Construction Of Transient Expression Systems Of Aquilaria Sinensis Protoplast And Plant MiRNA Functions Validation

In this study, we firstly induce callus using stem segments and leaves of A. sinensis, then separately treat callus and leaves of A. sinensis by changing the concentration of the enzyme solution, the amount of tissue, material handling and enzymatic hydrolysis time, and exploratorily build an effective protoplast free system for A. sinensis. After these, we extract the total RNA from A. senensis calli, and amplfy ASS gene using specific primers by RT-PCR and ligate to pEZS-NL and pFGC5941GFP vectors to construct two plant expression vectors: pEZS-NL-ASS-GFP、p5941-GFP-ASS. Three transient expression systems, agroinfiltration of tobacco leaves, particle bombardment of onion epidermal cells and PEG transformation of protoplasts isolated from A. senensis callus were adopted and compared. The expression of the fusion proteins were observed by a confocal laser scanning microscopy. The green fluorescence was observed in all the three systems but there was a little difference in their expression. Thereinto, it is highest in tobacco cells. Furthermore, the cytoplasm and plastid localization of the GFP fusion protein observed in protoplasts was comparatively clear and was consistent with studies in other plant species. The result demonstrated that the ASS was located in the cytoplasm and plastid and it might be more convinced to use the cells of the original species than other species for the subcellular localization study.Then, we adopted modified binary expression vectors pCAMBIA1200with inserted CAMV double35S promoter and pFGC5941GFP to transform the miRNA and potential target-GFP fusion protein respectively by agroinfiltration of the leaves of tobacco. Then the impact of the miRNA on the expression of the potential target could be validated by observing the fluorescence of the GFP fusion protein. To further confirm the effectiveness of this system, the arabidopsis miR393and its target AFB3, were adopted to construct pCAMBIA1200-35S-miR393and pFGC5941-GFP-AFB3vectors. Our previous studies found that it’s more efficient using Agrobacterium infection of tobacco leaf method than the others in three transient transformation methods. So pCAMBIA1200-35S-miR393and pFGC5941-GFP-AFB3vectors were co-transform into tobacco leaf by agroinfiltration and the pFGC5941-GFP-AFB3were transformed alone as the control. By a confocal laser scanning microscope, the green fluorescence could be observed in the nuclear of the control tobacco cells. However, in the co-transformed tobacco cells, no green fluorescence could be observed, demonstrating that miR393had inhibited the expression of AFB3. So this transient expression system we constructed could be used for the in vivo validation of plant miRNA functions and as a simple and speedy system, could supply efficient evidence to the miRNA function.This study successfully build a protoplasts free system from leaves and callus for A. sinensis,.and acquire the transient expression of target genes in protoplasts isolated from A. sinensis callus using PEG. After comparing the system with Agrobacterium injection tobacco leaves and gene gun bombardment onion epidermal cells, which are two plants transient expression systems, we choose Agrobacterium injection tobacco leaves and two smaller binary expression vector, and construct a plant miRNA function validation system, which could be used to study the relationship of miRNA and target gene in A. sinensis and all plants.