Cloning Of CBF1Gene From Neosinocalamus Affinis And Its Effects On Improving Cold Resistance

CRT-binding factor/Dehydration responsive element binding (CBF/DREB1) is a small family of transcription factors which plays an important role in response to cold. It controls expressions of genes involved in cold-resistance of plant. In our study, a Neosinocalamus affinis CBF1gene (NaCBF1) was cloned by RACE. Then, a NaCBF1overexpression vector and an RNAi expression vector were generated. Furthermore, the NaCBFl overexpression vector (pBI-NaCBF1) was transformed into tobacco by A. tumefaciens-mediated transformation, which lays a foundation for studying the mechanism of cold-resistance of bamboo. Main results are as follows:1. According to several known CBF1cDNA sequences, a pair of degenerate primer was designed. Then, a conserved sequence of CBF1gene was cloned from Neosinocalamus affinis. With the cloned fragment as the template, a full-length CBF1gene sequence from Neosinocalamus affinis was obtained through5’RACE and3’RACE, and designated as NaCBFl (Genbank:JN896707). The results of bioinformatics analysis indicated that the length of NaCBFl was663bp, encoding221amino acids with the conserved amino acid blocks PKRRAGRTKFKETRHP and LNFADS.2. In order to analyze the function of NaCBF1, an RNAi plant expression vector (pBI-NaCBF1-CBFl) and an overexpression vector (pB1-NaCBFl) of NaCBF1gene were generated. Then, the pB1-NaCBFl vector was transformed into tobacco by A. tumefaciens-mediated transformation. Several transformation plants obtained were further confirmed by PCR and RT-PCR testing. The results showed that the expression of NaCBF1gene in OE1, OE2and OE3lines of transgenic tobaccos were higher than that of untransformed wild type tobaccos.3. In order to detect the effects of NaCBF1overexpression on cold-resistance of transformed tobacco, wild-type and transgenic tobaccos were treated by the different temperature treatments (25℃,15℃,4℃,0℃and-2℃) with different times (0h,3h,6h,12h and24h). The results showed that under chilling or cold stress, the levels of maximal photochemical efficiency of PS II (Fv/Fm), actual photochemical efficiency of system Ⅱ (ΦPS Ⅱ), electron transport rate(ETR), photochemical quenching (qP) and potential activity of PS Ⅱ (Fv/F0) in the overexpressing NaCBFl transgenic tobacco were significantly decreased compared to wild type tobacco. However, the level of non-photochemical quenching (NPQ) in transgenic tobacco was higher than wild type tobacco under chilling stress or cold stress, which indicated that the cold resistance of transgenic tobacco with overexpressing NaCBF1was improved compared with wild type tobacco. The results also showed that, under the conditions of chilling-stress or cold-stress, the contents of relative conductivity(RC) and MDA in transgenic tobacco plants were lower than that of wild-type tobacco plants, which resulted in the decrease of electrolyte leakage and the extent of the membrane lipid perioxidation. Furthermore, in all stresses, the transgenic tobacco plants with overexpression of NaCBFl always sustained higher activities in SOD, POD and CAT, which resulted in more increase of scavenging active oxygen and less injury. Under chilling stress or cold stress, in transgenic tobacco plants with overexpression of NaCBFl gene, more prolines, soluble sugars and soluble protein were accumulated compared with wild type tobacco plants, which were favorable to maintain the function of cell membrane and improve cold resistance in transgenic tobacco.4. Correlation analysis showed in the transgenic tobacco plants with overexpression of NaCBF1gene, POD activity was significantly correlated with chlorophyll fluorescence parameters. This result indicated that under the condition of chilling or cold stress, higher photosynthetic capacity in the transgenic tobacco plants with overexpression of NaCBF1gene was correlated with increased POD activity.