【作者】 郭玉双；

【导师】 周雪平；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 活性氧(Reactive oxygen species, ROS)是植物体内正常代谢的信号小分子,在植物生长发育和抗逆反应的信号转导中具有重要作用。但是,过多的活性氧积累会严重影响植物的生长发育,造成氧化胁迫。谷氧还蛋白是一类小分子的氧化还原蛋白,能够调节蛋白质的氧化还原状态,从而维持蛋白质的活性,在植物抗氧化胁迫及生长发育反应中具有重要作用。近年来,越来越多的谷氧还蛋白基因被克隆,其分子功能被阐明。本研究从番茄中克隆得到了一个新的CGFS类谷氧还蛋白基因,并在番茄中利用病毒诱导的基因沉默技术以及在拟南芥中利用超量表达技术研究了其在植物抗氧化、干旱及高盐胁迫中的作用。根据已发表的水稻和拟南芥的CGFS类谷氧还蛋白基因的序列信息,采用同源克隆和电子克隆的方法,从番茄中分离得到了一个新的CGFS类谷氧还蛋白基因,命名为S1GRX1。该基因是从番茄中分离得到的第一个谷氧还蛋白基因,编码的蛋白质有292个氨基酸,其结构具有谷氧还蛋白基因家族保守的结构域和典型的CGFS催化位点。通过Southern杂交分析表明,SlGRX1在番茄在基因组中以单拷贝的形式存在。采用Real time PCR的方法分析了SlGRX1基因在番茄中的表达模式,发现该基因能够在番茄的根、茎、叶片和花瓣中表达,其中叶片中的表达量最高,根和茎中的表达量相对较低,同时也证明该基因能够受外界逆境条件如氧化、干旱和高盐的诱导表达。构建了SlGRX1和GFP的融合植物表达载体,将SlGRX1融合到GFP蛋白的C端以及N端,采用农杆菌浸润法,在本氏烟叶片的表皮细胞瞬时表达重组蛋白,通过激光共聚焦显微镜观察SlGRX1蛋白的亚细胞定位,结果表明,SlGRX1能够定位在植物细胞的细胞核和细胞质中。构建了基于双生病毒卫星DNAβ的SlGRX1的沉默载体,并将该载体导入农杆菌菌株EHA105中,以中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus, TYLCCNV)为辅助病毒,在番茄中沉默了SlGRX1基因,以接种TYLCCNV和空载体的番茄作为对照。SlGRX1基因沉默番茄与对照相比对氧化和盐胁迫更为敏感,主要表现为叶绿素降解速度加快；在干旱胁迫下,SlGRX1基因沉默番茄与对照相比则更容易出现黄化和萎蔫的症状,叶片内的相对含水量有明显的下降。这些结果表明,SlGRX1基因在植物的抗氧化、干旱和高盐反应中具有重要的调控作用。将SlGRX1基因构建到植物表达载体pBin438中,采用农杆菌介导的花序浸蘸法对模式植物拟南芥进行遗传转化,获得了2个单拷贝的纯合T3代转基因株系Y1、Y2,同时将pBin438空载体也转化到拟南芥中作为研究的对照。通过分析株系Y1、Y2与对照在氧化、干旱和高盐胁迫下的表型,进一步明确了SlGRX1基因的功能,无论拟南芥在离体生长条件下还是土壤生长条件下,SlGRX1基因超量表达的株系Y1、Y2对氧化、干旱和高盐的抗性都明显高于对照,说明超量表达SlGRX1基因能够提高植物对上述逆境的抗性。为了进一步分析了SlGRX1基因在提高植物抗逆性可能的作用机理,利用Real time PCR技术分析了拟南芥抗氧化、干旱和高盐反应中的Marker基因在Y1、Y2和对照中的表达差异。结果显示,拟南芥的Apx2、Apx6和RD22的表达水平有明显的上调,表明SlGRX1基因有可能通过提Apx2、Apx6和RD22的表达水平实现其功能。更多还原

【Abstract】 Reactive oxygen species (ROS) are redox signals essential to many physiological processes in plants. But high concentration of ROS can cause damage to macromoleces thus disrupt normal signaling in plant. Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases that play a crucial role in plant response to oxidative stress. Recently, studies have extended our knowledge on the physiological and molecular functions of Grxs in plant. In the present study, a novel CGFS type Grx gene was isolated from tomato and its functions were validated using loss-of-function and gain-of-function approaches by virus induced gene silencing (VIGS) of the gene in tomato and over-expression of the gene in Arabidopsis.Based on the sequence information of CGFS type Grxs from Arabidopsis and rice, a cDNA encoding a protein containing the consensus Grx family domain with a CGFS active site was isolated using homologue cloning and in silio cloning methods. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that SlGRX1 is expressed ubiquitously in tomato including leaf, root, stem and flower and can be induced by oxidative, drought and salt stresses.To investigate the subcellular localization of SlGRX1 protein in plant cells, the SlGRX1 protein was fused to both C-terminus and N-terminus of GFP and the construct was transiently expressed in Nicotiana benthamiana leaf epidermal cells then monitored by capturing GFP fluorescence. Our results suggested that SlGRX1 protein was localized in both nucleus and cytoplasm.SlGRX1 gene silencing vector DNAmβ-SlGRX1 was constructed based on the geminivirus satellite DNAmβinduced silencing system and then introduced into Agrobacterium tumefaciens strain EHA105. Agrobecterium harboring recombinant silencing vector DNAmβ-SlGRX1 and TYLCCNV (Tomato yellow leaf curl China virus) infectious clone were co-agroinoculated into tomato plants. Meanwhile, tomato plants co-agroinoculated with TYLCCNV and empty silencing vector DNAmP was used as the control. VIGS mediated silencing of SlGRX1 in tomato led to increased sensitivity to oxidative and salt stresses with decreased relative chlorophyll content, and reduced tolerance to drought stress with decreased relative water content. These results suggested that SlGRX1 was required for plant resistance to oxidative, drought and salt stresses.The coding sequence of SlGRX1 was then cloned into the binary vector pBin438 to produce pBin438-SlGRX1 in which the SlGRX1 gene was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The plasmid pBin438-SlGRX1 was then introduced into A. tumefaciens strain EHA105. Transformation of Arabidopsis was performed using the floral dip method. The empty vector pBin438 was also transformed into Arabidopsis as the control. Two independent homozygous lines (Y1, Y2) with a single copy of SlGRX1 were allowed to grow for 3 generations for further studies. We found over-expression of SlGRX1 in Arabidopsis plants enhanced lines Yl and Y2 the tolerance to oxidative, drought and salt stresses in both in vitro and in soil-grown conditions.Expression levels of some oxidative related ascorbate peroxidase (Apx) genes Apx2, Apx3, Apx4, Apx5, Apx6, and well-known drought/salt stress marker genes RD29A, RD22 and RAB18 in Y1,Y2 and the control plants were analyzed by qRT-PCR to explore the possible mechanism of the enhanced abiotic stress tolerance in SlGRX1 over-expression plants. We found that over-expression of the SlGRX1 gene in Arabidopsis can activate the expression of the Arabidopsis Apx2 and Apx6, indicating that the SlGRX1 gene may positively regulate the Apx2 and Apx6 genes to improve plants resistance to oxidative stress.更多还原