Transgenic Research Of Porphyra Yezoensis

Porphyra is the leading cultural species of the seaweed cultivation and the main export sea product in China. Recently, there is an increasing demand for good Porphyra cultivars with the enlargement of Porphyra cultivation scale. Porphyra is feasible for cultivation and its development is easy for regulation. The technique for preparation and regeneration of Porphyra protoplasts is perfect and the protoplast is a good material for research in molecular level. Therefore, it is necessary and viable to develop Porphyra genetic engineering research and breed new Porphyra cultivars via transgenic technique. Porphyra is also expected to be a bioreactor for foreign protein production because of the feasibility of cultivating of this alga. In this study, protoplast was used as receptor for transgenic research in order to construct an effective transgenic system for Porphyra yezoensis.The prerequisite of the traditional breeding and bioengineering research of Porphyra is the construction of pure lines. The wild P. yezoensis was collected from Qingdao and used to prepare protoplasts by enzyme digestion. The pure line PY-qingdaol was constructed by cultivating the protoplasts. The 18S rDNA of the PY-qingdaol was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed based on these sequences using neighbor-joining method. The results indicated the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.In order to establish the eclectroporation condition for P. yezoensis protoplasts transformation, confirm the effective promoter and test the feasibility of 18S rDNA used as foreign gene integration site, a targeting vector pQD-GUS was constructed containing a portion of the 18S rDNA of P. yezoensis and transformed it into the same strain protoplasts by electroporation. The results showed that CaMV35S promotor iseffective for transient expression of GUS gene in P. yezoensis. Electroporation is a useful method for foreign genes enter into P. yezoensis. The good condition for electroporation is that field strength was set at 2KV/cm and pulse length was set at 0.3 ms. GUS gene expression was greatly increased when 18S rDNA was used as homologous sequences to direct homologous recombination.In order to establish the suitable genetic selectable marker gene, two targeting vectors pQD-CAT-control and pQD-CAT-Enhancer were constructed which contained a portion of the 18S rDNA of P. yezoensis and cat gene promoted by SV40 and CaMVSSS respectively. The vectors were transformed into P. yezoensis by electroporation. The results showed that SV40 and CaMVSSS were effective promoters for cat gene expression in P. yezoensis. The targeting vectors containing the portion of the 18S rDNA of P. yezoensis could fulfill the stable expression of cat gene in P. yezoensis. Chloramphenicol could be used as selection pressure for positive transformant when cat gene was used as selectable marker gene.The mouse TRAIL cDNA was cloned and sequenced. A targeting vector pQD-TRAIL-CAT containing the portion of the 18S rDNA of P. yezoensis was constructed with TRAIL as foreign gene and cat gene as selectable marker gene. The vector was transformed into P. yezoensis protoplasts by electroporation 0 It was proved that foreign genes have incorporated into genome of P. yezoensis after preliminary selection with chloramphenicol.Cryopreservation of protoplasts of P. yezoensis by vitrification was studied. The results showed that VS6 (10% DMSO, 30% glycerol, 10% sucrose ) is seemed to be a preferable vitrification solution for P. yezoensis protoplasts. The best vitrification protocol using VS6 involves: loading with 25% VS6 for 5 minutes at 0 癈, dehydrating with ice cold concentrated VS6 for 3 minutes, immediately immersing in LN, and then warming in 40癈 water bath. Using this protocol, P. yezoensis protoplasts showed higher viability (66.5%) after Cryopreservation and could regenerate thallus.hi summary, the tra...