| Wheat is a monocots and an important crop from gramineae family, and the secondleading cereal crop in china. More than one third population in the world take wheat as themain food. Drought stress restrains wheat growth and yield. Drought stress may causevarious adverse effects on plant growth and development, such as dwarf plant, shorter rootlength, smaller leaf area and decreased biomass. Besides, proline content in leaf andelectrolyte permeation were significantly increased, associated with photosynthesis decreased,sometimes the whole plant is killed badly. The accumulation of waste proteins in cells wereincreased under stress conditions. Ubiquitination of proteins through ubiquitin/26Sproteasome system (UPS) is an important regulative manner in response to various abioticstresses in plants. Ubiquitin ligase E3, as a main determinant of substrate specificity in UPSwas involved in plant stress adaptability. F-box protein characterized by an F-box motif is asubunit of SCF complex, which works as determinant in substrate recognition. It is veryimpotant to investigate the relationship between F-box and stress tolerance in plants.In the present study, we isolated and characterized an F-box gene, TaFBA1, fromTriticum aestivum L. cv. Shannong16with stronger drought resistance. Sequence comparisonand expression analysis were analyzed. The full-length TaFBA1cDNA was subcloned intothe expression vector pBI121downstream of the CaMV35S promoter or RD29A promoter toform pBI121-35S-TaFBA1or pBI121-RD29A-TaFBA1constructs. The constructs weretransferred into arabidopsis plants and tobacco plants mediated by Agrobacteriumtumefaciens. And further functional analysis on the transgenic plants were analyzed in thiswork. The main results are listed as follows:(1) We isolated an F-box gene from wheat, named TaFBA1(GenBank ID: JN038382). TheTaFBA1contains a978bp open reading frame (ORF), and encodes a325amino acidspolypeptide of molecular weight36.6kDa. There are two conserved regions, an F-box motif and an AMN1（Antagonist of mitotic exit network protein1）motif in this protein.(2) We also isolated the promoter region of TaFBA1(GenBank ID: JN065654). The promoterfragment length is1104bp. Promoter sequence analyzed by PlantCARE showed that it hasTATA-box, CAAT-box and some cis-acting elements such as MYB binding site, cis-actingregulatory elements involved in MeJA, SA response, some light-induced responsive elementsand other transcription factor-binding sites.(3) p35S-TaFBA1-GFP fusion protein was constructed and transiently expressed in onionepidermal cells. It was observed with fluorescence microscope that the TaFBA1-GFP fusionprotein was found in the membrane, cytoplasm and nucleus.(4) qRT-PCR showed that TaFBA1mRNA was expressed in the leaves, stems, roots andspikes, and the transcript level in the leaves was higher than that in the roots and spikes, andin the stems, its expression level was minimal. NaCl, PEG and MV stresses led to asignificant increase of TaFBA1transcript level in wheat leaves. And exogenous application ofABA, SA, MeJA and ETH also increased the TaFBA1mRNA in wheat leaves.(5) A recombinant of prokaryotic expression vector pET-TaFBA1was constructed andtransformed to E.Coli. BL21. The strong induced fusion protein bands were collected intophosphate buffer (PBS) solution and were used to immunize mice to obtain antiserum. Thevalue of antibody reaches1:1500. Western blot analysis revealed the presence of the strongpositive protein signals corresponding to TaFBA1in transgenic plants. The results showedthat the expression of TaFBA1at protein level was induced by drought stresses and changedwith the treatment.(6) The full-length TaFBA1cDNA was subcloned into the expression vector pBI121downstream of the CaMV35S promoter to form pBI121-35S-TaFBA1constructs. Theconstructs were first introduced into Agrobacterium tumefaciens LBA4404and GV3101bythe freezing transformation method under the kanamycin selection pressure. The transgenicplants were verified by PCR and qRT-PCR. It was indicated that the TaFBA1gene had beenrecombined into tobacco and arabidopsis genome and the transgenic plants were obtained.(7) Compared to WT plants, overexpression of TaFBA1transgenic plants showed higherresistance to drought stress by increasing germination rate, leaf number, lateral number, rootlength and growth vigor. Under drought stress, the MDA content, ROS accumulation, relative electrical conductivity of transgenic plants were obviously lower than those of WT plants.These results showed that overexpression of TaFBA1alleviated the permeability ofmembrane, cell damage degree and oxidative damage under drought stress. Furthermore, netphotosynthetic rate (Pn) and relative water content (RWC) in WT and transgenic plantsdecreased markedly under drought stress, the decrease of Pn and RWC was more slower intransgenic plants compare to WT. The results indicated that overexpression of TaFBA1maintained high photosynthetic rate and strengthened the stress resistence of plants.(8) Overexpression of TaFBA1in tobacco conferred resistance to methyl viologen (MV)induced oxidative stress. Under MV treatments, the growth of TaFBA1-overexpressingtobacco plants was better than the WT plants, which including more green cotyledons andlonger root length. In addition, TaFBA1-overexpressing tobacco plants showed less ROSaccumulation and higher activities of antioxidant enzymes (SOD, APX, CAT and POD)compared to WT plants under MV treatments. Furthermore, the expression levels ofROS-related genes were changed by overexpressed TaFBA1.(9) Compared to WT plants, overexpression of TaFBA1transgenic plants showed highproportion of abnormal leaves. At the same time, transgenic arabidopsis plants exhibiteddecelerated growth and abnormal leaves compared to WT.(10) For further research, The expression vector pBI121-RD29A-TaFBA1was constructed,and the constructs was transferred into tobacco plants mediated by Agrobacteriumtumefaciens LBA4404under the kanamycin selection pressure. The transgenic plants wereverified by PCR. It was indicated that the TaFBA1gene had been recombined into tobaccogenome and the stress-induced expression of TaFBA1transgenic plants were obtained. Wefound that stress-induced expression of TaFBA1transgenic plants showed higher resistance todrought stress and oxidative stress compare to WT plants.In conclusion, TaFBA1plays an important role in the biological processes related toplant growth and development, adaptation to drought stress and oxidative stress. |
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