Cloning, Vector Instruction And Protokaryotic Expression Of CBF3 Gene In Vitis Amurensis

Low-temperature is an abiological intimidation factor which often affects plant growth process seriously. It is a research hotspot to use plant molecular biology techniques improving the plant cold tolerance ability in recent years. More and more cold-resistant genes was discovered, many related cold-resistance genes were transferred into plants and the transgenic plants with the ability of cold tolerance were reported, furthermore transferred the transcription activator of a COR(cold regulated) gene become a new pathway. CBF(C-repeat binding factor) gene recognize and specifically bound with cis-acting element of CRT/DRE(C-repeat/dehydration responsive element) in COR gene promoter region, induce the expression of CBF gene, thereby enhancing the ability of the cold resistance. CBF genes can improve plant resistant traits synthetically and it already has been one of research hotspots for molecular breeding.CBF3 (sequence of transcription factor) gene of Vitis Amurensis was cloned and analyzed by RT-PCR; prokaryotic expression vector pGEX-4T-CBF3 of CBF3 gene was constructed, fusion expression of the vector in Escherichia.coli was studied; plant expression vector pRCBF of CBF3 gene was constructed too. Main results of this Paper were as followings:1. The complete cDNA was cloned and analysed by designing a specific Primer (GenBank accession number the nucleotide sequence is EU672969), leaf total RNA of Vitis amurensis as template. The cDNA of CBF3 gene sequence was obtained and its molecular biology analysis was done. The gene size is 854bp, its coding area has 720 bases, it codes the protein which consists of a 239 amino acid remnant base. The protein relative molecular weight is 25914.7, its isoelectric point is 7.02, Aliphatic index is 59.29, Grand average of hydropathicity is -0.551. carried on the forecast analysis to the protein secondary and third structures of CBF3 was forecasted and analyzed, and its protein is unstable.2.Prokaryotic expression vector pGEX-4T-CBF3 of Vitis Amurensis was constructed, It was transformed into expressing host cell Escherichia.coli BL21(DRE3). The Bacteria was induced by 1mM IPTG, the result showed that: transformed bacteria expressed 52KDa fusion proteins after SDS-PAGE.3. The plant expression vector pRCBF3 of Vitis Amurensis was constructed, and it was transformed into agrobacterium LBA4404.