The Screening And Analysis Of A Gain-function Of Abiotic Stress Resistance Mutant Marabidopsis Thaliana

The phytohormone ABA plays crucial roles in various physiological processes during the plant lifecycle. It responses to environmental stress condition such as drought, cold and high salt by regulating thecomplex metabolic process of cell. ABA also plays a very important role in the process of plantdevelopment including seminal storage proteins and lipids synthesis, enhancing seed drought resistance,regulating dormancy and germination process, plant regeneration, etc.The main material unsed in this study comes from the gain function of mannitol insensitive mutant inthe ultra-induced expression of estradiol. Genetic analysis shows that the mutant, which we named gar16,phenotype can be stable genetic respectively, and the gene was cloned by TAIL-PCR. The result of BLASTanalysis shows that T-DNA was inserted into the promoter region of GAR16. In Arabidopsis, GAR16encodes an iron ion binding monooxygenase, belonging to CYP450superfamily. RT-PCR analysis showsthat the expression level of GAR16has been obviously weakened in the mutant. But the expression levelsof GAR16was increased about several fold compared with the wild type in the condition of estradiolinduction. In further experiments, we found the mutant have a certain degree of resistance to NaCl, ABAstress after estradiol induced, that is over-expression lines have a higher cotyledons incidence rate than wildtype plant. However, no matter in normal condition or in the condition of ultra-induced expression ofestradiol,the mutant and wild type plant have no evident difference in germination rate under various stress.It suggests the gene work in the post-germinative growth. Through the root elongation experiment wefound the root growth of over-xepression lines was more serious suppressed than wild type plant in thecondition of ABA or hydrogen peroxide stress, suggesting that GAR16gene may be involved in the processof hydrogen peroxide response to ABA regulating Arabidopsis root development. RT-PCR analysis resultsshow that the over-expression of GAR16restrains the upgrade of ABI2which is an ABA deregulate factorunder ABA stress. It explains over-expression lines have an insensitive phenotype under ABA stress on thepost-germinative growth. The expression of GAR16can not induced by NaCl, ABA, mannitol, SA ordrought, which suggests that the gene may exist a certain function redundancy. GUS staining analysis ofGAR16gene showed that this gene express in various organizations, including roots, stems, leaves, flowers and siliqua, especially in cotyledons. Fluorescence co-localization result of GAR16::GFP fusion proteinsusing transient expression in protoplast of mesophyll cells showed GAR16enzyme protein localized inperoxisome.