| Puccinellia tenuiflora is a perennial graminaceous plant that has a strong tolerance to salt and alkali soil conditions. It distributes widely in the Songnen alkaline grassland. The purpose of this study was to get genes related to alkali-salt tolerance from Puccinellia tenuiflora using the technique of suppression subtractive hybridization (SSH), and to compare the expression pattern of genes in salt-stressed plants with that of the control by technique of cDNA microarray. Puccinellia tenuiflora plants, which were naturally grown in saline-alkaline grasslands (soil pH at 9.5) in Qinggang county of Heilongjiang Province, were collected in May 2004, when salt concentrations near the soil surface rose because of higher water evaporation and less rainfall. These collected plants were used as the tester. Some Puccinellia tenuiflora plants were grown for eight weeks in soil at pH 5 to 6 in a greenhouse and used as the driver.The secondary PCR amplification products of SSH library were inserted in pGEM-T vector, and then transferred into competent JM109 cells. After screening by blue/white spot test and subsequent cell culture, the plasmid DNA was extracted. Sequencing PCR reaction of the plasmid DNA was performed using Dynamic ET Dye Terminator Cycle Sequencing Kit (Amersham). Purified PCR product sequences were read by MegaBACE 500 DNA Analysis Systems. The sequences were clustered by SeqClust 2.1 software to get rid of redundant sequences. The resultant sequences were compared with those available in NCB1 database to get some genes with predicted function.From the library, 2195 high quality sequences (longer than 200bp, 1012bp at the longest and 391 bp in average) were isolated. After clustering, we got 1041 unigenes. These genes were contigged by SeqClust 2.1 software, and we got 472 contig sequences. By running blastn program of NCBI. 207 contig sequences were found to have the homologous genes with the network database, and 265 ESTs without remarkable homology. Among 207 ESTs with homology. there were 103 ESTs with known function and 104 ESTs with unknown function. Function of these genes included active oxygen elimination, osmotic adjustment, gene regulation, transport of ions etc. We consider that some of these genes and their metabolisms are possibly involved in the process of salt tolerance in Puccinellia tenuiflora.Inserts were amplified from 1041 sequences of Puccinellia tenuiflora SSH library clones, using "nest primer I" and "nest primer 2R". and add 8 yeast specific junk DNA as external control, all above sequences were spotted on slide by Beijing CaptitalBio Corporation. Alkali-salt stress and non-stress Puccinellia tenuiflor's mRNA was labeled with Cy3- or Cy5-fluorescence dye respectively during reverser transcription and then mixed as targets.Following the direction of user manual of 3DNA 900?, we accomplish microarray hybridization. The array was scanned by ScanArray 4000? microarray scanner. For standardization, the Cy3 and Cy5 signal intensities were adjusted to fit external controls. Using SAM software to analyze the significance of difference of the microarray genes, we got 185 significant up-regulated genes and 8 significant down-regulated genes by setting the order field A=0.427. Among the 185 significant up-regulated genes, 103 genes were homologous with GenBank sequences and 42 of them were function-known. In function-known genes were included dehydrin, catalase, dehydrorbate reductase, glutathione-S-transferase, heat shock cognate protein 70, all of which were known to be related with stress tolerance. Among 8 down-regulated genes, 4 genes were significantly homologous with GenBank sequences but the function was not known and the other 4 were not significantly homologous with GenBank sequences.
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