Establishment and Identification of Cold-Stressed Normalized Full-Length cDNA Library of Ceratoides lanata

Abstract

Ceratoides lanata is the native sand-fixing plant in deserts of America Continent. As an even-green undershrub with extremely strong antifreeze capability, Ceratoides lanata had been considered as the winterfat and introduced into many countries including China. To explore the mechanism of cold resistance and to clone genes directly involving the strong antifreeze capability, a normalized cold-stressed full-length cDNA library was constructed from the cold-treated leaves of Ceratoides lanata by the method of DSN (duplex-specific nuclease)-based normalization and SMART (switching mechanism at 5' end of RNA transcript) full-length cDNA construction. The quality check showed that the titer of un-amplified cDNA library was about 1.4×106 cfu mL−1 and the average size of cDNA inserts was about 1 150 bp with a recombination rate of 100%. The result of sequencing 187 clones selected randomly revealed that the redundancy of the cold-stressed normalized full-length cDNA library is only 0.59%, and the ratio of full length is 46.3%. Among 187 cDNAs, 24 have homology with known genes or proteins, while the other 163 have no identity, suggesting that most cDNAs are new cold-involved genes from Ceratoides lanata. The analytic result showed that the cold-stressed normalized full-length cDNA library of Ceratoides lanata a was established successfully, and this will benefit the further study on the molecular mechanism of strong antifreeze capability and cloning of cold-resistant genes