| Plants inevitably suffer various environmental factors. Low temperature is one of the major environmental factors that limit plant growth, development and geographical distribution. So the plant chilling tolerance mechanism has been a hot spot in the study of plant resistance. Plants have evolved finely tuned low-temperature signaling and resistance mechanisms. When exposed to low temperature, plants respond rapidly and carry out protective processes, for example, physical adaptation (changes in plasma membrance lipid components and cell cytoskeleton remodeling), accumulation of osmoprotectants (soluble sugars, proline, betaine, etc) in cytosol, increased contents of various antioxidants (superoxide dismutase, catalase, ascorbinase), induced synthesis of proteins (anti-freezing proteins, dehydrins and heat-shock proteins) encoded by low-temperature-responsive genes, which reprogram the biological activities and establish a new metabolism balance for stressed plants and help survive the low temperature stress. To date, cold signaling and tolerance mechanism conferred by CBF transcription factors is the most understood and important pathway, which function in an ABA-independent manner. Following cold exposure, CBF genes are rapidly and transiently induced via an ABA-independent pathway, and their products activate CBF regulon to enhance freezing tolerance by specifically recognizing and binding to CRT/DRE cis-element in promoter region of cold responsive genes. CBF homologues have been identified in many plant species and overexpression of one CBF gene in transgenic plants gives rise to strong constitutive expression of CBF regulon and hence increased low temperature tolerance. In addition, CBF overexpression also increases the salt and osmotic stress tolerance in transgenic plants.The biologic membrane was regarded as the position which was firstly suffered damage under temperature stresses. The content of unsaturated fatty acid in plant membrane lipids was higher, the temperature of transition of membrane lipids was lower and the chilling tolerance of plants was higher. Contrarily, the temperature of transition of membrane lipids was higher and the thermal tolerance of plants was higher. In our study, the level of total unsaturated fatty acids in transgenic plants was higher than that in WT.Sweet pepper (Capsicum frutescens L.) is an important kind of condiments, because of its fruit, especially rich nutrition vitamin C content for all kinds of vegetables. Thus, the research of chilling tolerance mechanism is an important theoretical and practical value. The CBF3 functional homologue in sweet pepper, CfCBF3, is cloned and its expression modes and function are examined in detail:(1) Two degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from sweet pepper leaves according to the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. Then the 5′and 3′of the cDNA were isolated by 5′and 3′RACE. Then the full length of the sequence has been obtained.(2) p35S-CfCBF3-GFP fusion protein was constructed and transiently expressed in onion epidermal cells. It was observed with confocal microscopy that CfCBF3 is exclusively localized in the nucleus whereas the GFP can be detected in the whole cell. This result correlates well with the typical feature for canonical transcription factors.(3) Northern hybridization showed that the transcript of CfCBF3 was high in the leaf tissues. The expression of CfCBF3 was obviously induced by several kinds of stresses and changed with the treatment.(4) The full-length sweet pepper CBF3 cDNA was inserted into BamHI-SalI in botany expression vector pBI121, behind the CaMV35S promoter. The 35S-CaMV CfCBF3 constructs were introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method. The transgenic plants were selected by 5‰kanamycin. Then the kanamycin-resistant transgenic plants were further verified by PCR, Northern and Western hybridization. It was indicated that the CfCBF3 had been recombined into the genome of tobacco in parts of the transgenic plants.(5) A recombinant of prokaryotic expression vector pET-CfCBF3 was constructed and transformed to E.Coli. BL21. The strong induced fusion protein bands were collected into phosphate buffer (PBS) solution and were used to immunize mice to obtain antiserum. The value of antibody reaches 1:500. Western blot analysis revealed the presence of the strong positive protein signals corresponding to CfCBF3 in transgenic plants. The results showed that CBF3 about 19kDa could be detected significantly under chilling and drought treatment for 3h, then reaching maximum levels at around 6h. This might demonstrate that in response to chilling stress, translation of CfCBF3 follows rapidly after transcription.(6) The downstream genes ERD10B, ERD10C and P5CS was activated in the transgenic plants overexpressing CfCBF3. P5CS is the key enzyme in proline synthesis pathway, the changes of enzyme activity directly determines the amount of proline. Under normal conditions, the content of proline in transgenic tobacco were higher compared with wild type plants, which is correlated with the expression of P5CS genes. It has been suggested that proline accumulation can be as the physiological indexes in plant resistance, in addition, proline plays an important role in preventing enzyme degeneration, energy reserves and free radical scavenging. NtERD10B and NtERD10C, which function as target genes of CBF in transgenic tobacco were greatly elevated after 12h chilling stress. In the transgenic tobacco plants, CfCBF3 did act as a transcriptional activator thus caused expression of downstream genes.(7) The transgenic plants had higher chilling tolerance than WT plants. After germination and cultivation in different abiotic stress, the fresh weight of the transgenic plants was higher than that of WT plants. DAB and NBT staining assay were detected in the leaves of plants. The results indicated that the staining intensity was much higher in wild-type leaves than that in transgenic leaves. The contents of H2O2 and O2 in WT and transgenic plants were examined accordingly. These data confirmed the observations better.(8) Analysis of fatty acids composition from consecutive experiments showed that the percentage of unsaturated fatty acids did not reveal markedly differences between WT and transgenic plants under control conditions. After 12h chilling stress, the level of total unsaturated fatty acids in transgenic plants was higher than that in WT in all the six membrane lipids, with a marked increase in PG. The percentage of unsaturated fatty acids of PG in NT5, NT27 and NT38 increased from 32.41, 41.94 and 39.84 to 61.15, 70.46 and 74.86%, respectively. However, in WT the fatty acid content remained almost the same (43.11% and 43.24%, respectively). After chilling stress (0℃) for 12h, the relative electric conductivity increased in both WT and transgenic plants. However, there was less ion leakage from transgenic lines than that from WT. Fv/Fm decreased obviously in WT plants after 24h chilling stress (0℃) whereas all the transgenic plants were less affected. The Fv/Fm of NT5, NT27, NT38 and WT decreased by 10.7%, 11.9%, 9.5% and 20.5% of initial value, respectively. This demonstrated that overexpression of CfCBF3 alleviates the degree of PSII inhibition.(9) We tried to find out the interaction proteins with CfCBF3 using the yeast two-hybrid system in low temperature response way.
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