Functional Analysis Of Genes Coding Fructan Biosynthesis Enzymes In Wheat And Tabacco

Fructan, with a varieties of physiological functions and biological activity, is water-soluble and viscous polysaccharide polymer, which was fructosyltransferase catalyzed by sucrose and fructose molecules connected to one or more. In various abiotic stress conditions, fructan is one of the important regulators involved in plant resistance to stresses, by means of releasing soluble fructose and regulating cell osmotic pressure. As an important crop worldwide, wheat metabolisms fructan in whole growth period for energy storge and abiotic atress response. Wheat transformation efficiency is limited at some extent by its low regeneration frequency. Functional analysis of genes coding fructan biosynthesis enzymes (FBEs) Ta1-SST, Ta6-SFT, and Ta1-FFT in wheat and improvement of regeneration efficiency of wheat by adjusting medium composition will help further improvement of this crop on drought or salt resistance, and provide more theoretical basis for clearifying function of this metabolite in plant.Referenced our previous results, Ta1-SST and Ta1-FFT genes were isolated from wheat cultivar Yangmai 6, and two recombinant expression vectors of pBI121-Ta1-SST and pZP211-Ta1-FFT were constructed in this study. The three fructan biosynthesis enzymes encoding genes carried on three binary expression vectors of pBI121-Ta1-SST, pZP211-Ta1-FFT and pBI121-Ta6-SFT, respectively, were co-transformed into tobacco cultivar NC89 by Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR and ELISA, tested for abiotic stress resistance, and analyzed for different physiological indicators. The results showed that the tolerances of the seven types of transgenic tobacco plants to drought, low temperature, and high salnity were significantly improved in comparision with the wild type. Contents of fructans, proline, malondialdehyde, and soluble sugar in the all transgenic tobacco types were higher than it in wild type tobacco. Morever, the plants containing three or two FBEs genes showed stronger tolerance to stresses of water, salt, and low temperature, and produced more physiological metabolites of fructans, proline, malondialdehyde, and soluble sugar comparing with the plants with one alien gene. The higher content of fructan was 675.6 mg g^-1 which is the transgenic tobacco plants with Ta1-SST + Ta6-SFT gene and 140.7 mg g^-1 which is the transgenic tobacco plants with Ta1-SST + Ta1-FFT + Ta6-SFT gene; Content of proline, 184.4μg g^-1 and 201μg g^-1 was transgenic plants with Ta1-SST + Ta6-SFT gene and Ta1-SST + Ta1-FFT + Ta6-SFT gene; The lower content of MDA was 12.9μmol mg^-1FW which is transgenic plants with Ta1-SST + Ta6-SFT gene and 14.9μmol mg^-1FW which is transgenic plants with Ta1-SST + Ta1-FFT + Ta6-SFT gene; 0.262 % was the highest content of soluble sugar of transgenic tobacco plants with Ta1-SST + Ta6-SFT gene, the second was 0.234 % the content of transgenic tobacco plants with Ta1-SST + Ta1-FFT + Ta6-SFT gene. These results suggest that Ta1-SST + Ta6-SFT gene is the best gene, it plays a main role in the experiment.At the same time, RNA interference vector pANDA-Ta1-FFT-RNAi for silencing the fructan synthase gene of Ta1-FFT in wheat was transformed into wheat plants with into wheat by Agrobacterium-mediated transformation using the immature embryos and mature embryos as explants in this study. Three kinds of T1 transgenic plants achieved in our group, including the T1 plants with pANDA-Ta1-SST-RNAi and pANDA-Ta6-SFT-RNAi obtained previously, were detected together by PCR and qPCR. The marker gene nptII was detected sably in the transgenic wheat plants. qPCR analysis revealed that the expression levels of the target genes in transgenic plants was relatively lower than them in wild type, indicating that the gene silencing effect was obvious, and the lowest gene expression of positive plants is 0.11 of the controlled plant. In addition, over-expression vectors of pRTL2-Ta1-SST and pRTL2-Ta1-FFT with fructan synthase genes were transformed into by particle bombardment using the immature embryos as target materials, three types of T1 transgenic plants including the plants with pRTL2-Ta6-SFT obtained previously were confirmed by PCR for bar gene and FBEs. It was concluded that the target genes were successfully transferred into wheat plants and inherited in wheat stably.By testing different types of plant growth regulators, increasing the dosages of CuSO4 and iron source in culture medium, wheat regeneration system for the immature or mature embryos culture was optimized in order to further improve transformation efficiency of this plant. The results showed that the optimal concentrations of dicamba, 2,4,5-T, picloram when used alone were 2.5 mg L^-1, 3.0 mg L^-1, 3.0 mg L^-1, respectively. In the presence of 2.0 mg L^-1 2,4-D and 0.3 mg L^-1 ABA in the callus induction medium, the best regeneration efficiency from the immature was appeared, improving the callus quality and the green seedling numbers dramatically. There was no difference of the efficiencies for embryogenic callus induction and green shoot induction when the concentration of CuSO4 was employed from 0.1 to 4.1μmol L^-1 , however, CuSO4 at 6.1μmol L^-1 was toxic for mature embryos of wheat. Regeneration efficiency from the mature embryos of CB037 was obviously improved on the media containing 40μmol L^-1 iron source. The optimal concentration of iron source were 40 to 50μmol L^-1 when the immature embryos of CB037 were used as explants, both of callus differentiation and green shoots induction on this level were higher than those in the level of 20μmol L^-1(at random). For the immature embryo culture of Xinchun9 and Bobwhite, the ideal concentration of iron source was 20μmol L^-1.