Studies On Cloning And Transformation Of CBF1 Gene Of Vitis Amurensis

Freezing is one of adverse environmental conditions which occurs frequently and brings great harm, many tree fruits have different degree harms in response to low nonfreezing temperatures, sometimes the whole plant is killed badly. A recent advance in understanding cold acclimation in Arabidopsis was the discovery of the C-repeat/dehydration responsive element binding factor (CBF)cold response pathway , it make molecular biology of freezing tolerance and gene engineering have a great breakthrough.CBF transcription activator is associated with CRT/DRE DNA regulated element which locates in cold induced gene generator , and it is switch of cold regulated (COR) protein expression and can induce a series of COR protein expression , accordingly, it can improve plant's freezing tolerance, this article cloned a cold-induced transcription factor CBF1 and analyze it's sequence , constructing expression vector of transcription factor CBF1 of Vitis Amurensis. moreover, researching expression of transcription factor CBF1 in transgenic tobacco and Vitis Amurensis. The main results are as follows:1 .Designing a pair of merge primer by comparing CBF1gene of many plant which is published in GenBank. cloning interval fragment of transcription factor CBF1 gene by reverse transcription-polymerase chain reactin (RT-PCR) and achieving a fragment which length is 758bp. moreover, designing two pairs of single primers in interval fragment of targeted gene and cloning 5' and 3'domain which is not coding domain of CBF1 gene by Reverse PCR. achieving a fragment which length is 752bp , and combining with interval fragment is 910bp. This fragment of 910 bp is predicted full length .Designing two pairs of single primer in coding domain and not coding domain of predicted full length respectively and amplifying for genome and cDNA of Vitis Amurensis , Sequencing result of this two PCR products are the same in coding domain , this demonstrate transcription factor CBF1 haven't intron in coding domain , I discover similarity of amino acids and nucleotide of transcription factor CBF1 with transcription factor CBF1 of Vitis Amurensis is 98.1% and 97.6% respectively .this demonstrate I clone transcription factor CBF1 of Vitis Amurensis successfully.It is registered in GenBank, Number is DQ517296.2.Constructing expression vector of transcription factor CBF1 of Vitis Amurensis by 35S promoter , removing off GUS of pBI121 Plsmid by Xba I and Sac I restriction site and resign a pair of single primer which have Xba I and Sac I restriction site , amplifying cDNA of Vitis Amurensis by this pair of...