Identification Of A Novel Selectable Marker Gene DsdA And Functional Analysis Of OsGLK1 Transcription Factor

Via homologous recombination, chloroplast transformation technology can achieve specific genome editing of chloroplasts. Meanwhile, chloroplast genome is characterized by high copy number, and the polycistronic expression in plastid can organize multiple transgenes together for effective expression. More importantly, in most angiosperm plant species, plastid genes are maternally inherited. Therefore, chloroplast transformation can effectively overcome the deficiencies of traditional nuclear transformation, such as pollen drift, gene silencing, position effect and difficulties for multiple gene co-transformation and so on. Chloroplast transformation has become an important technology in plant breeding. To date, plastid transformation has been widely applied in dicots, however, relatively fewer studies of chloroplast transformation have been reported in monocot. Inefficient transformant selection is considered to be one of the important obstacles to the implementation of plastid transformation in monocot. Therefore, the discovery of more candidate genes with screening potential is very critical to the development of chloroplast transformation in monocot.Our study proves the feasibility of using dsdA(encoding D-serine ammonia lyase) as a selectable marker in both tobacco chloroplast and rice nuclear transformation systems. And it proposed that dsdA gene can be used as an effective candidate gene for the rice chloroplast transformation. The results are summarized as follows:1. By using biolistic bombardment, dsdA gene was site-specifically integrated into the tobacco plastid genome and displayed a high level of expression. The maternal inheritance of the homoplastomic plants were demonstrated by germination of the seeds from T1 crosses.2. The effective screening concentrations of D-Ser for seed germination, callus regeneration and foliar spray were 10 mM, 30 mM and 75 mM, respectively. Three assays proved the feasibility of using dsdA gene as a selectable marker in the chloroplast transformation of tobacco.3. Calluses from the identified homozygous transgenic rice lines with high-level expression showed significant tolerance to D-Ser(up to 75 mM), whereas the wild-type calluses did not regenerate and turned necrotic. This assay illustrated that dsdA gene can be potentially applied to the nuclear transformation of rice.4. The statistical analysis of chlorophyll content, plant growth and root development with the dsdA transgenic lines and the wild-type indicated that the expression of the dsdA in rice had no side effects to agronomic characteristics.5. Seed germination of five rice varieties were shown to be strongly inhibited at 10 mM D-Ser and fully inhibited at 15 mM D-Ser, which indicated that D-Ser can be widely used as a non-antibiotic selective agent in different rice varieties.Proplastids in callus and chloroplasts in green tissues are at different stages of plastid development and differentiation, and have different gene expression regulation patterns. The problem of callus differentiation and development is another important obstacle to the implementation of plastid transformation in rice. It has been reported that OsGLK1 regulates chloroplast development, and ectopic overexpression OsGLK1 induces chloroplast development in non-green rice cells. To further illustrated the molecular mechanism of transcription factor OsGLK1 in regulating rice chloroplast development, in this study a series of research vectors have been constructed and transgenic materials were identified, which will thus help to the establish the monocot chloroplast transformation system. The results are summarized as follows:1. The prokaryotic expression vector has been constructed, IPTG inducing assay showed that the GLK1 fragments can be efficiently expressed in E.coli, which will be helpful for protein purification and antibody preparation.2. The 35S::OsGLK1:GFP vector has been constructed and introduced into cultivar ZH11, the homozygous transgenic lines with single-copy insertion were identified from the 58 positive transformants in order to explore OsGLK1 transcription factor binding sites by Ch IP-seq.3. The agronomic traits investigation in over-expression and RNAi transformants showed no significant differences compared with the wild-type, which indicated that overexpression OsGLK1 or interference OsGLK1 expression alone didn’t affect the plant growth and development.