Molecular Cloning And Primary Functional Analysis On Gene CpABC From Chimonanthus Praecox (L.) Link

ATP-binding cassette (ABC) transporter constitute a large, diverse and ubiquitous superfamily. The majority of ABC genes encode membrane-bound proteins that participate directly in the transport of a wide range of molecules across membranes and can be broadly categorised as importers or exporters, depending on the direction of transport relative to the cytoplasm. Plants are notable for their particularly large complement of ABC proteins, which is associated with metabolic versatility and the need to establish steep concentration gradients of solutes across cellular membranes. The sessile nature of plants and their capacity for photosynthesis also demands an increased requirement for detoxification and associated transport functionsChimonanthus praecox (L.) Link, a shrub originated from China, blossoms lordly in deep winter, possessing many advantages, such as resistance of coldness, drought, and secateur. Among them, chilling tolerance is its main characteristic. As long as the temperature isn't below -15℃, it can safely live through the winter. In our rearch, a ABC transporter gene (CpABC) was cloned from imonanthus. praecox flower, and expressed in Escherichia coli,then we mensurated the effects of its' activity and stability at the different temperature and pH. The main results are as follows:1. Cloning of CpABCWe have found the EST sequence with the size of the 707bp, by rough randomly selection from cDNA library sequencing and clone the cDNA by the technology of hiTAIL-PCR and RACE, BLASTX comparison showed that which has high homology with the ABC transporter.so we presume it as ABC transporter, named CpABC. Sequence analysis showed that the sequence of CpABC have 2818bp, which contains an ORF frame of 2145bp. encoding 714 amino acids. 2. Construction of Plant Expression VectorWe link the fragment of CpABC ORF to cloning vector pMD-19T and digest it and expression vector pCAMBIA2301g by double restriction endonuclease BamHI and SacI,then recycle the target gene and link to expression vector pCAMBIA2301g.The identification of PCR and enzyme cutting show that the construction of the recombinant pCAMBIA2301g/CpABC plasmid could be confirmed.3. Construction of prokaryotic expression vectorDesign the primer with the BamHI and SacI sites,then cloned the CpABC gene through PCR.Digest the CpABC and prokaryotic expression vector PET-28a by double restriction endonuclease BamHl and SacI,then recycle the target gene and link to expression vector PET-28a. Digestion and PCR verification showed that the prokaryotic exprssion vector with the purpose of gene has been successfully constructed.4. Prokaryotic expressin vector PET-2Sa/CpABC expressed in E.coli BL21Under the propriate induce condition, we tranplanted the prokaryotic expressin vector into E.coli to induced CpABC gene'expressing in E.coli Transetta (DE3), and then proved the expression of CpABC through the SDS-PAGE detection on the recombinant protein.5.Expression analysis of CpABC geneThe expression quality of CpABC is little during blossom bud and increases gradually from bud to flowerlet, then decreases with the flower develop, but it is highest during senescence。The expression quality of CpABC is also decreases gradually from outside to inside organism,which shows the close relation between CpABC and the flower development.