Screening And Identification Of Regulator Involved In Hypocotyl Phototropism To Blue Light In Arabidopsis

To better adapt to the complex and ever-changing external environment, plants form a uniquemechanism to respond to external stimulis in the long-term evolutionary process, the phototropismmovement is an adaptive response to capture more light source. The discovery of phototropins provide thebasis to explore the signal transduction mechanisms of the blue light-dependent to phototropic bending. Atpresent, hypocotyl bending mechanism mediated by PHOT1in weak blue light is relatively clear, thephototropin PHOT1is blue light-activated and then interacts with its downstream signal proteinNPH3,RPT2and PKS1, regulates the activation and localization of the auxin-influx and auxin-effluxtransporters PIN1and AUX1, which lead to asymmetric distribution of the auxin between the illuminatedand shaded side of the hypocotyls and induces phototropic bending. But in strong blue light, PHOT1andPHOT2are functional redundancy in the blue light-dependent to phototropic bending, it limits to explorethe different mechanisms mediated by PHOT1and PHOT2.In this experiment, phot1which was mutagenized by EMS was used as material. Material wasirradiated with blue light100μmol m-2s-1for12h to screen the mutants which were not phototropic bending.We had found five potential mutants which were named as e20, e102, e176, e199and e215. The fivemutants were hybridized with Ler, statistics of segregation ratio between F1and F2showed that e199wasrecessive single gene mutation. Further study on e199, We located the gene on MSJ1BAC which was infifth chromosome in Arabidopsis by gene cloning. DNA sequence analysis showed that AT5G64330.1which was NPH3was mutated. Compared with wild type, e199had a point mutation (G-to-A) at1517bp inthe gene DNA. That is the codon encoding the tryptophan becomed the termination codon and earlytermination of translation. The mutant phenotype analysis displayed that NPH3mediated the phototropicbending of blue light-dependent and involved in regulating leaf curl, but did not participate in blue lightinduced stomatal opening reaction. In the experiment, we constructed NPH3gene overexpression, GFP andGUS transgenic plants to confirm whether the mutation results in the hypocotyl does not bending to thestrong blue light and to determine NPH3positioning in tissues and cells. In addition, we constructed frontof the point of the mutation NPH3gene overexpression and GFP transgenic plants to determine whether the mutation affects its positioning and function in cells. In order to further explore the relationship betweenNPH3and Ca2+, we builded aequorin transgenic plants to to observe the change of Ca2+concentration inthe e199mutant. At the same time, e199was hybridized with auxin positioning related DR5-GUS,DR5-GFP and auxin transportation related PIN1-GFP, AUX1-GFP transgenic plants to determine thedistribution of auxin in strong blue light, to provide a basis for further revealing the possible role of NPH3in PHOT2signaling pathway.