Functional Analysis Of Protein Phosphatase 2C Gene(ZmPP2C) In Zea Mays Roots And Its Genetic Transformation

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.