Applications Of Cell-penetrating Peptides On Algal Exogenous Substances Transduction

Algae are a class of single-cellular or multi-cellular photosynthetic autotrophicorganisms, which can be divided into macroalgae and microalgae based on theirmorphological characteristics. Due to their large biomass, rapid and sustainablegrowth, high efficiency of photosynthesis, algae have prompted broad interest in therecent years and played an essential role in food, medical, chemical or energy areas.Currently it has no simple and efficient way to deliver exogenous substances intoalgae to achieve regulation of the algal metabolic pathways. Cell penetrating peptides(CPPs) are a class of proteins which can cross cell membrane spontaneously. They arealso effective molecular tools that can transport exogenous biologicalmicro-molecules or macro-molecules substances into different cells. Although theyhave been fully applied on mammalian cells, cell penetrating peptides are rarely usedin the research of algae. In this study, we took common macroalgae and microalgae asour research objects and utilized cell penetrating peptides to conduct two followingresearch:1. It has always been a challenge to implement efficient exogenous substancestransduction into macroalgae. Yet, no study had been taken to apply CPPs onmacroalgae. In the present study, we utilized cell penetrating peptidenona-arginine (R9) to conduct translocation experiments on three common marinemacroalgae Ulva prolifera, Porphyra yezoensis and Laminaria japonica. Theimages were taken on the confocal laser scanning microscope. Then, thephotosynthetic efficiency of R9-treated thalli were determined by Dual-PAM. Theresults showed that R9can successfully get across the cell barriers of threemacroalgae and can also get into the cells of different generations of Porphyraand Laminaria. The efficiency of transductions was closely related to thestructures of cell walls. It had no significant change in Fv/Fm, ETR(I) and ETR(II)of macroalgae after being treated with R9, which demonstrated that R9had nonegative impact on the photosynthesis of macroalgae. This study has established anew method of importing exogenous substances into macroalgae, which laid a cornerstone for future investigations of macroalgae breeding, moleculartransformation and exogenous substance intake mechanisms.2. Current implementations of importing macromolecules by CPPs into microalgaehave only achieved on protein level; transductions of other macromolecules, suchas nucleic acid substances, have not yet been verified. Here, we tooknona-(L)-arginine (R9) to conduct CPPs and CPPs-mediated siRNA translocationson two marine algae (Dunaliella salina and Phaeodactylum tricornutum) and twofresh water algae (Chlamydomonas reinhardtii and Chlorella vulgaris). The resultshowed that fluorescent labeled R9can get into all four algae. The R9/siRNAscomplexes can successfully translocate into D. Salina, while other three algaedemonstrated no apparent siRNA internalizations. It may be due to the lack of arigid cell wall in D. Salina whose cell barrier is devoid of cellulose, and thus leadto a weaker blocking effect on CPPs/siRNA complex. These results indicated thatcell penetrating peptide R9could be applied as a new tool to further algaltransgenic and gene silencing research, especially on those algae which areresistant to high salinity.In summary, the cell-penetrating peptides can implement sufficient transductions ofexogenous substances into macroalgae and microalgae. They can also prompt thosebiological molecules of lower permeability to get into the algal cells. The applicationsof CPPs on algae have great significance in improving algal traits, exploring specificalgal gene function, achieving algal molecular transformations and regulations ofalgal internal metabolic processes.