| High photosynthetic efficiency breeding in crops is an important aspect of improving crop yield since it was proposed in 1980 s. Discovering high photosynthetic efficiency related genes in crops by molecular biology and molecular genetic methods, and creating high photosynthetic efficiency crop cultivars via gene engineering method have being vital contents in modern agricultural research. In previous research, a large number of wheat(Triticum aestivum L.) genes were found to show differential expression at m RNA level by microarray analysis. In this study, 6 target genes(Ta23008, Ta24695, Ta27787, Ta92165, Ta119251, and Ta106078) were chosen for genomic functional analyses, using barley stripe mosaic virus(BSMV)-mediated virus-induced gene silencing(VIGS) in bread wheat cv.Xiaoyan 54(XY54). Generally considering the differential phenotypes of these 6 BSMV:gene infected plants, Ta92165(Ta SCL14) was picked up and further dissected in detail of biological function by silencing of Ta SCL14 in wheat and overexpression of Ta SCL14 in Arabidopsis and wheat. Some main results have been achieved as follows:1. 6 target gene fragments(size in 160-200 bp) were isolated from c DNA of XY54, inserted in ? vector of BSMV, transcribed into viral RNA in vitro, and used to infect XY54. After emerging of stripe symptoms in leaves of the BSMV-infected plants, the expression of target genes was analyzed to confirm gene silencing effect by RT-PCR(reverse transcription PCR). The expression of target genes was lower in respective BSMV:gene plants than in control plants(BSMV:GFP), indicating successful silencing of target genes.2. To examine reponses to photooxidative stress, changes of Fv/Fm(maximal photochemical efficiency) and P.I.(photosynthetic performance index) in leaves of the BSMV-infected plants were analyzed under high light with normal(HLNT) or low temperature(HLLT) stress, as well as light with N-(3,4-dichlorophenyl)-N',N'-dimethylurea(DCMU), methylviologen(MV) or hydrogen peroxide(H2O2) treatment. Compared with control, the photooxidative resistance was lower in BSMV:Ta23008 and BSMV:Ta92165 plants under HLLT, DCMU, MV, and H2O2 treatments; in BSMV:Ta24695 plants under HLLT andH2O2 treatments; in BSMV:Ta27787 plants under HLLT, DCMU, and H2O2 treatments; in BSMV:Ta119251 plants under DCMU treatment; in BSMV:Ta106078 plants under DCMU, MV, and H2O2 treatments. These results suggested that these 6 target genes are involved in the regulation of photooxidative resistance in wheat.3. To analyze the completeness of cellular membrane in leaves of the BSMV-infected plants, the malondialdehyde(MDA) content and electrical conductivity were measured. The MDA content in leaves of the BSMV:Ta23008, BSMV:Ta24695, BSMV:Ta92165, BSMV:Ta119251, and BSMV:Ta106078 plants was higher than in control plants; the electrical conductivity in leaves of the BSMV:Ta23008, BSMV:Ta24695, BSMV:Ta27787, BSMV:Ta92165, and BSMV:Ta106078 plants was higher than control plants. These results suggested that silencing of the target genes results in injury to cellular membranes, which might be an important reason of exhibiting lower tolerance to photooxidative stress.4. Judging from the changes of leaf colour in the BSMV-infected plants under DCMU treatment, effects of gene silencing on accumulation of chlorophyll were evaluated. The blenching phenomenon in leaves of the BSMV:Ta23008, BSMV:Ta24695, BSMV:Ta92165, and BSMV:Ta106078 plants was more serious than control after 6 days of DCMU treatment, indicating that Ta23008, Ta24695, Ta92165, and Ta106078 might be involved in the regulation of chlorophyll accumulation.5. After 38 days of post-inoculation by virus, the biomass accumulation of BSMV-infected plants was measured to determine the effect of gene silencing on plant growth. The fresh weight of BSMV:Ta23008, BSMV:Ta92165, BSMV:Ta119251, and BSMV:Ta106078 plants was significantly lower than control, indicating that Ta23008, Ta92165, Ta119251, and Ta106078 are related to biomass accumulation in plant.6. A GRAS gene named TaSCL14 was cloned from