To Establish The System Of Genetic Transformation Of D. Salina

Dunaliella is a genus of unicellular eukaryotic green alga, which would be extraordinarily amenable to the study of plant stress resistance and theβ-carotene biosynthesis. Genetic transformation is the necessary and desirable technique for the molecular study in D. salina. Achievements related to establish such a system were shown as below:1. An electroporation procedure was developed for delivering foreign DNA to D. salina cells. Based on the numbers of individual colonies cultured on a semi-solid culture medium, about 50% of the cells survived after electroporation at EFS of 1000V/cm in the presence of 50 mmol/L NaCl and 1.4 mol/L glycerol. In the cells electroporated with pSP124S, the retention and expression of the ble gene was detected at least 72 hours after the electroporation. Individual resistant progeny colonies were isolated and Southern analysis of these colonies showed strong evidence supporting the integration of foreign DNA in some colonies and presence of free-formed foreign DNA in other colonies 6 months after continuous sub-culturing. By careful analysis, it was found that about 90% of the progeny cells from one single colony lost the resistance phenotype after every one sub-culturing step.2. D. salina NR (nitrate reductase) cDNA and gene were isolated from the cDNA and genomic libraries. Sequence analysis suggested that DsNR had 75% identity and 89% similarity with those NR genes from other green algae in the coding region. Northern analysis demonstrated that the transcription of DsNR reached the highest-level 6 hours after nitrate induction. A transformation vector containing the DsNR cDNA was constructed and was justified to be functional by complementing a NR gene defective Chlamydomonas reinhardtii mutant.