| Abiotic stresses are among the major factors that limit crop growth and yield.Maize, an important dry-land crop, is the second major food crop in China. It is widelyused as food, animal feed and industrial products. Abiotic stresses such as drought andhigh salinity are the principal causes of maize yield loss in most of the corn-producingareas in China. Traditional plant breeding has achieved significant results by breedingfor resistance to abiotic stresses, but the process is rather time-consuming and expensive.Plant biotechnology is an attractive alternative by allowing for direct introduction of asingle gene into a desirable genotype. Gene discovery and functional genomics effortshave revealed that productivity and adaptation of plants to abiotic stresses are controlledby a wide range of genes.In recent years, pre-mRNA splicing factor in plant has been of particular interestfor improving abiotic stress tolerance of crops. In this research, ZmSKIP, a maizehomologue of pre-mRNA splincing factor SKIP has been isolated and its function inresponse to abiotic stress has been investigated in transgenic tobacco plants. The resultswill provide an important basis for breeding maize resistant to abiotic stresses. The mainresults are as follows:1. ZmSKIP encoded a protein of609aa. The deduced amino acid sequence fromthe nucleotide sequence of the cDNA showed considerable homology with knownmembers of the SNW SKIP protein family. The S-N-W-K-N motif, which is absolutelyconserved in SNW SKIP protein family, was also found in ZmSKIP protein. ZmSKIPshowed the highest homology (85%) with SKIP in rice. Also, a putative bipartite nuclearlocalization signal motif was found. Based on its amino acid sequence features, ZmSKIPseemed to be encoded by only one gene in the maize genome and can be clearlyassigned to the SNW SKIP family of transcriptional regulators.2. ZmSKIP was induced at the transcriptional level by various abiotic stressesincluding drought, high salinity, and abscisic acid (ABA) in maize. Transgenic tobaccoplants expressing β-glucuronidase (GUS) reporter gene under the control of the ZmSKIPpromoter exhibited GUS activity in all tested tissues, and GUS expression wassignificantly induced by NaCl, mannitol, polyethylene glycol (PEG) and ABA.3. In order to confirm the sub-cellular localization of the ZmSKIP protein, weexpressed a ZmSKIP-GFP fusion protein under the control of the CaMV35S promoter in tobacco protoplasts. Fluorescence microscopy demonstrated that ZmSKIP wastargeted to nucleus.4. To further investigate whether ZmSKIP enhances tolerance to abiotic stress intransgenic plants, the coding region of ZmSKIP was inserted into the vector under thecontrol of the CaMV35S promoter and then transferred into tobacco plants. Comparedto wild type (WT) plants，the root growth of transgenic tobacco plants overexpressingZmSKIP was less inhibited under MS media supplemented with300mM mannitol,200mM NaCl or0.1mM ABA. In soil, transgenic tobacco plants overexpressing ZmSKIPwere more tolerant to drought, high salt and ABA stresses.5. The antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD) andcatalase (CAT) activities), malondialdehyde (MDA) content and relative electricalconductivity (REC) of T1transgenic tobacco plants overexpression ZmSKIP and WTtobacco plants were examined under various abiotic stresses. Compared to WT plants,transgenic tobacco plants overexpressing ZmSKIP displayed higher antioxidantenzymes, lower MDA content and REC under drought, manitol, NaCl and ABAtreatments.6. Stress-responsive genes were assayed at the transcriptional level in WT andtransgenic plants after12h mannitol, NaCl and ABA treatments by qRT-PCR analysis.CAT transcript level in transgenic tobacco plants was up-regulated under mannitol,NaCl and ABA treatments, and was significantly higher than that in WT plants. APXtranscript level was up-regulated under abiotic stresses, while it was down-regulated inWT plants. PR5transcript level of WT plants was down-regulated under abiotic stresses,howere; it was significantly up-regulated under mannitol stress in transgenic plants.7. The results of the fluorometric GUS assays on ZmSKIP promoter, as well as thephysiological activity assays, plant germination and growth experiments showed thattransgenic tobacco plants overexpressing ZmSKIP have not significant comparing toWT tobacco plants under exogenous salicylic acid (SA) treatment. ZmSKIP was notsignificantly induced at the transcriptional level by exogenous SA in maize.The above results indicate that overexpressing of ZmSKIP improves abiotic stresstolerance in tobacco plants and ZmSKIP can be a good candidate gene for improving thestress tolerance of transgenic plants... |
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