Integration Of Microarray And Genome Analysis In Alkaline-stressed Glycine Soja

The western Songnen Plain of China, which has 3.73million ha of sodic land, is one of the three major contiguous sodic soil regions in the world. Both sodium and bicarbonate ion pose a toxicity hazard. High pH leads a change of soil physical properties. Through genetic engineering technology of alkali - resistant capability of modified crop is urgently needed. Mining the alkaline resistant genes and understanding its molecular basis is the premise to breed crop plants with enhanced tolerance to high saline-alkaline. The wild soybean (Glycine soja) line used in this study is a ideal material to integrate the functional genomics of alkaline tolerance, which can germinate and set seed in the sodic soil at pH9.02.By Affymetrix? Soybean GeneChip?, we investigated transcriptional profiling in Glycine soja grew in saline and alkaline soil in Baicheng city, Jilin Province, stress response genes were screened. Integrating genome data, genes regulated by alkaline stress were identified and clustered so as to show the co-expression characteristics of stress response genes. Gene function category, location in chromosome and upstream motif were analyzed to investigate alkaline stress response mechanism of plants. This will provide the theoratical basis to elucidate the molecular mechanisms of plant stress response and provide functional genes for plant saline and alkaline tolerant molecular breeding. The main results were summarized as following.1. Alkaline stress-responsive genes expression profile in wild soybean50mmol/L NaHCO3 was used to simulate the alkaline stress. Wild soybean G07256 was stressed and RNA was isolated. The transcriptome profiling was established in wild soybean using Affymetrix soybean gene chip. This profiling includes 7 time points (0, 0.5h, 1h, 3h, 6h, 12h, 24h) and two organs (root and leaf). There were 23741 transcripts in roots and 22200 transcripts in leaves.The Genechip results were tested using real-time PCR. 14 candidate genes'transcription changes accorded with the genechip results. This results showed that the genechip results are reliable.2. Screening of alkaline stress response genesRankProd statistical method was used to identify the significance of differences between the gene expression levels of two time points (p < 0.01, pfp < 0.15). The gene whose expression level changed significantly between two time points was regarded as alkaline stress response gene. 2493 and 2310 stress response transcripts were found in root and leaf separately. Function enrichment test suggested that the differentially expressed genes were related to metabolism processes in roots as well as metabolism and membrane translocation in leaves. 28 key genes were identified in the regulatory network.The number of alkaline response genes shown a peak at 6h. Genes involved in cell structure, desease and defense, secondary metabolism and transcription (WRKY family was enriched in transcript factor) were early responded in root. Genes involved in cell structure, proterin synthesis, energy, and secondary metabolism were early responded in leaf. 28 key genes were identified in gene regulation network.3. Characteristic of wild soybean co-expressed genes regulated by alkaline stressClustering analysis of short time series gene expression data was conducted to reveal the co-expression characteristics of alkaline stress response genes. The co-expression characteristics (p < 0.001) of 11 classes of genes and 10 classes of genes were found in roots and in leaves seperately. Co-expressed genes showed co-functions and common motifs. Co-expression characteristics and its distribution in chromosome have no correlation. 21 motifs were found in up-stream of co-expression genes. 12 motifs were known cis- elements.4. Identification of alkaline stress response miRNA38 probe groups that can represent miRNA precursor were found by BLASTn miRNA precursor, probe groups and soybean gene sequences. 11 miRNAs expressed differentially in roots and leaves, seperately. The report about 7 miRNAs was not found, these miRNAs was regarded as new stress response miRNAs. By predicting miRNA target genes, transcriptional factors and kinase were found being regulated by miRNA.5. Screening of alkaline tolerant candidate genesThrough correlation analysis of alkaline stress response gene and molecular marker, a unknown gene and a gene with serine-threonine protein kinase activity. Two salt tolerant marker gene, 435 unkown genes, 9 soybean or wild soybean specific genes were screened to be candidate genes for the further crops'stress tolerant research according to expression pattern, function notation, domain of stress response genes.