Amhsa32and AmCSDP From Ammopiptanthus Mongolicus Enhance Abiotic Stress Tolerance In Transgenic E. Coli And Tobacco Plants

Extreme temperature, salinity or drought are the environmental factors that affect growth and distribution of plants. To be successful in the survival game of nature, plants need to cope with adverse abiotic stresses by activating multiple protection mechanisms. Ammopiptanthus mongolicus,as, the only evergreen broad-leaf shrub in the northwest desert of China, shows very strong resistance to drought, salt, low and high temperature. In this study, we gained AmHsa32and AmCSDP gene from cold-and drought-induced EST database of Ammopiptanthus mongolicus.The characterization of AmHsa32coding a heat stress associated32kD protein was analyzed by bioinformatics, including amino acid properties, protein structure, phylogenetics and function. The BLAST algorithm identified AmHsa32with high similiarity to Hsa32from Arabidopsis (74%amino acid identity). We checked mRNA expression levels of AmHsa32under abiotic stress by Real-Time PCR analysis. The results represented that AmHsa32was strongly induced by heat shock, and up-regulated by cold, drought and salinity. To further study the biological function of AmHsa32, the open reading frame was subcloned into PEA30a. When expressed in bacteria, recombinant AmHsa32conferred tolerance to high temperature stress. We tested the seed germination under cold and heat stresses, basic thermotolerance and acquired thermotolerance in wild type and transgenic Nicotiana Benthamiana. The above results showed that wild tobacco were sensitive to abiotic stress. Salt tolerance was examined, the transgenic plants grew better under0.3mol/L NaCl.AmCSDP contains540bp ORF encoding a polypeptide of180amino acids and exhibits greater sequence similarity to DNA-directed RNA polymerase Ⅱ subunit RPB7in Medicago truncatula.(79%amino acid identity). AmCSDP consisting of a C-terminal cold shock domain belongs to cold-induced protein family. To further confirm the function of AmCSDP, we constructed a fusion vector successfully, and the recombinant plasmid was transformed to E.coli BL21.We purified the fusion protein to gain the antibody and analyzed expression of AmCSDP in A.mongolicus by western blotting. Moreover, the transgenic tobacco plants had higher germination rates than the wild types at4℃or heat stresses. Our results suggested that AmCSDP responded to abiotic stresses.