Construction Of PCAMBIA1300-AFH16-GFP Recombinant Plasmid And The Screening Of Homozygous Transgenic Arabidopsis

The main cause of enhanced UV-B radiation in earth is caused by the thinning of the ozone layer.Study shows the increasing global radiation dose of UV-B year by year seriously threaten the life activity of all surface organisms.To plants that can not move autonomously,UV-B radiation has great influence on morphogenesis,physiological and biochemical,metabolism.Therefore,the study of UV-B radiation on plant response has far-reaching scientific significance and application prospect.The cytoskeleton of plant has been in the process of dynamic depolymerization and polymerization.It is an important structural component that can regulate the growth and development of cells and external stimulation.In many cells,the cytoskeleton plays a key role in the process of metabolism,such as cell growth,cytoplasmic streaming,the movement of organelles and cell division,and the implementation of these functions need some specific auxiliary protein to adjust the dynamic behavior of cytoskeleton protein.Formin protein is thought to be a multidomain actin-binding protein,there is growing evidence that formin protein can also bind with microtubule and may act as a bifunctional protein to involve in cellular processes.Compared with formin protein in mammalian and yeast,formin protein in plants is poorly understood.Study shows that AtFH16 in Arabidopsis belongs to class?formins,and it has been suggested that it may participate in the process of cell division.Our research group has been committed to the study of plant response to enhanced UV-B radiation,and found that the dynamic changes of the actin cytoskeleton involved in“partition-bundle division” phenomenon.Based on a comprehensive literature analysis,we determined that the change in the response of microfilaments may be dependent on the Formin family proteins.In order to verify this hypothesis,we do thefollowing:(1)In this study,GFP as report gene was chosen the first fusion gene to construct the fusion expression vector pCAMBIA1300-GFP-AFH16,then after the success of sequencing,the recombinant vector was transferred into agrobacterium,and the recombinant plasmid was transformed into Arabidopsis by using agrobacterium mediated method and screening homozygous transgenic plants.(2)At the same time,the recombinant expression plasmid was injected into the tobacco epidermal cells to be transient expression,then observed the tobacco epidermal cells existing green fluorescence by laser scanning confocal microscope,which further illustrated the plasmids were successfully constructed.(3)Throuth the comparison of the phenotype between the wild type plants and transgenic plants,transgenic plants showed root elongation,leaves increased,which indicated that AtFH16 may participate in and promote cell division.(4)Then we observed the subcellular localization of AtFH16 in Arabidopsis thaliana and found it mainly localized in the cytoplasm.The construction of the vector and the acquisition of transgenic plants provide an important experimental material for the research group to further verify the occurrence of AtFH16 and “partition-bundle division”.At the same time,it provides a certain basis for the research group to study the dynamic changes of microfilament cytoskeleton after UV-B radiation.