Application of suppression subtractive hybridization (SSH) to cloning differentially expressed cDNA in Dunaliella salina (Chlorophyta) under hyperosmotic shock

Two subtracted cDNA libraries of Dunaliella salina (Volvocales, Chlorophyceae) under different hyperosmotic shock were constructed using the suppression subtractive hybridization (SSH) method. The mRNA isolated from algae grown without stress was used as a "driver," and the mRNAs isolated from algae 16 h (short-term treatment) or 7 d (long-term treatment) after salt stress were used as "testers." The differentially expressed cDNA fragments in D. salina under salt stress were identified by screening these 2 libraries. Two cDNA fragments, D27 and D114, were identified from clones p27 and pL114 after the long-term treatment. Three cDNA fragments, D21, D39, and D88, were identified from clones pSh21, pSh39, and pSh88 after the short-term treatment. The homology analysis revealed that D27 was highly similar (91%) to the subunit V of PS I reaction center in Chlamydomonas reinhardtii. D21 was similar to fructose-1,6-diphosphate aldolase (78.4%). After searching GenBank with the sequences of D39, D88, and D114, no similar sequences were found. Northern analysis revealed that the expression levels of all 5 cDNAs were increased significantly after salt stress. This means that SSH can be used in cloning differentially expressed cDNAs in D. salina under salt stress. The expression of D27, D21, and D88 was de novo induced by salt stress, and the expression of D114 and D39 was increased from a relatively lower level; this indicates that all 5 cDNAs might exert an influence on the alga under hyperosmotic shock.