【作者】 刘丽霞；

【导师】 李德全；

【作者基本信息】 山东农业大学 ， 植物学， 2008， 博士

【摘要】 ZmPP2C(AY621066)是本实验室从玉米根系中分离的一个蛋白磷酸酶基因,其编码区长度为873 bp,编码含有290个氨基酸残基的蛋白质。为了研究ZmPP2C功能,我们首先将ZmPP2C与GFP在洋葱表皮内融合表达,发现该基因是在核内表达;然后又利用花序浸染法转化拟南芥,通过基因组PCR、半定量RT- PCR以及Western blot分析证实,该基因已经成功转入拟南芥染色体中并在蛋白质水平上表达。我们对获得的T3代转基因拟南芥纯合体,在逆境胁迫处理下,进行种子萌发、根的伸长以及各种生理指标的测定。并将该基因成功转化玉米。主要结果如下:1.玉米中ZmPP2C基因与拟南芥等其他植物中的同源性比较利用Invitrogen软件包中的alignX软件,对分别来自于所有拟南芥中蛋白磷酸酶,以及与拟南芥、玉米、大麦、水稻、苜蓿等已经研究过的PP2C蛋白进行了分子聚类分析,分别构建了ZmPP2C蛋白的系统进化树。进化树分析表明,ZmPP2C与冰叶日中花植物中的MPC9同源性较高。与拟南芥中PP2C类蛋白的F类同源性较高。2.玉米ZmPP2C基因的表达分析对玉米幼苗进行甘露醇模拟干旱和高盐胁迫处理,提取根系总RNA进行Northern杂交,结果表明,干旱和高盐胁迫均对ZmPP2C基因的表达影响不明显,且表达水平随着胁迫时间延长略微下降;而在逆境恢复阶段,基因表达量又逐渐恢复到正常水平。3.玉米ZmPP2C基因的亚细胞定位构建表达载体pBI121- ZmPP2C-GFP,将ZmPP2C-GFP在洋葱表皮细胞内融合蛋白表达。在洋葱表皮细胞的细胞核上观察到绿色荧光,表明ZmPP2C定位于细胞核。而在对照细胞中的细胞膜、细胞质和细胞核中都有表达。这与TMpred软件对ZmPP2C跨膜结构分析一致。4.转ZmPP2C基因拟南芥的鉴定构建表达载体pBI121-ZmPP2C ,利用花絮浸染法转化野生型拟南芥(Col-0)。通过半定量RT-PCR和Western blot方法,在转录和蛋白质表达水平上鉴定了5株高表达的T3代转化植株(L1～L5)。5.过量表达ZmPP2C基因抑制拟南芥在胁迫下的萌发率和根的伸长将转基因植株和野生型拟南芥播种在含有不同浓度的氯化钠和甘露醇的MS培养基上进行萌发试验。7天后,过量表达ZmPP2C基因的拟南芥相对于野生型其萌发率和根的伸长都受到抑制。而在对照状态下其萌发率和根的伸长没有明显差别。6.转ZmPP2C基因拟南芥在逆境胁迫条件下的生理变化为了检测转ZmPP2C基因拟南芥在胁迫条件下的变化,我们测定了一系列和植物逆境胁迫有密切关系的生理指标,包括净光合速率,游离脯氨酸的积累,MDA含量,膜透性以及失水速率等。结果表明,未处理的转基因和野生型拟南芥的这些指标没有明显变化。但是胁迫处理后,不管是野生型还是转基因植株净光合速率都下降,但野生型拟南芥下降的更明显;转基因拟南芥中Pro积累的量远远低于野生型;而转基因植株中MDA含量和失水速率均高于野生型。这些结果表明,过量表达ZmPP2C基因降低了转基因拟南芥对逆境胁迫的抗性。7.逆境胁迫相关基因的RT-PCR分析为了检测盐胁迫和干旱胁迫下转基因和野生型拟南芥胁迫相关基因的表达情况,选取胁迫相关基因RD29A, RD29B, P5CS1, P5CS2, ABI1和ABI2用于研究。结果表明,相对于野生型拟南芥,在高盐或干旱胁迫12到24小时内,转基因拟南芥中RD29A, RD29B, P5CS1, P5CS2表达量降低或较慢。在高盐胁迫条件下,ABI1和ABI2的表达量明显降低。在干旱胁迫下,ABI1的表达量基本不变,而ABI2表达量明显降低。8.转基因拟南芥植株降低了对ABA的敏感性在对照培养基上,转基因拟南芥和野生型拟南芥发芽率没有明显差别,几乎都是100%萌发,而在含有ABA的培养基上,转基因拟南芥的萌发率明显高于野生型,即转基因拟南芥对ABA的敏感性降低,根的伸长也是如此。9.过量表达ZmPP2C基因降低了转基因玉米的抗旱性构建表达载体pCAMBIA1301-ZmPP2C,以农杆菌介导法和基因枪法同源转化玉米齐319和18-599(红)自交系。PCR结果表明,ZmPP2C cDNA已经插入转基因玉米基因组中。RT-PCR结果也表明ZmPP2C已经在转基因玉米中表达。通过对T1代转基因玉米幼苗浇灌盐溶液和甘露醇溶液试验发现,在胁迫下转基因玉米幼苗地上部受胁迫症状较为严重。更多还原

【Abstract】 ZmPP2C ( Zea mays protein phosphatase type 2C ) is a protein phosphatase type-2c previously isolated from roots of Zea mays L. ( LD9002 ), which coded a protein including 290 amino acids. Sub-cellular localization of ZmPP2C protein with GFP ( Green Fluorescence protein ) was identified in Onion epithelial cell, which the results suggested that the protein was localized in cell nuclear. Arabidopsis of constitutive expression of ZmPP2C under the control of the Cauliflower Mosaic Virus ( CaMV ) 35S promoter were identified by PCR, semi-quantitative PCR and Western blot analysis. In this study, the transgenic Arabidopsis plants were treated by NaCl and mannitol, and tested seeds germination, root growth and other physiological parameters. At last we transfored ZmPP2C gene in maize. The main results are as follows:1. Phylogenetic analysis of ZmPP2CWe constructed a phylogenetic tree based on the amino acid sequences of PP2C domains of Arabidopsis and other plants PP2C proteins. The results suggested that ZmPP2C protein is more similar to MPC9 in Mesembryanthemum crystallinum, and has a high genetic homology with PP2Cs of class F in Arabidopsis.2. Endogenous expression analysis of ZmPP2CTo test whether ZmPP2C expression is regulated by abiotic stress, V2 to V3 leaves from the maize seedlings were subjected to different stress treatments, and total RNA samples were analyzed