Study On Molecular Mechanism Of Salt Tolerance And Cloning Of Salt-Related Genes In Puccinellia Tenuiflora

Puccinellia tenuiflora, a halophyte with high capacity of salt resistance, is an excellent target for plant salt tolerance research. A cDNA library was constructed from the leaf of P. tenuiflora treated with NaHCO₃ (450 mmol/L, 48 h). After cDNA sequences were obtained through randomly selected sequencing process, expressed sequence tags (ESTs) annotation and cDNA microarray analysis were carried out to investigate the gene expression patterns of P. tenuiflora under NaHCO₃ and drought stresses.Primary titer of the library was 1.0×10⁶ pfu/mL, and the rate of recombination was 95%, with an average inserts size of 0.8 kb, which were determined by both PCR and sequencing analysis. A Total of 2 972 high quality sequences were generated from 3 168 sequenced clones which were selected randomly from the library. Clustering analysis caused 496 contigs (from 1 102 sequences) and 1 870 singletons, representing 2 366 non-redundant unique transcripts. The GenBank accession numbers of these transcripts are CN485355^CN487720.A Total of 660 cDNAs whose sizes lower to 250 bp were selected from the EST collection to be spot on glass slide as target genes and make cDNA chip, while cDNA probes were synthesized and fluorescent dye (Cy5-dCTP and Cy3-dCTP) labeled from total RNA isolated from control and NaHCO₃-treated plants respectively via RT-PCR. The cDNA microarray was made through hybridizing with target genes and probes on the slide.BLASTX search results for 2 366 ESTs showed that 1 274 sequences had high homologous to known sequences, while 618 ESTs showed low homologous to known sequences and remaining 474 sequences had no homologous to identified genes. 12 functional categorizations were created from 1 274 transcripts that were identified based on their protein functions. 4 groups' transcripts were significantly expressed: Metabolism, Transcription, Unclassified and Cell rescue/defense, which represented 18.84%, 12.48%, 11.22% and 9.66% of all classified genes, respectively. The results of elucidation in most abundantly expressed and salt-stress-related genes indicated that photosynthesis, transcription, transporters and cell rescue/defense components comprised more genes than others, implying that photosynthesis, water/ions transporting, transcription regulating and toxic substance scavenging occupied vital positions during salt resistance in P. tenuiflora. The results of cDNA microarray hybridization showed that total 25 genes represented differential expression patterns, including 17 down-regulated (salt repressed) genes and 8 up-regulated (salt induced) genes. These genes functions involved in various aspects such as signal transduction, transcript regulation, cell defense, and metabolism, suggesting important roles in salt resistance of P. tenuiflora. The microarray experiments also found 24 genes were down-regulated and 18 genes were up-regulated by drought stress. These genes were believed that associated in signal transduction, transcript regulation and stress responses. As a result, the preliminary gene expression profiles by whichP. tenuiflora responses to NaHCCh and drought stresses were establishedPlant salt tolerance is a complex system associated with multi-genetic coordinated operating. From the results described above, we can safely conclude that the characteristics of multiple genes expression under salt stress could be monitored and revealed by using the genomics approaches integrated ESTs method and cDNA microarray analysis, thereby make a foundation for thoroughly studying the molecular mechanism of salt resistance of/3 tenuiflora.