Cloning And Differential Expression Analysis Of Transcription Factor CsCBF Gene Family In Tea Plant(Camellia Sinensis)

Tea plant,an economic crop with weak cold resistance,is limited by low temperature in its introduction and cultivation,and cold spell in later spring will cause the spring tea to reduce its production even to no harvest.The CBF gene is the pivot of the plant’s cold resistance pathway and plays an important role in plant cold response.In this study,two new CsCBFs genes were cloned and identified from tea plants to study their expression patterns and construct the plant binary expression vector PBI121-CsCBF,which was transformed into tobacco then.The results were as follows:1.Based on transcriptome data,the complete open reading frames of two new CBF gene(CsCBF3,accession number: MH017428;CsCBF5,accession number: MH165878)were cloned,of which the length of CsCBF3 open reading frame was783 bp,encoding 261 amino acids.Besides the molecular weight was 28.06 kDa.The open reading frame of CsCBF5 was 744 bp in length and it encoded 248 amino acids.The molecular weight was 27.16 kDa.2.Five genes in the CsCBF gene family did not contain introns.CsCBF1 and CsCBF4 had no single nucleotide mutations(SNPs)between different varieties and their sequences were identical.CsCBF2,CsCBF3,and CsCBF5 contained 4,8 and 6 mutations,respectively,of which the mutation rates were 1/180 bp,1/98 bp,and 1/124 bp,respectively.3.Five gene promoters of the CsCBF gene family were cloned.All of the five CsCBF promoters contained MYC,adversarial response elements,hormone response elements,light-responsive elements,respectively.4.The four genes in CsCBF gene family(CsCBF1,CsCBF2,CsCBF3 and CsCBF5)all responded to low temperature stress,The maximum expression level was related to time.And the maximum expression of Shuchazao,the cold-resistant variety,was higher than Yinghong No.9,a variety with weak cold resistance.The same expression pattern existed in the varieties under the cold acclimation treatment,and the maximum expression level at 14 days under the cold acclimation treatment was higher than that under low temperature.5.In CsCBF gene family,CBF4 responsed to PEG osmotic stress.With PEG osmotic stress,the expression level reached the highest at 3h,and the expression level of Shuchazao was higher than that of Yinghong No.9.6.The expressions of CsCBF gene were different in different tissue of tea plant.CsCBF1,CsCBF3,CsCBF5 had the highest expression in old leaves,and CsCBF2,CsCBF4 had the highest expression in young leaves.Except for the old leaves and young leaves,their expression was not significant in roots.7.Transgenic tobacco plants of CsCBF2 and CsCBF5 were obtained.CsCBF2 and CsCBF5 overexpressed under normal growth conditions,and the expression level of the two genes further increased under low temperature treatment.