Library Construction Of Differentially Expressed Genes Of C. Bungeana On Freezing Stress And Study Of Arabidopsis Cold Sensitive Mutant C-l 2-41

Agricultural productivity is decreasing due to the effect of various environmental problems. In particular, cold stress adversely affects the growth and development of plants, and significantly constraints the spatial distribution of plants and agricultural productivity. To solve this problem, we use a combination of genetic, biochemical, genomic and proteomic approaches to analyze various levels of gene regulation so as to reveal the mechanisms of stress signaling and stress tolerance. Our long-term goals are to elucidate the signaling pathways used by plants in responding to cold stresses and to identify the key genes for modifying the responses of crops to cold stress which ultimately will lead to major contributions to agriculture. Chorispora bungeana (Chorispora bungeana, Uniprot Taxonomy No:238895) is a representative alpine subnival perennial herb in the Brassicaceae family which can survive chilling, even freezing temperature and frequent temperature fluctuations. Therefore it is a valuable material to investigate freezing tolerance. In this project, on the one hand, the differentially expressed genes of Chorispora bungeana under non-acclimated freezing stress were studied, and the dominant genes on freezing tolerance were identified, trying to reveal the mechanisms of freezing stress tolerance from the point of view of the transcriptomics. On the other hand, Map-based clone was carried out on the C-L 2-41mutant, which seeks to find the upstream genes regulating CBF2 (C-rerat Binding Factor 2), so that to improve the regulatory networks of Arabidopsis thaliana under cold stress. Below are the results of this project:1. The deferentially expressed cDNA library of Chorispora bungeana seedlings under freezing stress was constructed. We used a combination of cDNA array, real-time PCR and bioinformatics analysis to find 85 cold responsive genes in total. Of which 52 genes were up-regulated while 33 genes were down-regulated. These genes were sorted into 7 broad functional categories including stress/hormone stimulus response, photosynthesis, transcription factors, protein metabolism, ribosomal proteins, others and unknown genes. This suggested that a very complex series of molecular mechanisms are involved in the response to cold stress in Chorispora Bungeana.2. Functional analysis suggested that FIB and ACTIN cross-linking protein may act as the cold sensor. And DREB2A signal pathway combined with ABA signal pathway may play a critical role in the regulation of the COR genes (cold responsive genes). Meanwhile, the downstream cold responsive genes such as KIN2(COR6.6), STZ(ZAT10), GR-RBP8(Glycine-Rich RNA Binding Protein 8), etc., also played a very important role in cold tolerance.3. CbCINP1 (Chorispora bungeana Cold Induced Novel Protein 1) encodes a novel cold induced protein of Chorispora bungeana, which was identified from the deferentially expressed cDNA library under cold stress and cloned by RACE. It has 1176 bp nucleotides encoding 391 amino acids. Phylogenetic relationship analysis shows that it has a high homology with Arabidopsis unknown protein DUF677. Furthermore, a trans-membrane domain was predicted by motif analysis. Confocal microscopy shows that it actually localized at the nucleus and the plasma which is in accordance with the prediction. The transgenic Arabidopsis under over-expression of CINP1 shows increased cold tolerance. Hence, CINP1 may play a role in signal transduction and stabilization of plasma membrane under cold stress.4. Arabidopsis mutant C-L 2-41 can induce the expression of CBF2. Map-based clone reveals that there is a single base mutation of FRY2/CPL1 gene, and wild type FRY2/CPL1 gene can recover C-L 2-41 by genetic complementation. Except cold stress, it is also sensitive to drought and heat stress. These results laid the foundation for the function study of FRY2/CPL1.In summary, the deferentially expressed cDNA library of Chorispora bungeana under freezing stress was constructed and the functions of the deferentially expressed genes were analyzed. A novel cold responsive gene CbCINPl was cloned and studied. Which indicates a complex mechanism of cold tolerance exists in Chorispora bungeana. Meanwhile, Arabidopsis mutant C-L 2-41 was identified to be an allelic mutant of FRY2/CPL1 gene, which is also sensitive to drought and heat stress. These results will contribute to the study of both Arabidopsis and C.bungeana cold responsive mechanisms.