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Temperature is an important factor to affect plant growth and distribution. Periodic chilling injury often caused lower production of citrus fruits, decline in quality, even the death of the tree, causing huge economic losses. So far, the global citrus frost damage problem still has not been effectively resolved. Therefore the Cold cultivate citrus varieties become an urgent problem in the citrus industry. With the development of plant molecular biology, transgenic crop breeding technology, plant cold tolerance is a complex quantitative trait is the result of low temperatures polygenic collaboration. The use of a single cold resistance gene conversion plant, although to some extent to improve the hardiness of the transgenic plants, but the actual effect is not ideal. CBF is a transcription factor regulation of a range of plant cold acclimation gene expression, rapid synthesis at low temperatures, and the corresponding cis-acting elements CRT / DRE induction of cold-related gene expression, and thus the synthesis of carbohydrates, proline and membrane stabilizing protein and other substances to improve plant cold tolerance. Therefore, the conversion of citrus with CBF is expected to become an effective way to cultivate new varieties of citrus Cold. Trifoliate with strong resistance, dwarf tree, commonly used rootstock for citrus. Trifoliate citrus in cold most varieties can be resistant to -20 ° C. Therefore, the study of citrus cold resistance gene first study cold resistance gene Hovenia. It is necessary to increase the hardiness of citrus, in order to make better use of the isolated Hovenia CBF gene to understand its functional characteristics and expression patterns. The study of gene expression, we must choose the right promoter. It has been reported that the trifoliate CBF gene and its promoter sequence, the reference to this reported cloning trifoliate CBF and the start of its own promoter construct with the reporter gene GFP fusion gene, and build a fusion gene expression vector Agrobacterium-mediated transformation into tobacco and trifoliate observation trifoliate CBF spatial and temporal expression patterns. The main results are as follows: (1) build a fusion gene CG. Trifoliate DNA as a template, the the trifoliate CBF and its own promoter as a complete sequence of cloned amplification product 5 'end EcoRI restriction sites, and the 3' end of the introduction of the Sal Ⅰ restriction sites. PMV plasmid as a template, amplified GFP, the 5 'end of the introduction of the Sal Ⅰ restriction sites at the 3' end increase Bstp Ⅰ digestion sites. CBF with GFP sequences were amplified single-digested with Sal Ⅰ connection. Note fusion gene frameshift, the amplification products were sequenced to verify that for the purpose of fusion gene, named CG in this process. The total length of 2.4 kb, which is a by trifoliate CBF gene promoter, trifoliate CBF and GFP fusion gene composed. ② build fusion gene expression vector CG2301. Expression vector pCAMBIA2301-based carrier with EcoR Ⅰ, Bstp double digested pCAMBIA2301 fusion gene CG, T4 ligase connection, to construct fusion gene CG2301. The carrier containing the trifoliate CBF and GFP fusion gene CG by trifoliate CBF own promoter start expression for Kan resistance. (3) fusion gene expression carrier CG2301 using Agrobacterium-mediated leaf disk transformation in tobacco, 43 rooting resistant seedlings, was identified by PCR, 13 transgenic tobacco. (4) fusion gene expression vector CG2301 using Agrobacterium-mediated transformed epicotyl trifoliate resistant shoots 14 were identified by PCR, two transgenic trifoliate. ⑤ fusion gene expression patterns of CG in tobacco research. Transgenic tobacco treated with a temperature of -5 ℃ at 4 ℃ different time, was observed by fluorescence microscopy for the expression of the gene in different organs. The results showed that the fluorescence first expression in the roots (at 4 ℃ 2h, at -5 ° C for 15min can be detected), followed by the blade (4 ℃ 12h, -5 ℃ under 15min, a small amount of fluorescence), the stem in the expression Old (4 ° C 48h, -5 ° C for 30min of a small amount of fluorescence). Leaves fluorescence first appeared in the veins at the expression also increased with the increased processing time, If the leaf is wound around the wound will be detected a large number of fluorescent. Continue to strengthen with the the cryogenic processing time increase the amount of fluorescence in the roots and stems of the same.
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