Study Of The Gene Expression During The Course Of NaHCO₃ Treatment In P.talassica Komm×P.euphratica Oliv Using MRNA Differential Display

In order to analyze the molecular mechanism of salt and alkali tolerance in Populus euphratica Oliv, differentially displayed cDNA fragments were separated, and sequenced after the Reverse Northern Dot Blotting, as well as analyzed through the method of DDRT-PCR with the results as follows:(1) By DDRT-PCR, the result showed that some special genes were inducibly expressed under salt and alkali stress with 4% NaHCO3 solution for three days in leaves of Populus euphratica Oliv.(2) CTAB method is quite ideal to extract the total RNA from this species in DD-RT and the RNA isolated has good quality and integrity. It was also a quick and effective method for other trees.(3) mRNA .differential display system of Populus euphratica Oliv was established. Combining 3 anchored primers and 16 arbitrary decamers, differential display pattern of the total RNA from the leaves of Populus euphratica Oliv treated by NaHCOs and the control was shown. The process was made easier and safer when the method of silver-staining was applied instead of radiolabel test. Combined with reverse northern dot blotting, DDRT-PCR's serious shortcoming with high rate of false positives can be overcomed through at least two repeated tests independently.(4) 24 differentially displayed fragments were cloned, and four of them were confirmed as positive clones by reverse northern dot blotting and then was sequenced. By both analysis of nucleotide and deduced amino acid sequence similarity, T12C-P1 and T12A-AP4 had significant identity to atpH gene encoding ATPase subunit III and sodium channel family gene respectively which had been previously identified as salt-induced expressed gene; T12G-P6 had no similarity to known proteins; T12C-AP4 had high identity to WD-40 repeat protein family which is putatively involved in protein interactions.