Preliminary Analysis Of Transcriptional Response Of Puccinellia Teniflora Root Under Saline And Alkaline Stress

Puccinellia tenuiflora is one of the halophytes with high capacity of saline and alkaline resistance. In order to understand saline-alkali resistance mechanism of Puccinellia tenuiflora and isolate important genes associated with saline-alkali resistance of Puccinellia tenuiflora, in this study, physiological index of Puccinellia tenuiflora under saline-alkali stressed conditions had been determined, and suppressed subtractive hybridization libraries of Puccinellia tenuiflora roots under saline-alkali soil and 300 mM NaCl stressed conditions had been constructed respectively. Recombinant clones with the insert fragment larger than 500 bp were selected randomly and sequenced, bioinformatic analysis including sequence alignment, functional annotation and functional classification of expressed sequence tags in libraries was performed. The main results are as follows:1. Physiological index including root length, stem length, water content, proline content and anthocyanin content of Puccinellia tenuiflora was determined under 300 mM NaCl and 150 mM Na₂CO₃ stressed conditions during short time courses respectively. The results indicated that the growth of Puccinellia tenuiflora seedlings was slightly suppressed under the above stressed conditons, but seedlings still actively responded to stress.2. From the suppressed subtractive hybridization libray of Puccinellia tenuiflora roots under saline-alkali soil stressed conditon, 311 ESTs were sequenced, 289 of them were high-quality ESTs, sequences were assembled using phrap software to get a total of 199 meaningful unigene sequences. Blastn and Blastx analysis respectively showed that 141 (70.85%) unigene fragments were homologous (Blastn E-value-10, Blastx E-value-5) to the nucleic acid or protein sequences in database, the other 58 (29.15%) unigene sequences had no homology or low homology (Blastn E-value>e^-10, Blastx E-value>e^-5) with the sequences in database, which could be regarded as new genes.3. In the suppressed subtractive hybridization library of Puccinellia tenuiflora roots under saline-alkali soil stressed condition, ESTs were divided into three aspects including Biological Process, Molecular function and Cell component based on GO analysis. In COG functional classification, ESTs can be divided in to 9 categories: Translation, ribosomal structure and biogenesis; Amino acid transport and metabolism; Carbohydrate transport and metabolism; Energy production and conversion; cell wall/membrane/envelope biogenesis; Function unknown; Posttranslational modification, protein turnover, chaperones; Transcription; Signal transduction mechanisms.4. In the suppressed subtractive hybridization library of Puccinellia tenuiflora roots under saline-alkali soil conditions, several ESTs associated with saline-alkali resistance of plants were screened, such as WD-40 repeat protein, Zn⁺ finger protein, cysteine proteinase, cysteine proteinase inhibitor, sucrose synthase, vacuolar ATPase, glyceraldehyde-3-phosphate dehydrogenase and universe stress protein genes. The gain of the ESTs will lay an important foundation for cloning the full-length cDNA genes and exploring the mechanism of Puccinellia tenuiflora under saline and alkaline stress.5. In the suppressed subtractive hybridization libray of Puccinellia tenuiflora roots under NaCl stressed conditions, 68 ESTs were sequenced randomly, Blastx analysis showed that 40 ESTs were homologous (Blastx E-value-5) to the nucleic acid or protein sequences in database, the other ESTs had no homology or low homology (Blastx E-value-5) with the sequences in the database, which could be regarded as new genes.6. In the suppressed subtractive hybridization libray of Puccinellia tenuiflora roots under NaCl stressed conditions, several ESTs associated with salt resistance of plants were screened, such as MAPK, EF1A and CPI. The gain of the ESTs will lay an important foundation for cloning the full-length cDNA genes and exploring the mechanism of Puccinellia tenuiflora under salt stress.