Studies On Chloroplast Genetic Engineering And Plant Organelle Genomes Evolution

In plant cells, in addition to the nucleus with the genetic material DNA, two kinds of organelles, chloroplast and mitochondria also have a semi-autonomous self-replicable and heritable genome. With the advances in DNA sequencing technology, more and more plant nuclear genome, chloroplast genome and mitochondrial genome sequencing projects have been completed. Previous studies have shown that the genome structure and gene information in chloroplasts and mitochondria could provide important reference on the origin of plant species, evolution and the genetic relationship among different species. Meanwhile, the chloropolast genetic engineering has become a novel developmental field of plant genetic engineering as its advantages over nuclear transformation in the area of genetic improvement of crops and expression of therapeutic proteins. The structure and sequences analysis of chloroplast genome is the foundation of chloroplast genetic engineering. To some extent, this kind of research can not only provide experimental evidence for the mechanism of coordination gene expression between nuclear and organellar genomes, but helpful for understanding of the plant species origin and gene transfer events in the evolution process as well.Chloroplast genetic engineering technology offers a novel method to carry out sit-specific integration, highly effective gene expression, breeding for various resistant crops and creating bioreactor for biosynthesis of certain valuble proteins. In this study, two wheat chloroplast genes, atpB and rbcL, were chosen as homologous recombination fragment, with the nptII and gfp gene as selection and marker genes to construct site-specific integration vector for wheat chloroplast transformation. The fertile, homoloplasmic regeneration wheat plants were obtained by bombarding the immature inflorescences and scutella from immature embryos. PCR and Southern blot analysis of T0 plants demonstrated that gfp gene has successfully been integrated into the wheat chloroplast genome. And the expression of green fluorescent protein (GFP) was confirmed by confocal microscopy in the chloroplast of the T1 seedlings. The above results demonstrated that a stable genetic transformation system of wheat chloroplasts has been established.Using plant cell as a bioreactor to produce pharmaceutical proteins or vaccines, also has emerged as a hot research. In this research part, human interferonα- 2b (HuIFNα-2b) gene was transformed into the nuclear genome of tobacco leaves through the leaf disc dip method and the molecular analysis confirmed that the regenerated tobacco plants could express the active HuIFNα-2b protein. Tobacco chloroplast transformation vector containing the HuIFNα-2b gene was also successfully constracted and it would be applied to potentially express the HuIFNα-2b protein in tobacco chloroplasts.In this study, comprehensive statistics of nuclear and organelles genome information has been carried out among the sequenced species of Algae, Bryophytes, Pteridophytes, Gymnosperm and Angiosperm. The variety trend of the size of genome, gene-coding region and non-coding region during their evolutionary course was compared. In 174 species of which, their chloroplast genome sequenced, 26 species of nuclear genome sequencing were also completed. Aligning the various species of chloroplast genome and its nuclear genome and it was found that the higher of the species, the higher homology between the chloroplast DNA and its nuclear genome are. There are five species of Gymnosperm group whose homology were up to 100%. The similar analysis of 15 species of both sequenced mitochondrial DNA and nuclear genome showed the same trend with the chloroplast genome alignment. But the increasing extent in mitochondrion was not so obvious and the highest proportion was only 82.6%.The conserved genes in chloroplast genomes of algae, dicotyledons and monocotyledons species were statistically counted. The conserved genes in Algae but not present in Monocotyledons were compared with the nuclear genomes of Arabidopsis thaliana and Oryza sativa respectively, the majority genes of genetic system were successfully transferred, and considerable genes of the biosynthesis and photosynthetic system have lost. Statistics of the proportion and copy number of chloroplast and mitochondrial genes in their nuclear genome of Arabidopsis and Oryza sativa were conducted. On the basis of comprehensive analysis of these results obtained, we proposed the hypothesis that organellar genes have been transferred to their nuclear genome via the process of insertion in the non-coding region of the nuclear genome, followed by obtaining the function in certain conditions and then the corresponding genes in the organellar genome disappeared. By this way of evolution, the nucleus becomes the central supremacy in the cell gradually.