Establishment Of The Chloroplast Genetic Transformation System In Rice By Using Hygromycin B As Selection Pressure

Previous studies showed that DNA-containing cellular compartments in plant cells were the nuclei and chloroplasts. Although nuclear genetic transformation plays an important role in plant breeding, a series of disadvantages of this conventional method are appeared, including the low expression level of foreign genes, gene silencing, position effects, pollen dispersal, etc. Chloroplasts that have the function of photosynthesis in higher plants are ideal hosts for expression of foreign genes. Foreign genes integration into chloroplast genome occurs via homologous recombination between flanking sequences of target genes and homologous region in genomes, thus advoiding position effect and gene silencing. Moreover, chloroplast transformation provides a number of unique advantages that nuclear transformation could not match, such as multi-gene engineering in a single transformation event, high level expression and maternal inheritance of transgenes. To date, chloroplast transformation technology has become a valuable tool for the creation and cultivation of plants, especially for dicotyledons, and chloroplasts offer a viable alternative to conventional bioreactor systems. However, this emerging technology has not been established effectively in rice.Rice (Oryza sativa.L) is one of main staple foods in the world. If chloroplast transformation technology could be established effectively in rice and applied for rice breeding, it will be helpful to sustainable development of agriculture. This study is to establish the rice chloroplast transformation system, and the main contents can be stated as follows:1. Rice callus was found to be very sensitive to the hygromycin, chloramphenicol and herbicide PPT through sensitivity test. Therefore, these three agents could be empolyed for seletion, and the hpt gene, CAT gene and the bar gene are feasible selection marker genes.2. Two rice chloroplast genes, trn1and trnA, were selected as homologous recombination fragments. Promoter Prrn and terminator TpbsA both from tobacco chloroplast genome were chosen to regulate gene expression. The EGFP genes with a series of different leader sequences were used as reporter genes. All of these elments were constructed into a series of rice chloroplast fluorescence expression vectors. We examined the effects of different leader sequences on expression of EGFP gene in E.coli. The results showed that Prrn promoter alone can not facilitate fluorescent expression effectively, but the highest level expression of EGFP gene was achieved under Prrn promoter with T7g10leader sequence. The sequential order of fluorescence intensity was:Prm+T7g10Leader> Prrn+r bcL Leader> Prrn+atpB Leader> Prrn+psbA Leader>Prrn+RBS> Prrn. Then the vector pUIPEBA7including T7g10SD sequence was bombarded into Onion epidermal and Dunaliella salina cells, the transient fluorescence expression of EGFP gene confirming function reliability of the vector and applicability of transformation system.3. After then, hpt gene, CAT gene and bar gene were constructed into the vector pUIPEBA7, and named pCTE04, pCTE05, pCTE06, respectively. These three vectors were transformed into calli and immature embryos from four rice varieties by particle bombardment. PCR and Southern blot analysis of To plants demonstrated that hpt and EGFP genes have been integrated in chloroplast genome of TP309variety. RT-PCR results of T\plants demonstrated expression of the two genes at transcription level. And green fluorescence was detected in chloroplasts of T1transformants by confocal microscopy. The above results confirmed that the chloroplast transformation system has been established in rice by using hygromycin as selection pressure.4. Due to the fact that the transformation efficiency was low in rice chloroplasts, we investigated the impact of Co60γ irradiation on transformation efficiency of particle bombardment through nuclear transformation in rice. The result indicated that the transformation efficiency of callus irradiated by30Gy was two times higher than that of callus without irradiation. The above results pave the way for further improvement in chloroplast genetic transformation system of rice.