Related Genes And Proteins Expression Profile Of Leymus Chinensis Shoots Under Saline-Alkaline Stress

Leymus chinensis (Trin.) Tzvel., a high quality plant for grazing, adapts to alkaline-sodic soil conditions and distributes widely in the Northeast of China. This species has developed molecular and physiological mechanisms during the course of evolution, which involve lots of anti-stress genes and proteins to adapt stress conditions. To identify and characterize the complexity of this adaptation, it is important to identify, genes by large-scale sequencing of cDNA clones and related proteins. The cDNA clones and related proteins are expected to enrich the stress related molecular resources and reveal the molecular mechanism. In this dissertation, studies were made on the stress-related genes and proteins expression profile of Leymus chinensis shoots under saline-alkaline stress.1. The construction of subtraetive library of Leymus chinesis under saline-alkaline stressUsing cDNAs from Leymus chinensis seedlings grown on the natural habitat of saline-alkaline soil was regarded as tester and cDNA from seedlings in normal growth as driver, suppression subtractive hybridization (SSH) was employed to construct cDNA subtracted library. Of the 1920 cDNA clones showed high quality sequences. The size of inserts was 300-1000 base pairs as it was determined by PCR. In the subtractive library, 552 clones were non- redundant, of which 339 had homology to the known transcripts, and 209 were unknown or unclassified. The remaining 4 ESTs had no homology in the databases searched. The suggested functions ESTs which have the annotation in the database include metabolism, energy, protein synthesis, protein storage, disease/defense, signal transduction, transporter and transpon. Sequence comparison of these clones using BlastN led to the identification of several putative saline-alkaline stress related genes, such as monodehydroascorbate reductase (MDAR), gamma-glutamylcysteine synthetase (γ-ECS), thioredoxin reductase (TR), Ferrtin (FER), Aquaporin (PIP), Dehydrin (DHN) and 14-3-3 protein. The suggested function of the ESTs involved active oxygen elimination, osmotic adjustment, ion transport and regulation of the genes. The putative ESTs will serve as useful resource to demonstrate the molecular basis of saline-alkaline stress related genes and for the further study of gene expression in Leymus chinensis. We consider that some of these genes and their metabolisms are possibly involved in the process of salt tolerance in Leymus chinensis.2. Gene expression profile of Leymus chinensis under saline-alkaline stress detected by cDNA microarrayThe inserts amplified from 552 sequences of Leymus chinensis SSH library clones were spotted on slides to get cDNA microarray. Saline-alkaline stress and non-stress mRNA were labeled with Cy3- or Cy5- fluorescence dye respectively during reverse transcription and then mixed as targets. Microarray hybridization was accomplished following the direction of user manual of 3DNA 900^?. The array was scanned by ScanArray 4000^? microarray scanner. For standardization, the Cy3 and Cy5 signal intensities were adjusted to fit external controls. The microarry was hybrid for three times. Using SAM software to analyze the significance of difference of the microarray genes, 198, 197, and 192 significant regulated genes were detected in the three hybridizations, respectively. Among the significant regulated genes there were 3, 53, 0 down-regulated genes, respectively. In function-known genes included monodehydro-ascorbate reductase, gamma-glutamylcysteine synthetase, ferrtin, aquaporin, dehydrin, thioredoxin reductas, and 14-3-3 proteins, all of which were known to be related with stress tolerance. There is no report for other up-regulated genes.3. Expression of up-regulated genes, MDAR, FER, PIP, and 14-3-3s investigated by Northern blotting.Firstly, to blast the sequence of MDAR, FER, PIP, 14-3-3s with the related genes of other plants in database to design the primer. The special sequences of the four genes were labeled by the dig- mark to get the probe. The seedlings of treated with 200 mmol/L Na₂CO₃ for 2, 6,12, 24, 48 and 72h were used for extraction of RNA for Northern blot. The expression of four genes was detected in treatments. MDAR, FER, PIP, and 14-3-3s transcriptome increased with the duration of the treatment. But the genes showed different expression patterns. In 14-3-3s expression pattern, the largest amount was observed in seedlings treated for 12h and 24h, and then the expression amount decreased. The results indicated the genes induced by saline-alkaline stress. Yet the function genes and regulation genes showed different expression patterns.4. Protein expression profile of Leymus chinensis with two-dimensional liquid chromatographyThere were four groups of Leymus chinensis plants seedlings collected from the natural habitat (treatmentâ… ), seedlings poured with 200 mmol/L Na₂CO₃ after 72h (treatmentâ…¡), seedlings in the natural habitat planted in the non-saline-alkaline soil (controlâ… ), and eight- week-old seedlings grown in the greenhouse without any stress (controlâ…¡). Total proteins were extracted by TCA-acetone precipitation method. Proteins were fractionated in the first dimension using chromatofocusing (CF). Subsequently the fractions with pH value between 8.5 and 4.0 collected after first dimension separation were further fractionated by high-performance reverse phase liquid chromatography (HPRP). The pI/UV map showed that the protein expression profiling of Leymus chinensis was generated by ProteoVue software. In the experiment, four protein express profiles were got from samples of each of treatmentsâ… ,â…¡, controlâ… , andâ…¡.The protein expression profile maps showed clear bands and good-qulity fraction. The contribution of the protein fraction focused on pI range 6.1-4.0 and 8.5-7.0. The amount of protein in pI 6.2-4.0 was higher than that in 8.5-7.2, indicating mostly acidic proteins. But the proteins with pI in 6.2-4.0 had the similar hydrophilicity. The expression amount of neutral and basic proteins in treatments was higher than that of the controls.To get the differential proteins under the saline-alkaline stress, protein expression profiles of controls and treatments were compared by Deltavue software. Proteins which the peak height of controls and treatments was above two was decided the differential ones.There were 90 differential proteins, in each of controlâ… and treatmentâ… , and 46 in each of controlâ…¡and treatmentâ…¡. In pI 8.5-6.4, there were 58 differential proteins in controlâ… and treatmentâ… . The higher amounts of differential proteins in control were 37, and that in treatment were 21. And in controlâ…¡and treatmentâ…¡there are 23 differential proteins. The higher amount of differential proteins in control were 12, and that in treatment were 11.The peak height ratio of mostly differential proteins were about two, with some observations showing higher than five. Further studies remain to be made on such as the identification of differential proteins. The differential expression protein is important in order to understand the mechanism under saline-alkaline stress.