Cloning Of CBF Cold Responsive Pathway Related Genes From Vitis Amurensis (VaCBF) And The Corresponding Plant Express Vector Construction

CBF(CRT/DRE binding factor) transcriptional factor plays an important role in Arabidopsis response to cold stress.They can activate a number of stress tolerance genes.Though there are many cold-response pathways in plant,the CBF pathway have been studied extentively,and its cold-resisting function was confirmed firmly. So the CBF family genes are importment resources for plant cold-resisting genetic engineering.In this paper, two novel CBF family genes from Vitis amurensis(VaCBF) were cloned(GenBank accession number: DQ517297 and DQ517298) and named VaCBF2 and VaCBF3,respectively.The plant express vectors of them were constructed.The main results are as follow:1.A pair of degenerate primers was designed by comparing CBF genes of several plants which are published in Genebank.The degererate primers were used to isolate the conserved region with two length of 536bp and 527bp of the CBF genes from the cDNA of Vitis amurensis by polymerase chain reactin(PCR).2.Two pairs of specific primers from the conserved fragment of targeted gene were designed to isolate 5' and 3'ends of CBF genes by inverse PCR(iPCR),respectively.Two fragments of 1200bp and l000bp are obtained.After being spliced,two full length CBF genes from Vitis amurensis(VaCBF) were predicted.3.From the predicted full length sequence of VaCBF genes,a pair of specific primers was designed to isolate VaCBF from the genomic of Vitis amurensis,respectively.Two fragments of 954bp and 885bp are obtained.And the results showed that they are true the full lengh of CBF genes.So they are named VaCBF2 and VaCBF3,respectively.4.By DNAMAN software,homologous alignment was done of VaCBF2 and VaCBF3 with other genes from CBF gene family published in Genbank,the result showed that VaCBF2 and VaCBF3 have the conserved AP2 region as one of the characteristics of CBF gene family,which demonstrates we have cloned VaCBF2 and VaCBF3 from Vitis amurensis successfully.5.Analysis of the expression of VaCBF2 and VaCBF3 was done by Semi-quantitative RT-PCR.6.Using Gateway technology to construct the plant express vectors of VaCBF2 and VaCBF3.The new constructed plant express vectors were transferred to Agrobacterium tumefaciens EHA105.