XY54 genome by PCR. There was no intron in genomic DNA sequence of the Ta SCL14 gene. The predicted protein sequence of Ta SCL14 consists of 675 amino acids with a predicted molecular mass of 74.9 k Da and a p I of 6.45. Bioinformatic analysis indicated that Ta SCL14 belongs to the GRAS protein family and that the homological gene of Ta SCL14 in Arabidopsis is At SCL14. In order to determine the chromosomal location of Ta SCL14, a set of cv. Chinese Spring nulli-tetrasomic lines were used as templates for genomic PCR. Ta SCL14 is located on chromosome 4A. A 163 h GFP::Ta SCL14 fused protein recombinant vector was constructed, and then introduced into onion epidermal cells for transient activity analysis. The transient assay indicated that Ta SCL14 is located in nuclei.7. The expression of TaSCL14 was measured by Real-time PCR. TaSCL14 was expressed inroot, stem, leaf, glume, rachis and seed of wheat, with a strong expression in root, suggesting that Ta SCL14 might function in the regulation of various organs growth in wheat, especially in root growth. Ta SCL14 was significantly induced by high-light stress, implying a potential role in wheat tolerance to photooxidative stress.8. Silencing of Ta SCL14 in wheat resulted in an inhibition of growth in the BSMV:Ta SCL14 plants than the BSMV:GFP plants, which demonstrated by lower biomass, smaller leaf area, less number of tillers and leaves. To detect the possible reason of having poor growth in BSMV:Ta SCL14 plants, the photosynthetic capacity of BSMV-infected plants was evaluated. A lower photosynthetic rate, stomatal conductance, quantum efficiency, carboxylation efficiency, and Rubisco(Ribulose-1,5-bisphosphate carboxylase/oxygenase) activity were observed in the BSMV:Ta SCL14 plants as compared to the control plants. In addition, leaves of the BSMV:Ta SCL14 plants showed faster senescence rate than the BSMV:GFP plants induced by darkness treatment, indicating that Ta SCL14 might be involved in the regulation of leaf senescence.9. To further confirm the biological function of TaSCL14, a recombinant vector 35S::Ta SCL14 under the control of 35 S promoter was constructed, and then transferred into Arabidopsis wild type(WT) plants. Genotype analysis showed that the transgenic Ta SCL14 plants exhibited a better growth than WT plants, demonstrated by an increased of fresh weight, dry weight, leaf number and leaf area, as well as yield in the transgenic Ta SCL14 plants. Gas exchange experiments indicated that the photosynthetic rate and stomatal conductance were higher in transgenic Ta SCL14 plants than in WT plants. Compared with WT plants, transgenic Ta SCL14 plants showed stronger tolerance to photooxidative stress induced by high light, MV and H2O2. Moreover, transgenic Ta SCL14 plants showed superior root system because of its longer total root and more lateral root number. Cytological analysis showed that there was longer meristem zone size and matural cell length, as well as more meristem cell number in main root of transgenic Ta SCL14 plants, indicating the reason of having larger root system. Some genes related to plant growth and development, or related to stress resistance were up-regulated in transgenic Ta SCL14 plants, for example, NAC032(At1g77450), CYP81D11(At3g28740), AKR(At2g37770), GST25(At2g29420), MO1(At4g15760), UGT74E2(At1g05680), ASI3(At4g13180), DJ1A(At3g14990), At5g61820, ATAF1(At1g01720), At5g16980, NIT4(At5g22300) and At5g61950.10. Ta SCL14 was inserted into an overexpression vector under the control of ubiquitin promoter, and then transferred into immature embryo of wheat by gene shot gun method.The generation T1 of ransgenic plants showed better growth and higher photosynthetic rate and stomatal conductance than control plants. These results indicated that Ta SCL14 shows a potential role in the modification of plant genotype and improving the photosynthetic capacity in wheat. Other physiological and biochemical experiments of transgenic Ta SCL14 wheat are still being in progress. |
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