Characteristic of Differentially Expressed Genes in Cold-Stressed Suppression Subtractive Hybridization Library of Winterfat

As a high stress resistance psammophyte, winterfat, Krascheninnikovia lanata (Pursh) A. Meeuse & Smit, has been successfully introduced into many countries. The leaves and branches of winterfat grown in room temperatures (15-25°C) and under cold stress (-10-5°C) were used as the Driver and the Tester, respectively. Both Tester and Driver ds cDNA were prepared from their high quality mRNA, which were purified from their total RNA. Tester and Driver cDNA were separately digested to obtain shorter, blunt-ended molecules by RasI. Two Tester populations were created with different adaptors, while Driver cDNA had no adaptors. Differentially expressed genes were equalized and enriched by two round subtractive hybridizations using excess Driver population as compared with Tester population. The differentially expressed cDNAs were exponentially amplified by first round suppression PCR using the diluted hybridization product as template. The secondary PCR was performed using the first PCR product as template by nested primers to finally enrich the differentially expressed cDNAs, which consist of the SSH library of winterfat. These cDNAs were inserted into vectors and 362 cDNA clones were obtained. The sequencing results showed that some cDNAs of cold-stressed winterfat SSH library had relatively high homology with known stress resistance-related genes or proteins. Other cDNAs are new genes which are firstly reported. Our results lay a foundation for the cDNA cloning of valuable genes, including antifreeze, heat-resistant, drought-tolerant and alkali-salt-tolerant genes, and their transgenic applications.