Genetic Transformation Of Nicotiana Tabacum L.by An Apoaequorin-egfp Fusion Gene And Phenotypic Analysis Of Cyanobacteria PRX Rhythmic Marker

Continuous detection of the real-time changes in cytosolic Ca²⁺ by a non-invasive indicator,Aequorin,has been widely used in the studies on mammals and plants.The relatively lower quantum yields which generated from the Aequorin bioluminescence process have negatively affected the application of this method.When bonded by three cytosolic free Ca²⁺ the substrate,coelenterazine,the conformation changes of an Aequorin would lead to the emission of an unique quantum of blue light with a wavelength at 469 nm.For the reason that there is a precise quantitative relation between the amounts of Ca²⁺binding to Aequorin and the final quantum yields of blue light generated in the bioluminescence process,researchers have the possibilities to measure the dynamic changes of cytosolic Ca²⁺ concentration in target living cells by overexpressing the exogenous aequorin gene in them.However,limited by the difficulties in the detecting of blue light photons,it often caused the poor performance and lower efficiency in the dynamically detecting of intracellular Ca²⁺ by the directly using of Aequorin as probes.It is also the restriction for Aequorin in large-scale application to detect Ca²⁺ in living cells.To overcome these shortcomings in current Aequorin bioluminescence probe method,by cohesively expressing the coding genes of Aequorin and EGFP in tobacco,this work have established a new method to carry out the real time capturing and tracing of stable Ca²⁺ signals.The major results of this method following the strategy of BRET are listed as:?1?A more stable and robust florescence probe based on the principle of bioluminescent resonance energy transfer?BRET?,has been established as one of the improved experimental protocol in the detection of intracellular dynamics in Ca²⁺.?2?In this master dissertation,a binary plant expression vector harboring an apoaequorin-enhanced green fluorescent protein?egfp?fusion gene was first constructed and then was introduced into Agrobacterium tumefaciens LBA4404 competent cells.Subsequently,by PCR,RT-PCR detections and BRET imaging detecting in transgenic protoplast,the integration and expression of target fusion gene in transgenic tobacco are confirmed.?3?Finally,based on the results from laser confocal fluorescence microscopy images and in vivo bioluminescence traces,a new method was established based on BRET for the real-time detection of free Ca²⁺ in living plant cells by overexpressing this apoaequorin-egfp fusion gene in Nicotiana tabacum L.via an A.tumefaciens-mediated transformation method in this study.As a model organism for the study of biological circadian rhythm,Synechococcus elongatus has developed a posttranslational circadian clock comprised of KaiA,KaiB and KaiC proteins,which is different from eukaryotes.However,An ancient non-transcriptional oscillator?NTO?,the circadian rhythm from the redox states of Peroxiredoxin?2-Cys PRX?,has been proven to be highly conserved among various living organisms.In the mature erythrocytes of human,it was reported that the 2-Cys PRX protein was able to persist a circadian rhythm of redox states with a period about 24h in the absence of transcriptional regulation,whereas no other family member of PRXs had been found to exhibit similar circadian rhythms.In this present study,2 KaiC mutant cyanobacterium strains with changed Kai clock phenotypes were used as material for the detecting of the phenotypes of PRX-SO2/3 rythmic makers by Western blot with anti-prdx-SO2/3 as the first antibody.The result indicated that the maintenance of PRX-SO2/3 rythmic makers is independent to the intactness of Kai clock,but which could be interfered by the input pathway of Kai clock.