Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L.

The ability of Antarctic ice algae, Chlamydomonas sp. ICE-L, to survive and proliferate at low temperature and high salinity implies that they have overcome key barriers inherent in Antarctic environments. A full-length complementary DNA (cDNA) sequence of omega-3 fatty acid desaturase, designated CiFAD3, was isolated via reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The full-length of CiFAD3 cDNA contained an open reading frame of 1,302 bp with 5'-terminal untranslated region (UTR) of 36 bp and 3'-terminal UTR of 507 bp encoding a fatty acid desaturase protein of 434 amino acids. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known chloroplastic omega-3 fatty acid desaturase. Meanwhile, CiFAD3 sequence showed typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane spanning regions that were universally present among plant desaturases. Under different stress conditions, messenger RNA (mRNA) expression levels of CiFAD3 were measured by quantitative RT-PCR. The results showed that both temperature and salinity could motivate the upregulation of CiFAD3 expression. The mRNA accumulation of CiFAD3 increased 2.6-fold at 0°C and 1.8-fold at 12°C compared to the algae at 6°C. Similarly, mRNA expression levels of CiFAD3 increased 3.8-fold after 62‰ NaCl treatment for 2 h. However, CiFAD3 mRNA expression levels were partially decreased after UV radiation. These data suggest that CiFAD3 is the enzyme responsible for the omega-3 fatty acid desaturation involved in ice algae Chlamydomonas sp. ICE-L acclimatizing to cold temperature and high salinity in Antarctic environment.

To study the molecular mechanism of the Antarctic sea-ice alga in adaptation to polar sea-ice environments, the RNA was prepared for cDNA library construction of Chlamydomonas sp. ICE-L. Three different methods were tested to prepare total RNA from this psychrophilic, unicellular green alga rich in protein and polysaccharide. Lauryl sodium sulfate-based method allowed a most effective extraction of high-quality total RNA compared to the other methods. Total RNA extracted with this protocol was used for cDNA library construction. The recombination rate of constructed cDNA library was 98.60%, the primary titer was 7.15×10 6 pfu, and an average sequence length was 1.2 kb. These results show that with a high-quality RNA preparation, a cDNA library can be constructed successfully for Chlamydomonas sp. ICE-L.