Cloning And Functional Analysis Of The Promoter Of A Heat Shock Protein Gene CpHSP1from Chimonanthus-Praecox

The heat shock proteins (HSPs) are a type of proteins newly synthesized or increased in biological organisms or in vitro cells when they are stimulated by the stress factors such as:heat, cold, drought, ABA, heavy metals and other stress stimulations. According to the size of the molecular weight, HSPs can be divided to five categories:HSP100s, HSP90s, HSP70s, HSP60s and smHSPs. All types of heat shock proteins are widespread in plants, among which smHSPs is the most common type. smHSPs can be induced by lots of stress factors, such as:high temperature, low temperature, high salt, drought, hormones and mechanical damage; they also can be regulated by seed germination, anther development and fruit ripening process.The present study showes that smHSPs has molecular chaperone function, which prevents the accumulation of denatured protein and protect protein folding process independent of ATP. It plays an important role in environmenta stress resistance and plant growth and development.Chimonanthus praecox thrives in cold environments and blooms in low-temperature seasons with little rainfall. As a important adversities response protein.What HSPs participated in the adversity defense process play some crucial role remains a problem to be explored. Our research team has cloned a full-length sequence, named CpHSP1. The study showed that its expression has a strong stress response characteristics, a strong tissue-specific and time-specific.In order to further to explore the machanisms of CpHSP1genes involved in the Chimonanthus adversity response and the development process, using hiTAIL-PCR, upstream regulatory sequence of CpHSP1gene was obtained from Chimonanthus genomic DNA and the function of the promoter was preliminarily studyed. The experiment conclusions are as follows:1、The isolation of the Chimonanthus CpHSP1gene promoter sequenceOn the basis of the obtained CpHSP1gene sequences, using hiTAIL-PCR technology, we got the954bp promoter fragment by amplifying the unknown promoter sequence upstream of the CpHSP1gene.2、The online analysis of Chimonanthus CpHSP1gene promoterUsing the online analysis software in PlantCare, the promoter sequence has basic components of the TATA-box and CAAT-box, abscisic acid (ABA) components which are in response to plant abiotic stress and hormone response elements, G-BOX element which is in response to light and anaerobiosis, and the ABA response, HSE (thermal response element) and drought-related MBS components. The promoter also has a CATT-the motif (the light effect element), circadian related to the circadian rhythm and Skn-1_motif related to Endosperm expression.3、Constructing the plant expression vectorWe replaced the35s promoter of pCAMBIA-1300-221with the cloned promoter CpHSP1to drive the report gene GUS by digestion and connection.The results of PCR and Double Digestion made clear that the plant expression vector pCAMBIA-1300-221-CpHSPlpro had been structured successfully.4、Transformation of tobacco and Activity analysisThe vector pCAMBIA-1300-221-CpHSP1pro 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 roots, stems,leaves, flowers of the transgenic tobacco,indicated that the promoter had the activity to drive the report gene GUS and has a strong tissue specificity for Chimonanthus heat shock protein gene expression and regulation.