Development Of Plastid Genetic Transformation System In Pyropia Yezoensis

Pyropia yezoensis belongs to Rhodophyta, Bangiaceae, Pyropia. As a large, redalgae growing in the intertidal zone, P. yezoensis is abundantly farmed in the northerncoast of China. At present, P. yezoensis is considered as a representative modelspecies to study the intertidal algae about their physiological and ecological characters,as well as adapting mechanisms to environmental stresses. Establishing the genetictransformation system of P. yezoensis will not only provide an important platform forin-depth research of the mechanism that P. yezoensis responding to stresses in themolecular level, but also provide a new idea for genetic breeding of P. yezoensis.In this research, we committed to establishing the plastid genetic transformationsystem of P. yezoensis and the following results has been achieved:1. The psbA5’ and psbA3’ regulatory sequences were cloned from P. yezoensis plastidgenome and a enhanced green fluorescent protein reporter gene expression cassette(eGFP) was constructed, which were validated in E. coli. Under the fluorescencemicroscope, bright green fluorescence can be observed in E. coli cells transformedwith the eGFP cassette (psbA5’-EGFP-psbA3’).2. The site-specific integration vector pYV1for P. yezoensis plastid transformation,with chloramphenicol resistance gene (cat) expression cassette were constructed.The rrsB-trnI-trnA-rrsL sequence fragment from P. yezoensis plastid genome wascloned as the homologous recombination fragment. The E. coli cell transformed withthe vector pYV1can survive on the LB solid medium.3. Particle bombardment was firstly applied to introduce exogenous genes into P.yezoensis plastids. The expression vector pYG, containing the enhanced greenfluorescent protein reporter gene expression cassette (eGFP) has been bombarded intoP. yezoensis plastids of thallus cells.The bombardment parameters are as follows:1100psi,12cm bombardment distance and25inHg vacuum. The bombarded cells wereobserved under a fluorescence microscope. The results showed that bright green fluorescence was observed in some cells, suggesting that the pYG vectors have beensuccessfully introduced into P. yezoensis thalli cells, and the eGFP gene has beensuccessfully expressed.In this research, the developments of plastid genetic transformation techniquesin P. yezoensis were carried out for the first time. We provided the establishment ofplastid genetic transformation system in P. yezoensis with efficient endogenousregulatory sequences, selection gene expression cassette, reporter gene expressioncassett, as well as a plastid site-specific integration expression vector for stablegenetic transformation in P. yezoensis. The particle bombardment method to introduceexogenous genes into P. yezoensis plastids has been established preliminarily. Theseresearches will lay the foundation for completing the platform of plastid genetictransformation technology system in P. yezoensis.