| Abiotic stress is a major limiting factor for plant growth and crop production, and thus the adaptability of plants to abiotic stresses is being studied throughout the world. Fructans may play important roles in stress tolerance in plant species. Wild relatives of the wheat is characterized by high resistance to cold, drought, salinity, and barren conditions, which is a useful gene pool for wheat improvement. Therefore, isolation and functional analysis fructan related genes from wild relatives of the wheat has great theoretic and practical implications. In addition to the commercialization of genetically modified crops, the potential impact of crop transgene flow on the environment and food safety are now major concerns. Current bottlenecks that limit the commercialization of genetically modified crops include the presence of selectable marker genes and vector backbone sequences in plant genomes, which have affected the safety assessments for transgenic plants. It has been proposed that direct DNA transformation using selectable markers and reporter gene cassettes at different molar ratios provides a safe and efficient method for obtaining transgenic plants, but without introducing unnecessary DNA sequences.In this study, using Psathyrostachys huashanica, Dasypyrum villosum(L.) Candargy, Leymus racemosus, and Leymus mollis as experimental materials, genome walking and reverse transcriptase PCR(RT-PCR) techniques were employed to clone genes coding for sucrose:fructan-6-fructosyltransferase(6-SFT) from P. huashanica and D. villosum. In addition, rapid-amplification of c DNA ends(RACE) and RT-PCR techniques were employed to clone the genes coding for 6-SFT from L. racemosus and L. mollis, coding for fructan:fructan-1-fructosyltransferase(1-FFT) from P. huashanica, D. villosum, and L. racemosus, and coding for sucrose:sucrose-1-fructosyltransferase(1-SST) from P. huashanica. Multiple sequence alignments and phylogenetic tree analyses were also performed for these genes and among those with different origins. The fragments were cloned into p1300-35 SN at the Bam HI/Eco RI + Hind III site to construct p1300-35SN-fructan biosynthesis enzymes(FBE) vectors. The p1300-35SN-FBEs were then transferred into tobacco(Nicotiana tabacum L.) cv. W38 via Agrobacterium-mediated transformation and the transgenic tobacco plants obtained were tested to determine their drought, cold, and salt stress resistance capacities, as well as analyzing various physiological parameters related to fructan accumulation. A safe and efficient particle bombardment(biolistic)-mediated common wheat transformation system was established using the whole plasmid p AHC25 as the reference. Wheat immature embryo transformation was performed in parallel experiments using either p AHC25 and p Easy-Blunt-bar plasmids, or the corresponding minimal gene cassettes, which were purified from the same plasmid by restrictive digestion. The molar ratios of the selected and target gene varied from 1:1 to 1:3 in order to optimize the molar ratio and to develop a safe and efficient particle bombardment-mediated common wheat(Triticum aestivum L.) variety Kenong 199 co-transformation system. In this study, the main results obtained were as follows.1. The genes encoding FBEs were cloned from different materials. Four full-length c DNAs encoding 6-SFT were cloned from P. huashanica, D. villosum, L. racemosus, and L. mollis, which were designated as Ph-6-SFT(1951 bp), Dv-6-SFT(1863 bp), Lr-6-SFT(1863 bp), and Lm-6-SFT(1866 bp), respectively. Three full-length c DNAs encoding 1-FFT were cloned from P. huashanica, D. villosum, and L. racemosus, which were designated as Ph-1-FFT(1989 bp), Dv-1-FFT(1950 bp), and Lr-1-FFT(1989 bp), respectively. One full-length c DNA encoding 1-SST was cloned from P. huashanica, which was designated as Ph-1-SST(2001 bp).2. Plant expression vector construction of p1300-35SN-FBEs. The Ph-1-FFT, Dv-1-FFT, Lr-1-FFT, Ph-6-SFT, Dv-6-SFT and Lr-6-SFT genes were constructed in p1300-35 SN and six vectors were obtained.3. The FBEs genes were transformed into tobacco and molecular analysis of transgenic plants. FBEs genes were introduced into