Molecular Cloning And Functional Analysis Of CBF And ICE Transcription Factor Gene From Peach

As one of the most important fruits in the north of China,the cultivated area and yield of peach keep a steady growth.However,recently it is common to see that chilling injury and freezing injury of peach are caused frequency and severity by the extreme weathers.This paper in order to provide molecular information for the researches of studying the mechanism and enhancing of the cold resistance in peach,the CBF and ICE genes were isolated from some main peach cultivars,which are reported as the key transcription factors of the transduction path that response to low temperature.Then some of their appropriate information and functions were analyzed.The main findings are as follows:1.CBF gene（AEG64738）from pear was used as the information probe of amino acid sequence,based on the in vitro splicing of the peach EST data in the Gen Bank database and its highly homologous EST sequences,CBF-specific primers were designed.Then by using the DNA and c DNA of downy peach as the templates,the genomic DNA and c DNA full-length sequence of this gene were obtained,without introns,both length are 894 bp which encodes 229 amino acids.The 894 bp is including121 bp of 5 ’non-coding sequence,83 bp of 3’ non-coding sequence and 690 bp of the open reading frame.This gene contains the AP2 / EREBP functional domains of the CBF family and it was named as Pp CBF（Genebank: KC885952）.2.Using the amino acid sequence of the ICE gene（XP₀02264407）in grape as information probe,in the same way as above to design ICE specific primer,the DNA and c DNA of downy peach were used as templates,as a result we know that the full-length sequence of the genomic DNA is 1585 bp,and of the c DNA is 1160 bp which containing 3 introns,41 bp of 5 ’ non-coding sequences,36 bp of 3 ’ non-coding sequences,and 1083 bp of open reading frame,encoding 360 amino acids.Containing the b HLH functional domain of ICE family,and named the gene as Pp ICE（Genebank: KC885953）.3.The whole length of the CBF gene nucleotide sequence was obtained from the ‘Jinqiu’,‘Dajiubao’and other 14 peach cultivars,894 bp in length,encoding 229 amino acids.The sequence similarity in these varieties varies from 97.8% to 99.6 %.It shows that Pp CBF gene is highly conserved in these varieties.The homology analysis shows that the homology of its amino acid sequence with apple and pear are up to 84%and 81.3%,which means it has the closest genetic relation to them;while with rubber tree is only 66.1%.These results indicate that in the evolutionary process,the amino acid sequence encoded by the AP2 superfamily gene may be different from species to species.4.The ICE gene nucleotide sequence was obtained from the ‘Jinqiu’,‘Dajiubao’ and other 14 peach varieties,however the sequence length are obviously different between varieties.The ICE gene encodes360 amino acids in most varieties.But in ‘Dajiubao’,‘B6832’,‘Zaolu’ and ‘Jinqiu,because the internal base mutation occurs in ICE gene which leading to the translator’s early-termination,there only185,266,184 and 266 amino acid residues are encoded respectively.Genetic similarity of varieties’ amino acid ranged from 91.9% to 99.4%.Homology analysis shows the genetic relation of Pp ICE amino acid sequence of peach with the soybean and Medicago truncatula are closest,homology are 74.4% and72.9%;the farthest genetic relation is with rice,only 41.8 %.It shows that in the evolutionary process,the amino acid sequence encoded by the b HLH transcription factor may be different from species to species.5.Respectively constructed protein expression vector p PC86.CBF and the bait vector p RS315 His.S011 containing "CCGAC" core sequence,and transformed yeast y WAM2 strains by yeast one-hybrid method.The strains grew well in the three-missing medium,indicating that the CBF transcription factor of peach can identify and combine specific with the CRT / DRE cis-acting element in the promoter region of COR gene,then induced the expression of COR gene.6.The over-expressed vectors,P3301.Pp CBF and UN.Pp ICE were constructed,and transplanted into tobacco by Agrobacterium-mediated method.The PCR detection results show that the 9 of 11 plants are transgenic plants,the positive conversion rate is 81.8%.ICE transferred in tobacco has also been in the bud induction phase.7.The CBF positive transgenic tobacco seedlings were treated under the conditions of low temperature,drought and salt stress respectively,then the related physic-chemical parameters and phenotype changing were tested and investigated.The results show: the drought and cold resistances of transgenic plants are significantly better than the control plants from physic-chemical parameters and the appearance;the resistance to salt stress is no difference from the phenotype,but the physic-chemical parameters are slightly better than that of control.