Study Of Salt Tolerance Mechanism Of Puccinellia Tenuiflora Using Suppression Subtractive Hybridzation And CDNA Microarray

Puccinellia tenuiflora, a kind of halophyte with high capacity of salt resistance, is a dominant plant species in saline-alkali soil of Songnen plain. Leaves from NaHCO₃ treated(400 mmol/L, 6 h, 12 h, 24 h, 48 h)plants and non-treated control plants were used as "tester"and "driver", respectively. Suppression subtractive hybridization (SSH) was employed to construct 4 SSH cDNA libraries. Clones from the 4 libraries were selected randomly and sequenced. After cDNA sequences were obtained, expressed sequence tags (ESTs) annotation and cDNA microarray analysis were carried out to investigate the gene expression patterns of P. tenuiflora under NaHCO₃ stresse at different time.An average size of inserts in 4 SSH libraries is 302 bp in length, which is determined by both PCR detection and sequencing analysis. After removing the vector sequences, low quality sequence and sequence less than 100 bp, the final 1 775 effective ESTs were generated from 1964 sequenced clones. The GenBank accession numbers of these ESTs are EB104056-EB105830.According to reported MIP protein category standard of Arabidopsis thaliana, the ESTs of each SSH library can be categorized into metabolism, Photosynthesis, cell defence reaction, transport facilitation, signal transduction, transcription, protein synthesis and destination and novel gene etc. The results showed that the expression of genes in P. tenuiflora were significantly changed under NaHCO₃ stress at different time. For example, the category of cell rescue/defence were significantly expressed at NaHCO₃ stress 48 h and 6 h, which accounted for 10% and 8% of in 48 h and 6 h libraries respectively, but only 2% in the 24 h library;The catergory of transcription accounted for 4%, 2%, 1% and 4% in 6 h, 12 h, 24 h and 48 h libraries, respectively;Two groupes of protein destination and signal transduction have the largest amount (8%) in 48 h library, but it is only 2% in 24 h library. These results showed that the expression profiles of Puccinellia tenuiflora continually varied under salt stress.cDNA Microarrays were generated and employed to study the gene expression under NaHCO₃ stress. Totally, 142 significantly different expressed genes were identified. The results of cDNA micarray indicated that there 97% of genes in each library are signifcantly up-regulated in the library at the time they were subtractive hybridization. This result showed that the EST analysis to each SSH library could fully represent the genes up-regulated under NaHCO₃ strees at that time.cDNA microarray analysis showed that threr are 6 gene expression profiles in P. tenuiflora, among them, the expression profiles, which the amount of expression at 6 h and 24 h were higher than at 12 h, but at 48 h which was lower than at 12 h, accounted for 59.6% of alldifferenlt expressed genes.Salt tolerance in plants is a complex system associated with multiple genes coordinated action. Our research showed that the genes expression profiles could be obtained effectively through the application of EST analysis and cDNA microarray, and will make a solide foundation for systemly reveal the molecular mechanism of-alkali tolerace in P. tenuiflora.