by RNA gel blot. As shown in the result, endogenous ZmPP2C mRNA levels were low in Maize, and remained constant during the entire 3 day treatments with NaCl or mannitol treatment. Instead, they were up-regulated during the recovery process after the stress treatments were removed. This confirmed that the endogenous expression of ZmPP2C was not induced by salt and drought stresses.3. Sub-cellular localization of ZmPP2C proteinTo examine its subcellular localization, ZmPP2C was fused in frame to the 5’terminus of the green fluorescent protein (GFP) reporter gene under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S). The recombinant constructs of the ZmPP2C fusion gene and GFP alone were introduced into onion (Allium cepa) epidermal cells by particle bombardment. As shown in the result, the ZmPP2C-GFP fusion protein accumulated mainly in the nucleus, whereas GFP alone was present throughout the whole cell. Thus, ZmPP2C is a nuclear-localized protein, which is consistent with software predictation.4. Identification of the transgenic ArabidopsisThe coding region of ZmPP2C was introduced into the vector pBI121 under the control of the CaMV 35S promoter and then transformed into wild-type Arabidopsis (Col-0) by floral dip. Five independent T3 homozygous lines (L1 to L5) showed high expression of ZmPP2C at both mRNA and protein levels by semi-quantitative RT-PCR and protein gel blot analysis, thus identifying them as 35S::ZmPP2C transgenic plantlets.5. Over-expression ZmPP2C in Arabidopsis decreased plants salt and drought tolerance during germination and root growthTo examine the effects of ZmPP2C-overexpression in responding to salt and drought stresses, Arabidopsis seeds were germinated and grown on MS medium supplemented with different concentrations of NaCl or mannitol. After 7 days, seedlings with green and normal cotyledons were recorded. On normal MS growth media, ZmPP2C-overexpressed and wild type plants did show no obvious difference in the rate of the germination. However, with the addition of NaCl or mannitol, the germination of transgenic plants was significantly reduced. Similarly, under normal condition, transgenic and wild type plants also showed no obvious difference in the rate of root growth, but under NaCl or mannitol treatment, root growth of 35S::ZmPP2C plants was inhibited. 6. Changes in physiological parameters of Arabidopsis under salt and drought stressesTo investigate the effect of ZmPP2C over-expression in Arabidopsis on the physiological response to stresses, a panel of physiological parameters, including stress-related net photosynthesis rate, free proline and malondialdehyde (MDA) contents, relative membrane permeability (RMP) and loss of water, were examined. All these physiological parameters reflect normally plant response or tolerance to stress environment, thus serving as the plant physiological index under stress. As shown in the result, wild type and transgenic Arabidopsis under normal condition displayed similar rates of Pn over a range of light intensities, but the Pn of transgenic plants was significantly lower than that of wild type plants under NaCl or mannitol treatments, especially with the 500 or 700μmol m-2 s-1 light intensity. Under NaCl and mannitol treatments, free proline accumulation was increased