Establishment Of Rape BnAtg8-Arabidopsis Transgenic Lines And Function Study Of Its Autophagy

Autophagy(AP), plays important roles in plant growth and development. When cell or organelle are damaged, autophagy functions to degrade them so that the absorbable nutrients are recycled, which is important for the maintenance of cell homeostasis, auto-immunization and response to stresses. Autophagy is also required in plant normal growth and development, for example the reconstruction of development pattern. The research on autophagy was first started in yeast and then on animals. Different from the research in yeast and animals, the study of Autophagy in plant has just started and many questions required to be answered. In this study, we introduced Bn Atg8(from Brassica Napus L.) recombined with GFP(Green Fluorescent Protein) into Arabidopsis thaliana genome using agrobacterium-mediated floral-dip method abel and obtained three Bn Atg8-Arabidopsis transgenic lines. After that, the differences in morphology, physiology, biochemistry and molecular biology between the wild type, the transgenic plant, two autophagy mutants(atg5, atg7) and their wild type(WT) were analyzed and compared through fluorescence assay, MDC staining, RT-PCR to explore the role of autophagy in plant growth and development under normal or stress conditions. Main results were as follows:(1) Three Bn Atg8–Arabidopsis transgenic lines were obtained via PCR identification. Two of them expressed both GFP and Bn Atg8(named GFP-Bn Atg8 transgenic line) and the other one expressed BnAtg8 only(named BnAtg8 transgenic line).(2) RT-PCR analysis showed that that the expression level in the 60d-leaves was significantly higher than that in the 20d- and 40 day leaves, and the expression level in the 40d-leaves was significantly higher than that in the 20 day leaves(60d >40d >20d). Comparing the expression level of the BnAtg8 in the leaves of 20, 40 and 60 days old plant as well as the leaves, stems and siliques of 50 days old plant using semi quantitative RT-PCR showed.(3) Under laser confocal microscope, it was found that green fluorescence was distributed in all parts of the root tips of the Bn Atg8–Arabidopsis transgenic lines, and the size of the fluorescence intensity in different disectons was meristematic zone > elongation zone > maturation zone > root cap in turn. Furthermore, the GFP fluorescence intensity in the tetrad was found higher than that in uninucleate microspore during pollen development.(4) The fluorescence intensity in the transgenic plant was found higher than that in wild type when MDC staining was performed on the root tips.(5) The phenotype analysis showed that the leaf area as well as the lengths of primary root and hypocotyl of the transgenic plants were more than that of wild type, and that of wild type was more than that of autophagic mutant(atg5, atg7). In bolting time, wild type is later than transgenic lines, and transgenic lines is later than atg5 and atg7. Compared with the wild type, in plant height and seed yield. transgenic lines had no obvious change, atg5 and atg7 showed a decreases(6) Physiological analysis indicated that under salt stress, seeds germination rate of wild type was higher that of transgenic lines and atg5/atg7, and that of transgenic lines was higher than that of atg5/atg7.The results showed that the expression of Bn Atg8 has obvious effect on growth and development, tolerance to stress and autophagy activity in arabidopsis, which suggested that that autophagy plays important role in the regulation of plant growth and development. The results showed autophagy has a positive side and negative side to the growth and development of plants, only the right level of autophagy was conducive to plants.