|
Citrus often subject to frost damage, drought, salinity, and other environmental factors, stress, a serious impediment to the healthy development of the citrus industry. Therefore, the study of the mechanism of plant's response to environmental stress, the use of genetic engineering to improve stress resistance in plants, has become a key work of the citrus industry. CBF (C-repeat binding factor) is a transcription factor regulation of plant cold acclimation gene expression, expression induced by low temperature, combined with the corresponding cis-acting elements CRT / DRE (C-repeat/dehydrationresponsive element), and induction of cold-related genes the expression, and then the synthesis Cold sugars, proline and membrane stabilizing protein and other substances to improve plant hardiness. Therefore, the conversion of citrus with CBF is expected to become an effective way to cultivate new varieties of citrus Cold. The laboratory predecessors have built good CBF-GFP fusion gene of Agrobacterium expression vector CG2301. This test aims will build good expression vectors CG2301 transferred by Agrobacterium-mediated trifoliate orange (Poncirus trifoliata (L.) Raf.) And Ponkan (Citrus reticulata Blanc). The low-temperature processing of transgenic plants to detect the temporal and spatial expression of transgenic seedlings under a fluorescent microscope. The main results are as follows: 1) CG2301 subcellular localization: gene gun mediated transformation of onion epidermal cells under a fluorescence microscope to detect fluorescence is concentrated in the nucleus, indicating PtCBF located in the nucleus. 2) transfer the gene seedlings obtained: the fusion gene carrier CG2301 transferred by Agrobacterium-mediated trifoliate orange and ponkan, transgenic seedlings, some of which are genetically modified seedlings were potted and propagation. 3) CBF gene spatial expression: the transgenic trifoliate 4 ° C treatment, sensitive roots emitting strong fluorescence after 15min issued weak fluorescence, stems and leaves on the back. With the processing time lengthened, leaves, stems, fluorescence was gradually enhanced, the wing leaves and three fronds junction of the front of the blade, the petiole and stem junction and young leaves fluorescence was strong. The transgenic trifoliate fluorescent light-emitting portion is not mechanical homogenization, this test did not find that all the light emitting phenomenon of the whole plant or the entire blade. The fluorescence expression occurred in the thin roots, leaves and other organizations stem expression is relatively small. Cold resistance compared Citrus aurantium weak Ponkan 4 ° C treatment, a small amount of green fluorescence. Among them, the backs of the leaves at the main vein, stems fluorescence was stronger. 4) CBF gene expression: fluorescent protein the trifoliate transgenic seedlings with longer processing time rendering \Seedlings at 4 ℃ for 15min, the fluorescence of the fluorescent protein, as the processing time lengthened, strengthened and maintained a high level in a few hours (2 h, 4 h, 8 h). However, in the 16 h after treatment, fluorescence expression but weaker. As cold processing time continue to lengthen, fluorescence expression began to strengthen (20 h). 24h and 48 h fluorescent expression continued to improve. Cold resistance compared to the the Fructus weak Ponkan fluorescence intensity change with treatment duration of relationship is not obvious.
|