Construction Of CDNA Library Under Salt-stress And Cloning Of BADH Gene In Hordeum Brevisubulatum (Trin.) Link

Salt-stress caused by the salinization of soil is a main environmental factor restraining plant growth and yield, which severely restricts the development of modern agriculture. Studies on the molecular mechanism of plant salt- tolerant and cultivating new variety of plant resisting salt-stress are of great significance for developing and utilizing saline-alkali soil. Hordeum brevisubulatum (Trin) Link, an excellent forage with high nutritional value and yield, is of high performance great economic value. On the basis of previous researches on physiological and biochemical features of Hordeum brevisubulatum (Trin) Link and its salt-resistant mutant in our Lab, we probe into the salt-tolerant molecular mechanism of Hordeum brevisubulatum (Trin) Link, which is important for the further development of saline-alkali soil.In this study, Total RNA was isolated with TRIzol reagent from leaves of Hordeum brevisubulatum (Trin.) Link, which was treated with 2%NaCl (in Hoagland) for 3~6 days. After mRNA was isolated by cellulose volume, double strain cDNA was synthisized with cDNA Synthesis Kit of Stratagene. Then two adaptors of EcoR I and Xho I were linked to the cDNA, which could be cloned into the A, ZAP Express vector directionally. Gigapack III Gold Packaging Extract was provided by Stratagene. So a salt-stressed cDNA library of Hordeum brevisubulatum (Trin.) Link was constructed for the first time in the world, whose recombination rate was 96% and the capacity of library was 2X 106pfu. The detecting result indicate that the size of recombinations were between 0.5kb and 3kb. So a good expressed library was successfully constructed.It was found that betaine aldehyde dehydrogenase (BADH) gene is one of the stress-tolerant genes. Therefore, primers were designed according to homogeneity of BADH. The probe HbBL of 613bp was obtained by RT-PCR. The probe was DIG labeled after homologous comparison. The cDNA library of Hordeum brevisubulatum (Trin) Link under salt-stress screened by the probe and a positive cDNA clone obtained. The full-length of the cDNA was 1776bp with the open reading frame lacated from 85bp to 1602bp, which encoding 505 amino acid residues. Its 5' UTR is 85bp and 3' UTR is 174bp which contains polyA signal (AATAA) and polyA tail. The sequence was accepted by the GenBank and the accession number was AY188952.