Generation and application of a fluorescent probe for phosphatidic acid in Arabidopsis thaliana
[Objective]Phosphatidic acid(PA)serves as an important signal molecule involved in the regulation of plant growth and development and different responses to various stresses as well as a general precursor for glycerolipid biosynthesis.However,little is known thus far about the dynamic changes of PA in plant cells.This study attempted to construct a fluorescent probe that can effectively monitor the changes of PA in plant cells and use it to measure the changes of intracellular PA under saline-alkaline stresses.[Method]The corresponding nucleotide sequence for PA-specific binding domain within the Spo20p protein was fused to the green fluorescent protein gene.Transgenic Arabidopsis thaliana lines bearing the fusion gene under the control of the constitutive promoter UBQ10 was then generated via genetic transformation.The resulting fusion protein constituted a fluorescent probe specifically binding to PA.Subsequently,this probe was employed to monitor the changes of cellular PA under saline-alkaline stresses.[Result]Seven transgenic A.thaliana lines homozygous for single insertion of the fusion gene was generated.Real time quantitative PCR(RT-qPCR)analysis showed that expression levels of the fusion gene varied among different lines.Experiments with various exogeneous PA concentrations revealed that as the expression level of the PA probe increased,it could effectively monitor the changes of cellular PA in the root tips treated with 2 μmol·L-1 exogenous PA for 10 min,whereas this was not the case when the expression level of the probe was low,indicating that the sensitivity of the probe for PA detection is,to a certain degree,associated with its expression level.Based on this fluorescent PA probe,PA accumulation at the plasma membrane or in the intracellular space was evident in the root tips under saline-alkaline stresses for 5 min,implying that PA may play important roles in early plant responses to saline-alkaline stresses.[Conclusion]A fluorescence probe for effective monitoring of cellular PA was developed in this study.This probe can monitor the alterations in cellular PA level during early plant responses to saline-alkaline stresses,thereby providing a new tool to study early responses to various stresses.[Ch,7 fig.1 tab.36 ref.]
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