tobacco(Nicotiana tabacum L.) cv. W38 via Agrobacterium-mediated transformation, 113, 98, 101, 65, 69 and 72, a total of 518 transgenic T0 plants were screened out by the hygromycin resistance. The screened plants were tested by PCR and RT-PCR. In total, 321 putative transgenic tobacco plantlets were obtained and tested, where 64, 53, 55, 24, 38 and 43 a total of 277 plants were confirmed to be positive by semi-q RT-PCR analysis.4. Resistance identification in transgenic plants. The results of phenotypic analyses showed that the expression of these genes resulted in enhanced tolerance of drought, cold, and salt stresses. The results confirmed that 6-SFT genes were more effective in improving crop stress resistance compared to the 1-FFT genes. The stress resistance are not obviously different between the transgenic plants with same gene transfer from different species. By contrast, Ph-6-SFT and Dv-6-SFT gene is much better than others in improving crop abiotic stress tolerance.5. Analysis of the physiological parameters of the transgenic plants. Analyses of various physiological parameters under drought and cold stress showed that compared with non-transgenic plants, the tolerance of transgenic plants was associated with the accumulation of fructan, soluble sugars, and proline, but decreases in malondialdehyde(MDA). After drought and cold stress, significantly higher levels of fructan, soluble sugars, and proline were found in the transgenic plants, whereas there was no significant change in the MDA levels of the transgenic plants. By contrast, after drought and cold stress, a significantly higher level of MDA was found in the non-transgenic plants. However, there were no apparent differences in the carbohydrate and proline contents of the non-transgenic plants in all conditions. These results indicate that the expression of Ph-1-FFT, Dv-1-FFT, Lr-1-FFT, Ph-6-SFT, Dv-6-SFT and Lr-6-SFT genes can enhance the fructan concentrations and improve resistance to the oxidative stress generated by drought and cold in transgenic tobacco plants. Physiological parameters comparisons of transgenic plants with different gene transfer confirmed that 6-SFT genes were more effective in improving crop stress resistance compared to the 1-FFT genes. The accumulation of soluble carbohydrate, proline and malonaldehyde are not significantly different between the transgenic plants with same gene transfer from different species. By contrast, Dv-6-SFT and Ph-6-SFT genes are much better than others in increasing crop cold and drought stresses. Our results suggest that the Ph-6-SFT and Dv-6-SFT genes may be applied as a genetic tool for improving stress tolerance in other crops.6. The molar ratio of the selectable marker gene cassette and non-selected gene cassette was established for biolistic-mediated wheat co-transformation. Using minimal gene cassettes and the whole circular plasmids for biolistic-mediated wheat transformation, 3964 phosphinothricin-resistant plants were obtained, 70 of which were β-Glucuronidase(gus)-PCR positive, although only 56 plants exhibited clear blue staining indicative of gus expression. Eleven transgenic wheat lines transformed with p AHC25, 30 transgenic wheat lines transformed with gus plus phosphinothricin acetyltransferase(bar) gene cassettes, and 29 transgenic wheat lines transformed with gus plus p AHC20 were regenerated with transformation frequencies of 0.70%, 0.16-1.03%, and 0.09-0.98%, respectively. The combination of p AHC20/bar:gus at a molar ratio of 1:2 performed significantly better than that of plants with p AHC20/bar:gus at any other molar ratio. Southern blot analysis indicated that the gus gene integrated successfully into the wheat genome. The wheat transformation system may provide useful information for future transformation study.7. Plant expression vector was construsted for Ph-6-SFT and Dv-6-SFT genes and transgenic wheat plants were produced by biolistic-mediated transformation. Leaf GUS assay and PCR analysis confirmed the presence of transgenes in T0 generation. One independent transgenic wheat plant was produced for every vector. |
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