Eukaryotic Expression Of CBF3 Gene Of Vitis Amurensis In Pichia Pastoris And Genetic Transformation Of Tobacco

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.In this study, CBF3 (sequence of transcription factor) gene of Vitis Amurensis was cloned into pPIC9K to construct the recombinant expression vector pPIC9K-CBF3. The pPIC9K-CBF3 was introduced into Pichia pastoris; At the same time we made a research about introducing CBF3 gene of Vitis Amurensis into tobacco genome. Main results of this Paper were as folio wings:1. CBF3 gene of Vitis Amurensis was amplified by polymerase chain reaction (PCR), then digested and purified, was inserted into the eukaryotic expression vector pPIC9K in the end. Next the recombinant expression vector pPIC9K-CBF3 was introduced into Pichia pastoris. The microzyme was induced by carbinol, then the result of SDS-PAGE showed that: transformed bacteria expressed 32KD fusion proteins after SDS-PAGE, and 72h is the best time of inducing CBF3 gene2. The recombinant expression vector pRCBF3 was introduced into tobacco. we got six transgenic plants by PCR. The results of RT-PCR showed that three transgenic plants can express steadily.