Studies Of Two Arabidopsis Activation Tagging Mutants, Asr1 And Dai1, Related To Plant Defense Response And Development

Plant development is closely linked to its environment, and among the various environmental factors, stress is of most importance. Stress is composed of biotic stress and abiotic stress, the former means the damage caused by microbial pathogens, insects and weeds, and the latter indicates the adverse effects on plants by high-salinity, drought, flood, low temperature and high temperature, etc., and both are the main reason for the yield loss of major crops in the world. Therefore, understanding the molecular mechanism of plant adaptability to biotic and abiotic stress is not only theoretically meaningful, but also economically important. Activation tagging is a technique to acquire gain of function mutants based on gain of function mutation, and it has particular advantage in isolating and studying genes from gene family, genes controlling hard-to-test traits, and genes involved in QTL, etc. Moreover, activation tagging mutants usually are dominant, so their phenotypes can be observed in the T1 generation, which is very helpful to screen the mutants on purpose. As a result, it is feasible and promising to construct the model plant, arabidopsis, mutants pool, screen mutants related to stress, clone genes and study their functions.The main work of this dissertation is constructing Arabidopsis gain of function mutants pool, screening mutants related to plant defense response, cloning the genes and studying their functions. The main results are as following:(1) Arabidopsis in planta transformation were made by Agrobacterium tumefaciens carrying the activation tagging vector pSKI015 with herbicide-resistant Bar gene as a selective marker. About 12 000 independent transformant lines were acquired by the above method, from which two mutants related to plant defense response and development, asr1 (auxin and .salicylic acid responsive 1.) and dai1 (brought tolerant and obscisic acid insensitive 1) were isolated.(2) ASR1 gene was cloned by plasmid rescue, and transgenic confirmation was conducted in the wild type Col-0 background. ASR1 is a gene from early auxin-responsive GH3 gene family, and the protein encoded by this gene is overexpressed in the mutant, which results in the following morphological phenotypes including dwarf, epinastic rosette leaves, and restrained lateral root formation, etc. Meanwhile, ASR1 may modify the plant defense signaling molecule salicylic acid in vivo, and therefore asr1 mutant is more susceptible to the infection from the pathogen Pseudomonas syringae.(3) Protokaryotic expression vector of ASR1 was constructed and then effectively expressed in E. coli. The corresponding multiple-clone antibody of ASR1 was acquired. Western blotting result indicates that ASR1gene is overexpressed both in transcription and in translation with the higher expression level in homozygotes than in heterozygotes. In addition, the expression vector of ASR1 and GFP fusion protein, pCAM-ASRl-GFP, was constructed, and the ASR1: GFP fusion protein was located in the cell wall shown by onion epidermal transient expression system.(4) Preliminary analysis of stress-related phenotypes of dai1 indicates that the dai1 mutant is more resistant to osmotic stress caused by high-salinity or drought than the wild type Shahdara, and dail is insensitive to the plant stress hormone abscisic acid. The DAI1 gene is located on the arabidopsis No.2 chromosome by TAIL-PCR.