be added to our basic understanding of plant biology and have the protential to suggest a novel approaches to improve the cold tolerance of crops.1. We isolated a cDNA clone, designated SlCZFP1 (for Solanum lycopersicum Cold Zinc Finger Protein 1), from tomato by analyzing the previous microarray data of the low temperature transcriptome of tomato. SlCZFP1 encodes a novel TFIIIA-type zinc finger protein, which contains two typical zinc finger motifs, CX2-4 CX3FX3QALGGHX3-5H, and a potential nuclear localization signal (NLS). The SlCZFP1-GFP fusion protein was localized to the nucleus in a transient expression assay. Expression of SlCZFP1 was strongly induced by cold stress, dehydration and salt treatment, but not by abscisic acid (ABA). Overexpression of SlCZFP1 in transgenic Arabidopsis and rice induced constitutive expression of cold-regulated (COR) or cold-responsive genes and conferred enhanced tolerance to freezing or cold treatments for non-acclimate transgenic plants, compared with wild type plants. However, there was no obvious enhancement observed in drought and salt tolerance. Our data suggest that SlCZFP1 plays an important role in plant responses to cold stress by regulating cold-responsive gene expression and that SlCZFP1 might be a useful gene for improving cold tolerance in crop plants.2. We also isolated another cDNA clone, designated SlCMYB1 (for Solanum lycopersicum Cold MYB 1) from tomato. SlCMYB1 encodes a R2R3-MYB family gene which contains a conserved DNA-binding domain composed of two repeat motifs. The SlCMYB1-GFP fusion protein was localized to the nucleus in a transient expression assay. Expression of SlCMYB1 was not only strongly induced by cold and salt, but also induced by dehydration and abscisic acid (ABA). To understand function of SlCMYB1 in plants under stress conditions, we generated transgenic plants in tomato and rice over-expressing SlCMYB1 under control of the CaMV 35S promoter. We were able to produce 12 tomato and 16 rice transgenic lines, respectively. Preliminary results showed that overexpression of SlCMYB1 in rice induced constitutive expression of the cold-responsive gene OsP5CS1 and OsWCOR413 and might be conferred enhanced tolerance to cold treatments for non-acclimate transgenic plants, compared with wild type plants. Due to the lack of transgenic rice seeds, further assay for drought and salt were undergoing. Analysis of abiotic stress tolerance in SlCMYB1overexpressing transgenic tomato plants will be conducted in further study.更多还原