Construction And Transformation Of Expression Vector Into Haematococcus Pluvialis By Electroporation

Haematococcus pluvialis is one of the important species of microalgae with great economic value. This algae can accumulate high content of astaxanthin up to 4% dry weight under stress conditions. For further improvement of the content and yield of astaxanthin from Haematococcus pluvialis, there are still some key techniques need to be overcomed, for example, to increase the cell density and to enhance the regulation of metabolic pathway for astaxanthin biosynthesis. It is essential to improve the biological characteristics by genetic engineering, aiming a high-cell -density cultivation and high effective accumulation of astaxanthin. To date, there are few reports on genetic transformation of Haematococcus pluvialis, and no report on the successful transformation of foreign genes into Haematococcus pluvialis by electroporation.In the present work, 5'-flanking region ofα-tubulin gene with promoter activity was isolated firstly by Genome Walking method, then the expression vector containing the 5'-flanking region and Sh ble gene was constructed followed by establishment of the transformation method for Haematococcus pluvialis by electroporation. The main results are shown as follows:1. Based onα-tubulin mRNA sequence of Haematococcus pluvialis,α-tubulin genomic fragment was obtained by PCR amplification. Sequence analysis showed it contains four exons and three introns.2. 5'-flanking region ofα-tubulin gene containing 914 bp in the upstream of translation initiation codon ATG was isolated by Genome Walking method. The analysis results based on promoter database indicate that it contains various cis-acting elements. Compared with tubulin promoters from Chlamydomonas reinhardtii and Volvox carteri, several elements playing an important role of regulation in tubulin promoters from Chlamydomonas reinhardtii and Volvox carteri were found, including 16-bp consensus sequence, TATA box and G+C-rich sequence.3. The expression vector pLSX containing the 5'-flanking region ofα-tubulin gene and Sh ble gene was constructed.4. The expression vector pLSX was introduced into Haematococcus pluvialis by electroporation. The effect of cell growth phase and electroporation conditions on cell viability was investigated systematically, and the results show that the cell viability during the early log phase was obviously higher than that of the mid-log phase or late log phase by electroporation under the same conditions. A cell viability of approximate 50% was achieved by using square wave pulses with 600 V/cm and 2 ms, or by using exponential decay wave pulses with either 500 V/cm, 5.3 ms or 600 V/cm, 2 ms.5. The transient expression of Sh ble gene in electroporated cells was confirmed by RT-PCR, indicating that the isolated 5'-flanking region ofα-tubulin gene has promoter activity. The constructed expression vector and the established transformation method by electroporation in this study will play an important role in the transgenic research of Haematococcus pluvialis.