条斑紫菜pyropia yezoensis质体遗传转化体系构建-条斑紫菜条斑萤叶甲质体遗传转化体系构建.docxVIP

万方数据 万方数据 Development of Plastid Genetic Transformation System in Pyropia yezoensis Abstract Pyropia yezoensis belongs to Rhodophyta, Bangiaceae, Pyropia. As a large, red algae growing in the intertidal zone, P. yezoensis is abundantly farmed in the northern coast of China. At present, P. yezoensis is considered as a representative model species to study the intertidal algae about their physiological and ecological characters, as well as adapting mechanisms to environmental stresses. Establishing the genetic transformation system of P. yezoensis will not only provide an important platform for in-depth research of the mechanism that P. yezoensis responding to stresses in the molecular level, but also provide a new idea for genetic breeding of P. yezoensis . In this research, we committed to establishing the plastid genetic transformation system of P. yezoensis and the following results has been achieved: The psbA5 and psbA3 regulatory sequences were cloned from P. yezoensis plastid genome and a enhanced green fluorescent protein reporter gene expression cassette (eGFP) was constructed, which were validated in E. coli. Under the fluorescence microscope, bright green fluorescence can be observed in E. coli cells transformed with the eGFP cassette (psbA5-EGFP-psbA3 ) . The site-specific integration vector pYV1 for 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 was cloned as the homologous recombination fragment. The E. coli cell transformed with the vector pYV1 can survive on the LB solid medium. Particle bombardment was firstly applied to introduce exogenous genes into P. yezoensis plastids. The expression vector pYG, containing the enhanced green fluorescent protein reporter gene expression cassette (eGFP) has been bombarded into P. yezoensis plastids of thallus cells.The bombardment parameters are as follows: 1100psi, 12