Cloning And Stress Resistance Of RcCBF2 Gene From Rhodiola Crenulata

Stresses such as low temperature,drought and high salinity not only adversely affect photosynthesis and respiration,but also affect osmotic regulation of plants,leading to plant death in severe cases.In the process of plant evolution,various resistance mechanisms to abiotic stresses have emerged to protect plants from injury.CBF is a class of transcription factors related to stress,which can recognize and bind to CTR/DRE cis-acting elements in promoter region COR of genes related to various stress responses.CBF is a kind of transcription factor related to stress.It can recognize and combine CTR/DRE cis-acting elements in promoter region COR of various stress-related genes,activating the expression of downstream stress-resistant genes and regulating multiple stress-resistant genes at the transcriptional level.In this study,RcCBF2 gene was cloned from Rhodiola crenulata by PCR for the first time.Through bioinformatics analysis,construction of plant expression vector,transformation of Arabidopsis thaliana by inflorescence dissemination and stress resistance research,the results are as follows:1.In this study,RcCBF2 gene was cloned from R.crenulata by PCR.The similarity of RcCBF2 protein between R.crenulata and Polyhairy tomato(ACB45088.1)was 63%.2.Bioinformatics analysis of RcCBF2 protein sequence showed that the open reading frame of RcCBF2 protein was 77 bp,the number of amino acid residues was 231,the coefficient of instability was 47.49% and the theoretical isoelectric point was 4.93.It was predicted that RcCBF2 protein was acidic.The lowest hydrophilicity score of RcCBF2 protein was-2.844,and the highest hydrophobicity score was 1.789.It was predicted that RcCBF2 protein was hydrophilic.RcCBF2 protein had no signal peptide,trans-peptide and transmembrane domain.The main structure of RcCBF2 protein in R.crenulata was alpha-helix(36.36%)and random curl(46.75%),followed by elongation chain(12.55%)and finally by beta-folding(4.33%).The RcCBF2 protein contained AP2 domain,and the tertiary structure of RcCBF2 protein was predicted by homology modeling.3.The target gene was linked to the PHB vector by double digestion,and the plant expression vector was constructed.Agrobacterium tumefaciens GV3101 was transformed into Arabidopsis thaliana by inflorescence impregnation transformation method and the transgenic Arabidopsis pure lines were obtained.4.By recording the characteristics of roots,leaves and flowering period of wild type and RcCBF2 transgenic plants under normal growth conditions and measuring the chloroplast content,it was indicated that overexpression of RcCBF2 gene could delay plant senescence.The root length of wild-type plants was twice as long as that of transgenic plants.RcCBF2 transgenic plants had more lotus-shaped cluster leaves than wild-type plants,and their flowering period was later than wild type plants,and their chlorophyll content was higher than that of wild-type plants.The senescence process of RcCBF2 transgenic and wild type plants showed that transgenic plants could delay senescence.The leaf appearance time of RcCBF2 transgenic plants was not much different from that of wild type plants,but the leaf wilting of RcCBF2 transgenic plants is later.5.It was found that the survival rate of RcCBF2 transgenic plants was higher than that of wild type plants under both cold and non-cold acclimation conditions.Relative electrical conductivity of RcCBF2 transgenic plants was lower than that of wild type plants under freezing stress at-4?,suggesting that overexpression of RcCBF2 gene improved the freeze resistance of plants.6.Through the dehydration resistance experiment,it was found that the weight of RcCBF2 transgenic plants was 27.1±2.2% of the initial weight,which was higher than that of wild-type plants 6.7±1%.This indicated that the overexpression of RcCBF2 gene enhanced the tolerance of plants to dehydration.7.Through salt tolerance treatment,it was found that the root growth rate of RcCBF2 transgenic plants was higher than that of wild-type plants,and the ratio of RcCBF2 transgenic plants to the initial weight was higher than that of wild type plants,which indicated that the overexpression of RcCBF2 gene enhanced plant resistance to salt stress.