Transcription Level Change Of The Aquaporin Gene CpAQP1from Chimonanthus Praecox And Analysis Of Its Promoter Activity

Zhang Mi(2009) had cloned two aquaporins CpAQP1and CpAQP2, and then they have finished the construction of the plant expression vector through Agrobacterium-mediated transformation of Nicotiana tobacum var. xanthi by way of leaf disc and the regenerated tobacco plants were obtained, and the function of two aquaporins were analyzed by stress treatment to the transgenic.The result indicated that the transgenic tobacco plants improved more tolerability than the wild ones.We could study the change of the gene expression and analysis the prmoter to proving the role of the gene in resistance and other physiological functions. So,the RT-PCR method was used to analysis the expression of CpAQP1in different organs, different developmental stages and different stresses to proving the function of CpAQP1by quantitative analyses in our research.At the same time,the promoter fragment of1082bp upstream from the5’ upper of CpAQP1was cloned from the genomic DNA from Chimonanthus praecox by hiTAIL-PCR. Bioinformatics analysis revealed that the sequence contained the basic cis-elements,such as TATA-box、CAAT-box and some elements involved in the plant abiotic stress and light response.And then plant expression vector PBI121-CpAQ1pro was successfully established.The result of stable expression by a grobacterium tumefaciens mediated method indicated that the promoter fragment had the function to drive reporter gene.1. Expression analysis of the aquaporin gene CPAQP1The result of RT-time PCR indicated that the expression of CPAQP1were dissimilar between different tissues. CpAQP1transcripts were detected more abundant in petals than in other tissues during blooming.Moreover, CpAQP1was hardly expressed in non-stressed leaf but strongly induced under the various abiotic stresses such as high temperature, low temperature, salinity, ABA and heavy metel. All these transcriptional expression analysis results suggested that CpAQPl plays a significant role in plant development、cell aging and abiotic stress responses.2. Cloning the promoter of CpAQP1Three nested primers have been designed near the5’side of CpAQPl.The promoter fragment of1082bp upstream from the5’upper of CpAQPl was cloned from the genomic DNA of Chimonanthus praecox by hiTAIL-PCR.3. Sequence analysis of the promoter CpAQP1proA lot of cis-regulatory elements of the promoter sequence have been predicted by PlantCARE.The result indicated that CpAQP1pro had the basic transcription elements such as TATA-box、CAAT-box,besides5UTR Py-rich stretch、CGTCA-motif、G-BOX、 OBP-1site、TC-rich repeats and so on.4. Constructing the plant expression vectorWe replaced the35s promoter of PBI121with the cloned promoter CpAQP1pro to drive the report gene GUS by digestion and connection.The results of PCR and Double Digestion made clear that the plant expression vector PBI121-CpAQP1pro had been structured successfully.5. Transformation of tobacco and Activity analysisThe vector PBI121-CpAQP1pro was brought in tobacco genome by the way of leaf disc through Agrobacterium-mediated transformation.The result of GUS histochemical stain,which was from the leaves of the transgenic tobacco,indicated that the promoter had the activity to drive the report gene GUS.