in both transgenic and wild type plants, but the increase was lower in the transgenic lines. The increase in MDA contents was also more significant in the transgenic lines than that in wild type plants. RMPs increased in both wild type and transgenic plants in response to increasing concentrations of NaCl, however, the increases were elevated more in the transgenic plants. The over-expressing ZmPP2C plants also showed faster water loss in detached rosette leaves under conditions of dryness and airiness. These results indicated that ZmPP2C-overexpression increased plant sensitivity or decreased their tolerance to salt and drought stresses, namely, 35S::ZmPP2C Arabidopsis has a lower tolerance to salt or drought stress compared with wild type plants.7. Detection of expression of stress responsive genes in Arabidopsis by semi-quantitative RT-PCRTo elucidate the molecular mechanism of ZmPP2C in response to stresses, we examined the expression of a panel of known stress-responsive genes by RT-PCR. In the result, RD29A, RD29B, P5CS1 and P5CS2 were all up-regulated in both the wild type and transgenic plants under salt stress, but the increases in the transcription level of these genes were smaller in the transgenic plants than those in the wild type plants. Under mannitol treatment, changes in the expression patterns of all these genes were similar to those under salt treatment except that P5CS2 expression had no difference between wild type and transgenic plants. Also, ABI1 and ABI2 expressions were obviously different between wild type and the transgenic plants; they decreased significantly in the transgenic plants under both salt and drought stress treatments for 24 h. These results suggested these genes in the the transgenic plants had a slower response to stress than that in wild type plants, and ZmPP2C may be involved in the regulation of stress-regulated gene expressions.8. The transgenic Arabidopsis responses to ABA during germination and root growthTo investigated the sensitivity of 35S::ZmPP2C Arabidopsis in response to ABA during germination and root growth, exogenous ABA were added in culture medium at a range of doses (0-10μM). Exogenous ABA displayed an inhibitory effect to the germination of Arabidopsis seeds of both wild type and the transgenic plants compared with those of untreated, however, the ABA’s inhibitory effect were attenuated in the transgenic plants. Namely, the 35S::ZmPP2C plants were more insensitive to ABA compared with wild type plants. Similarly, ABA at 0.5-1.0μM also inhibited the root growth of both the wild type and the transgenic plants, but the transgenic plants were less sensitivity to ABA than wild type plants. These results indicated that over-expression of ZmPP2C gene in Arabidopsis endowed the plant with less sensitivity to exogenous ABA.9. Over-expression ZmPP2C in Maize decreased plants drought toleranceTo investigated the further founction of ZmPP2C in Maize, we constract pCAMBIA1301- ZmPP2C vector, and transferred into maize Q319 and 188-599 (red) using Agrobacterium tumefaciens and particle bombardment transformation. Some transgenic progeny was produced and used for simple drought tolerance test. The result suggested that the transgenic plants had a lower stress tolerance than the non-transformation plants.更多还原