Cloning, Bioinformatics Analysis Of CBF₂Gene In Different Cold/chilling Resistance Grapes And Construction Of Plant Expression Vector

Low-temperature is an a biological intimidation factor which often affects plant growthprocess seriously.It is a research hotspot to use plant molecular biology techniques improving theplant cold tolerance ability in recent years. More and more cold-resistant genes were discovered，many related cold-resistance genes were transferred into plants and the transgenic plants with theability of cold tolerance were reported，further more transferred the transcription activator of aCOR（cold regulated） gene become new pathway.CBF（C-repeat binding factor） gene recognizeand specifically bound with cis-acting element of CRT/DRE（C-repeat/dehydration responsiveelement） in COR gene promoter region，induce the expression of CBF gene，thereby enhancingthe ability of the cold resistance.Although the research relay to cloning and function of CBFs in grape has been reported,And the difference relay to function and gene expression of CBF₂in different varieties of grapehas not been reported yet. In this study, with Cabernet Sauvignon、Beta and Vitis amurensis asthe materials, The full-length gene of CBF₂was amplified from the genomic DNA in threedifferent chilling resistance gapes. Then the method of bioinformatics was also applied toanalyze the Sequencing results, And through the comparison with the experimental results ofCBF1in different chilling resistance gapes in our lab, We found out that: The difference in firstseven amino acid residues from CBF₂gene may lead to the results why different varieties ofgrapes have different chilling resistance ablity. In this study, we also have constructed the CBF₂gene plant expression vector of Vitis amurensis for the next step to establish efficient genetictransformation system for grape experimental basis. The main results of this study are as follows:â’ˆA DNA fragment was cloned from the genome of three different varieties ofgrape:Cabernet Sauvignon、Beta and Vitis amurensis by polymerase chain reaction （PCR） usingthe primers designed following CBF₂genes from Gene Bank. Sequence analysis showed that thethree cloned DNA fragments all were969bp long and enconding253amino acids and98.28～99.21%identical to CBF₂genes from GeneBank. Moreover, they showed one AP2/EREBP DNAbinding domain and two signature sequences of the CBFs family proteins （PKK/RPAGRx KFxETRHP and DSAWR） in the deduced amino acid sequence. These results indicated thatthree varieties of grape CBF₂gene fragments are cloned in this study.2.The results of CBF₂protein primary structure analysis in three varieties of grape, theformula are C1201～1214H1895～1919N353～359O372～375S9～11, the molecular weight are27.49～27.85KD,the theoretical pI are9.34～9.67, the aliphatic index are662.13～64.86, the grand average ofhydropathicity are-0.542～-0.452, the instability index are52.54～60.19, this classifies theproteins are unstable protein. The results of analysis from the homology of amino acid sequences,physico-chemical property and hydrophobicity and hydrophilia showed there is some differenceamong the high chilling resistance gape Vitis amurensis and Beta, and a Clonal Race of CabernetSauvignon：The comparison of sequence homology with the sequence published on Gene Bankshowed that the worst one is Cabernet Sauvignon; And the first seven amino acid residues fromCabernet Sauvignon were totally different with Vitis amurensis and Beta; This diference alsoresulted the higher hydrophobicity in first fifteen amino acid residues from CabernetSauvignon;Through the comparison with the experimental results of CBF1in our lab, we foundout that: The homology of CBF1from different chilling resistance gapes is higher than CBF₂.Because of these analysis results, we may infer that the difference of CBF₂sequences and genemutation should lead to the different chilling resistance in different varieties of grape.3. Based on the vector PBI121with promoter CaMV35S, the plant expression vectorpBI121-CaMV35S-CBF₂was constructed successfully.Further the reeombinant expressionvector pBI121-CaMV35S-CBF₂was introduced into LBA4404and regenerated plant system wasestablished, which could offer the basic researching data to improve the cold-tolerance ability ofgrape.