Cloning And Characterization Of Cold-induced Gene Introns And The AmCIP Promoter In Ammopiptanthus Mongolicus

Ammopiptanthus mongolicus is the only evergreen shrub in northwest of China. It can survive -30℃or even lower temperature in winter. We previously reported three cold-induced genes, AmCIP AmLEA and AmPI, and confirmed that these genes play important role in enhancing the ability of thermotolerance and cold-resistance in transgenic E.coli which expressed the above genes.In this study, alternative introns were cloned with the primers designed according to AmCIP AmLEA and AmPI cDNA sequences, AmCIP and AmLEA lacked the constitutive dinuceotides(5'-GT and AG-3') actually with splice site (5'CA-A A3', 5'AG-GC3') and rich with AU. Furthermore, the AmCIP promoter was cloned via Genome walking assay which include at least four core cis-acting elements such as DRE,ABRE,repeat A and repeat D. Another putative cis-acting element, the sequence TAACCA complementary to MYB(TGGTTAG), may be a trans-acting factor binding site. For analyzing its function, the AmCIP promoter was fused with reporter gene GFP and GUS simultaneously in a single vector and then was introduced into tobacco by agrobacterium tumefaciens-mediated gene transfer method.