Identification And Acquisition Of TaCBF2 Heterologously Transformed Begonia Semperflorens With Cold Resistance

Begonia semperflorens is an ornamental plant of the Begonia family.It is a perennial evergreen herb with green leaves and red flowers,and is widely used in flower mirrors,flower beds and other landscape plantings because of its bright colors.However,as it is native to Brazil in South America,it likes warm and humid environment and does not tolerate severe cold,so the promotion and application of B.semperflorens in northern areas is limited to a certain extent.CBF(CRT/DRE-Binding Factor)transcription factors are an important class of transcription factors that respond to low temperature in plants.They specifically bind to the CRT/DRE cis-acting elements in the promoters of cold-regulated(COR)genes under low temperature stress to activate and initiate the expression of a series of functional resistance genes and promote the synthesis of proline,glycans,membranestabilizing proteins and other substances,thereby enhancing the cold resistance of plants.In order to produce low-temperature tolerant varieties of B.semperflorens,the TaCBF2 gene in Dongnong Winter Wheat 1 wheat was cloned in this study,and the overexpression vector of TaCBF2 was constructed using the p Can G plasmid.p Can GEGFP-TaCBF2 overexpression vector and p Can G-EGFP null vector were transferred into wild-type B.semperflorens by Agrobacterium infestation of leaf disc method,respectively.Three overexpression strains,OE8,OE9,OE24 and the null strain,were obtained by identification and screening.The relative conductivity of each strain was measured and the low-temperature semi-lethal temperature of each strain was derived by combining the Logistic equation.A series of physiological and biochemical indices such as antioxidant enzyme activities,proline and soluble sugar contents,conductivity,etc.of wild-type and no-load strains of B.semperflorens were measured,and the relative expression of TaCBF2 gene and related transcriptional regulatory genes were determined.The relative expression of TaCBF2 gene and the expression of related transcriptional regulatory genes were determined.The results were as follows:1.The TaCBF2 gene(accession number AY951945)was successfully cloned from Dongnong Winter Wheat 1 wheat,and the CDS region of the gene is 678 bp long.p Can G-EGFP-TaCBF2 overexpression vector was constructed,and the TaCBF2 gene was stably expressed in B.semperflorens using Agrobacterium tumefaciens-mediated genetic transformation,and was identified and screened by Kanah(Kan)resistance and PCR.Three overexpression strains OE8,OE9,OE24 and a null strain EM were identified and screened by Kanan resistance screening and PCR assay,respectively.2.Leaves of five strains,wild-type WT,no-load EM and three overexpression strains OE8,OE9 and OE24,were taken and their relative conductivity was measured at 6℃,3℃,0℃,-3℃,-6℃,-9℃,-12℃,-15℃and-18℃.The low-temperature semilethal temperatures of each strain of B.semperflorens were found by fitting Logistic equations as-1.9℃ for wild-type WT,-2.16℃ for no-load EM,-4.51℃ for OE8,-4.25℃for OE9 and-4.39℃ for OE24℃,no-load EM-2.16℃,OE8-4.51℃,OE9-4.25℃,and OE24-4.39℃.The results indicated that overexpression of TaCBF2 transcription factor in B.semperflorens quadrangularis improved the cold resistance potential of B.semperflorens quadrangularis.3.A total of five strains,wild-type WT,null-loaded EM and three overexpression strains OE8,OE9 and OE24,were subjected to low temperature treatment at 4°C.By observing the phenotypes of the five strains before and after the low temperature treatment,it was observed that the wild-type and null-loaded showed severe chlorosis under low temperature stress,while the three overexpression strains showed relatively mild damage symptoms.The chlorophyll concentration was measured and analyzed,and it was found that under normal conditions,the chlorophyll content of the five strains did not differ significantly;however,at 15 d of low temperature treatment,the chlorophyll concentration of the three transgenic strains was significantly higher than that of the wild type and the null,and there was no significant difference between the null EM and the wild type WT.It showed that overexpression of TaCBF2 gene in B.semperflorens could attenuate the degree of chlorophyll damage by low temperature stress.4.A total of five strains of wild-type WT,null-loaded EM and three overexpression lines OE8,OE9 and OE24 were subjected to low temperature treatment at 4°C.It was found that the Fv/Fm of each strain gradually decreased under low temperature stress,and the Fv/Fm of the three transgenic lines was higher than that of wild-type Arabidopsis after 15 d of stress,indicating that the transfer of TaCBF2 gene could alleviate the damage caused by low temperature on the maximum photochemical efficiency of B.semperflorens.In addition,the antioxidant enzyme activities(superoxide dismutase SOD and peroxidase POD)of the three overexpression lines were significantly higher than those of the wild-type and null plants after low temperature stress,indicating that the transfer of TaCBF2 gene could improve the ability of B.semperflorens to scavenge reactive oxygen species in adversity;the osmoregulatory substances(proline Pro and soluble sugar SS)of the three overexpression lines were significantly higher than those of the wild-type and null plants.The contents of osmoregulatory substances(proline Pro and soluble sugar SS)in all three overexpression lines were significantly higher than those in wild-type and no-load plants,indicating that the transfer of TaCBF2 gene could improve the osmoregulatory ability of B.semperflorens under stress;the conductivity and malondialdehyde(MDA)contents in all three overexpression lines were lower than those in wild-type and no-load plants,indicating that the biofilms of the three overexpression lines were less damaged than those in wild-type and no-load plants.The above results indicate that overexpression of TaCBF2 gene can reduce the degree of damage under low temperature stress in B.semperflorens.5.Real-time quantitative q PCR analysis showed that the expression of all genes in response to low temperature were up-regulated to different extents during the low temperature treatment,especially in the three overexpression lines,which were significantly higher than those in the wild-type and null plants.The above results indicate that the trans-TaCBF2 gene can be stably expressed in B.semperflorens and can respond to low temperature stress,which can activate the expression of various functional genes related to low temperature stress in B.semperflorens,and then cause the synthesis of a series of cold-related substances in the plant,and finally improve the cold resistance of B.semperflorens.