Differentiated Analysis Of CBF₁ Transcription Factor Gene Sequence In Different Varieties Of Grape And Construction Of Plant Expression Vector

Plants subjected to low temperature stress in varying degrees of damage occur, serious and even lead to death of the whole plant. A recent advance in understanding cold acclimation in Arabidopsis was the discovery of the CBFs (CRT/DRE binding factors)cold response pathway,it make molecular biology of freezing tolerance and gene engineering have a great breakthrough. CBFs(C-repeat binding factors) genes can recognize and bound with cis-acting element of CRT/DRE(C-repeat/dehydration responsive element) in COR genes promoter region specifically, induce the expression of COR genes, thereby enhancing the ability of the cold resistance. CBFs genes can improve plant resistant traits synthetically and it already has been one of research hotspots for molecular breeding.In this study, with Vitis amurensis,Beta , Ln33,SO4 and Pinot Noir as the materials, low-temperature induction of transcription factor CBF₁ gene were cloned from them and analyzed their sequences,then constructed the CBF₁ gene plant expression vector of Vitis amurensis for the next step to establish efficient genetic transformation system for grape experimental basis. The main results of this study are as follows:⑴A DNA fragment was cloned from the genome of five different varieties of grape, Vitis amurensis,Beta,Ln33,SO4 and Pinot Noir by polymerase chain reaction (PCR) using the primers designed following Vitis vinifera CBF₁ genes. Sequence analysis showed that the five cloned DNA fragments all were 756bp long and 98.28～99.21% and 98.39～98.8% identical to the Vitis vinifera CBF₁ gene in nucleotide and deduced amino acid sequences respectively. Moreover, they showed one AP₂/EREBP DNA binding domain and two signature sequences of the CBFs family proteins (PKK/RPAGRxKFxETRHP and DSAWR) in the deduced amino acid sequence. These results indicated that five varieties of grape CBF₁ gene fragments are obtained in this study.⑵The results of CBF₁ protein primary structure analysis in five different varieties of grape, the formula are C_1191～1202H_1899～1908N_347～350O_378～380S₁₂, the molecular weight are 27.57～27.69kD, the theoretical pI are 6.97～8.41, the aliphatic index are 65.74～66.14, the grand average of hydropathicity are -0.576～-0.564, the instability index are 48.34～52.22, this classifies the proteins are unstable protein; and the results of CBF₁ protein hydrophilic/hydrophobic,signal peptide,transmembrane structure analysis showed that the five proteins are hydrophilic and non-secretory protein, no signal peptide and transmembrane domain exists. The results showed that the CBFl genes in five varieties of grape have no significant difference.⑶Construction of the CBF₁ gene plant expression vector of Vitis amurensis by CaMV35S promoter. pMD18-CBF₁ cloning vector and the pBI121 expression vector were digested by restriction endonucleases BamHI and SacI,then retrieve the CBF₁ gene and the expression vector without GUS gene. pBI121 expression vector will be connected with the CBF₁ gene and transformed into E.coli DH5αcompetent cells. After PCR and digestion identification by BamHI and SacI, the results proved that the vector was constructed successfully.