Differential Expression And Function Analysis Of Genes In Populus Simonii×P.nigra Under NaCl Stress

To study physiological response of Populus simonii×P.nigra to salt stress, SOD activity, POD activity, MDA content and relative content of chlorophyll were measured at 6 h,1 d,2 d, 4d,6d,8d under 0,75,150,250 mM NaCl stress. As the salt concentration increased and the stress time continued, POD and SOD activity increased gradually and then decreased. MDA content was increased and chlorophyll content was decreased gradually. By Morphological observation of Populus simonii×P.nigra leaf, it was found that water loss and petiole softening was serious under NaCl sress at the second day. Under continued stress condition, leaf was wilting and yellow, brown, crimping and brittle. This also showed that Populus simonii×P.nigra was not salt-tolerant plant. It had a certain tolerance to low concentrations of salt but high concentration of salt was harmful.To identify genes involved in salt stress responses, we carried out cDNA-AFLP analysis on leaves under normal growth and NaCl stress. Selective amplifications with 64 primer combinations allowed the visualization of about 4407 transcript-derived fragments (TDFs). 2027 of TDFs were differentially expressed.996 TDFs were up-regulated and 1031 TDFs were down-regulated.161 differentially expressed TDFs were excised from gel, eluted and re-amplified.121 TDFs were re-amplified and 107 TDFs sequenced successfully, yielding 86 unique sequence. These TDFs were submitted to the Genbank as accession number of GW672587-GW672672. They were further classified into 10 functional categories based on their putative functions, i.e., unknown protein(18.6%), metabolism(17.44%), regulation of transcription(12.79%), signal transduction(11.63%), cellular biosynthesis(10.47%), transport (4.65%), cellular catabolism(4.65%), response to stress(5.81%), photosynthesis and redox (6.98%), development process(6.98%).PsnRZF gene was induced in Populus simonii×P.nigra under NaCl stress. The 1061 bp full length cDNA of ring zinc-finger gene was isolated by rapid amplification of cDNA ends (RACE), including a 184 bp 5'untranslated region, an 82 bp 3'untranslated region and a 795 bp open reading frame encoding 264 amino acid residues. The molecular weight of deduced protein was 30.25 kDa with a theoretical pI of 8.04. Real-time PCR revealed that PsnRZF gene was expressed differentially in roots, stems and leaves. The expression level of PsnRZF gene in leaves and stems was increased gradually. The peak value was appeared at the 6th day in leaves and 4th day in stems under NaCl stress. The expression level of PsnRZF gene in roots was not changed significantly under NaCl stress.To verify PsnRZF gene function, the plant expression vector with PsnRZF gene was constructed, and transformed tobacco was performed by agrobacterium-mediated method. Physiological analysis showed that PsnRZF gene may play different roles under different growth conditions. Over expression of PsnRZF gene is harmful to plant under normal condition. But over expression of PsnRZF gene under salt stress can relieve the damage of membrane lipid oxidation.