Transgenic Research Of Porphyra Yezoensis Protoplasts

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. But there have been only a few reports concerning the introduction of foreign genes to Porphyra. Transient expression of GUS genes has been accomplished in P. miniata and P. yezoensis under control of CaMV 35S promoter. Although the 35S promoter has a wide host range and is widely used in many plant cells, this promoter was originally derived from a higher plant virus. Therefore, in order to establish a reliable gene delivery system in P. yezoensis, it is necessary to use endogenous promoter sequences derived from Porphyra genes.In order to investigate the characteristic of Porphyra gene and find endogenous promoter, beta-tubulin gene of P. yezeoensis is selected as interest gene for investigation according to the successful example that endogenous beta-tubulin promoter has often been used for foreign gene transformation in other alga. The sequence of beta-tubulin gene and its flanking sequence is to be cloned, and the regulatory functions of its flanking sequences and the method for introducing foreign gene are to be study by transient expression in P. yezeonsis protoplasts.P. yezoensis PY-qingdao l is pure line constructed in previous research, and used in this experiment. Amino acid sequences and nucleotides sequences of beta-tubulin of eukaryotes are aligned to find the conserved sequence. Degenerate primers are...