Studies On Molecular Mechanisms Of DNA Mismatch Repair In Arabidopsis

Cross-breeding between the different genotypes or varieties of plant species is one of the most important methods for the crop breeding, and is also the main way to cultivate new varieties. A principal object of Cross-breeding is to integrate a variety of desirable traits from different species or varieties, and DNA homologous recombination during meiosis is essential in this process. Previous studies have shown that the DNA mismatch repair system (MMR) plays a vital role in DNA recombination during mitosis and meiosis. Therefore, in-depth study on plant DNA MMR mechanism will provide valuable information for the crop breeding. However, comparing to the related studies on mammals and prokaryotes, the research process on DNA MMR in plant cells is still far behind. To date, little is known about plant DNA MMR system. Researches concerning in molecular mechanisms of DNA MMR in plant cells have not been emerged, which might be obstacled by lacking of a suitable assessing tool to demonstrate it experimentally. Here, We establish the DNA MMR assessment research system and are able to attest the activity of DNA MMR in vivo and in vitro. Our results demonstrate that Arabidopsis cells possess the DNA MMR activity. The major findings are shown in below.1. Results from in vitro DNA MMR assay using nuclear extracts from Arabidopsis leaves showed that the DNA MMR activity of plant nuclear extracts is very low. The occurrence of double-strand breaks at the nick of DNA subtrate makes it difficult to assess the repaired mismatched DNA product. However, the suceeded assay was finally fulfilled after modifying the assessing methods.2. The T-T and G-Deletion mismatched DNA base pair was suscessfully incroprated into the CDS of LUC (luciferase) reporter gene, thus the mismatched DNA substrates were created and prepared for the assay. Through assessing these mismatched DNA substrate in Arabidopsis protoplasts, we successfully detected the repaied LUC activity in Arabidopsis cells.3. Using LMDA (Ligation-Mediated DNA Detection Assay), we were able to determine a trace of single base change from the DNA mixture. Results showed that the Arabidopsis nuclear extracts from protoplasts could repair the A-C mismatch DNA substrate in vitro. Further studies revealed that AtMsh2plays important role in MMR activity, and results demonstrated that this in vitro MMR prodcess is also nick-directed.In addition, we established a "quick-dirty assay" for screening potential molecular targets in studying gene expressions. A transformation system using PCR amplified DNA fragment (PCR-fragment) was successfully established. Thus, the PCR-fragment based transient expression system (PCR-TES) was applied for determining the potential signaling components, such as CDPKs and AITR1, which might be involved in the ABA signal transduction pathway. The major findings are highlighted in below.1. PCR-fragments can be transformed into plant cells and expressed successfully. The expression properties of PCR-fragments in plant cells are similar to those of expressing plasmids. Results showed that PCR-TES is suitable for transient expression assay in protoplasts, and the transformation efficiency can be up to70%; which might produce higher transformation yields when comparing to transformations using plasmids. Moreover, results indicated that PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.2. Undertaken PCR-TES, we screened potential candidates of CDPK family members which might be involved in the ABA signaling, and determined that phosphorylation of ABF2by CPK4could be mediated by ABA-induced PYR1and ABU, demonstrating a crucial role of CDPKs in the ABA signal transduction.3. Primary analyses on AITR1in response to ABA have been carried out. AITR1belongs to family of CHC4H3-type zinc finger transcription factors. The nuclear localized AITR1 could bind to A/T-rich c/s-element and functions as an transcription repressor through its C terminal. The expression of AITR1could induced by ABA.The proAITR1-GUS strongly expressed in the meristem and developing organs. Overexpression of AITR1results in hypersensitive phenotype to ABA during seed germination, indicating that AITR1may play a positive regulatory role in the ABA signal transduction.