| Low temperature is a major environmental constraint that limits plant growth,development and geographic distribution.In order to adapt to the external environment,plants have evolved multiple response mechanisms to slow down the damage caused by low temperatures.Low temperature causes changes in gene expression and triggers regulation of physiological and biochemical in plants,such as changes in cell membrane lipid composition,soluble substances,and accumulation of antifreeze proteins.At present,The ICE-CBF-COR signaling pathway is clear in the response of plants to low-temperature response mechanisms.CBFs are relatively conserved in monocots and dicots,regulating the expression of most cold-responsive COR genes and acting as a central pivot in cold signaling pathways.ICE1 as a key transcription factor upstream of CBFs,directly regulates the transcription of CBFs.This study explored the function of BcICE1 gene and screened for interaction protein by yeast two-hybrid in non-heading Chinese cabbage.The main results are as follows:1.BcICE1 gene contains a 1338 bp open reading frame encoding 466 amino acids with a relative molecular mass of 47.40 kDa and an isoelectric point of 5.42 in the non-heading Chinese cabbage.Multi-sequence alignment of amino acids and phylogenetic tree results indicate that BcICE1 protein is closely related to the ICE1 protein of Brassica napus,Brassica campestris and Arabidopsis thaliana.The BcICE1 protein is localized in the nucleus and responds to cold stress and drought stress.Overexpression of BcICE1 gene can enhanced the cold resistance of Arabidopsis thaliana.Meanwhile,the expression of cold stress related genes AtCBF3 and COR15A in BcICE1-overexpressed plants was significantly higher than that in wild type plants,and the electrolyte permeability was significantly lower than that in wild type plants.2.The expression of BcICE1 gene was highest in leaves and lowest in stems of non-heading Chinese cabbage.TYMV-induced BcICE1 gene silencing decreased gene expression by 48%compared with control plants.After 48 hours of low temperature treatment,the BcICE1-silenced non-heading Chinese cabbage showed obvious symptoms of wilting compared with the control plants.Meanwhile,the expression of BcCBF3 and BcCOR15A in BcICE1-silenced plants was significantly lower than that of the control plants under low temperature treatment.3.The cold-induced yeast cDNA library of non-heading Chinese cabbage was constructed.The average conversion rate of the library was 1.94×106 transformants per μg pGADT7-Rec.The average titer of the obtained library was 1.34×107 CFU·mL-1,and the recombination rate was 100%.The length distribution of the insert ranged from 400 bp to 2,000 bp indicating that the cDNA library of non-heading Chinese cabbage was successfully constructed for yeast hybridization.Self-activation and autotoxicity assays showed that BcICE1 full length(FL)has no autotoxicity,but has a self-activation and self-activated region at the N-terminus of BcICE1 amino acids(F1).The C-terminal(F2)and(F3)of BcICE1 have not self-activation.The bait vector was constructed with the(F3)region of BcICEl amino acid,and the BcHSP40 protein was obtained from the library.The yeast plasmid co-transformation demonstrated BcICE1 interacted with BcHSP40. |
