Study Of New Non-antibiotic Based Marker Systems And Plastid RNA Editing In Rice

Plastid transformation technology has unique advantages and broad application prospects. Unfortunately, so far, plastid transformation technology in rice has not been established. For the reasons of lack of an effective screening system and the limited of optional selection marker in the nature, it has become one of the important bottleneck in the development of rice plastid transformation. The screening system of dsdA (dao1) /D-amino acid has various advantages, as in this non-antibiotic based selection system, D-amino acid is cheap and widely available, especially relatively non-toxic to animals and microbes. In this study two new selectable marker gene dsdA and daol which come from Escherichia coli and Schizosaccharomyces, respectively, were synthesized by codon-optimization, and then introduced into japonica rice cultivar zhonghua 11 (ZH11) and tobacco Nicotiana Tabacum cv. Petit Havana through agrobacterium mediated gene transformation and the particle gun-mediated biolistic, respectively. Transgenic plants were identified by using molecular biology and genetic analysis methods, which allow us to evaluate the feasibility of dsdA ／ daol as new selection marker genes. Furthermore, this work will lay the foundation for setting up a reliable rice plastid transformation system. The main results are as follows:1、Chloroplast transformation system of tobacco was established successfully in our laboratory, homogenous transgenic plants were obtained.2、The transformation vector of pZF75-dsdA, pZF75-daol were constructed and introduced into tobacco leaves by bombardment. Then through primary,1st generation and 2nd regeneration selection, homoplastomic plants are expected to obtain later on.3、By testing the optimum screening concentration of D-amino acid for tobacco leaf tissues, it is shown that the optimum screening concentration of D-Ala is 5 mM, while the optimum screening concentration of D-Ser is 30 mM.4、The construct with 2 selectable marker genes (dsdA and daol) were introduced into japonica rice cultivar ZH11 through agrobacterium mediated gene transformation. Southern hybridization of the DNA transformants showed,7 single-copy plants with dsdA gene and 6 single-copy plants with daol gene were obtained. By expression levels detection of the single-copy transformants and the germination texts of To generation seeds, the results showed:for daol gene, there were two single-copy gene transformants (numbered as 6 and 7), which showed high expression levels and consistent with seed germination testing on D-Ala. For dsdA gene, in all single-copy transformants, the expression levels of dsdA were relative low, seed germination testing showed the resistance to D-Ser.RNA editing is the phenomenon of nucleotide insertion, deletion or replacement during RNA maturation, such that the RNA sequence does not match that of its DNA template, and a variety of alterations of RNA primary sequence were arisen from. In higher plant, RNA editing mainly occurs in chloroplast and mitochondria. It has been shown that RNA editing efficiency is not only corresponds to the development stages and, also varied at different editing sites. A small differences in editing efficiency can have significant functional consequences. Therefore, it is an important way of post-transcriptional modification process, and is closely related with the plant growth and development. Compared with the large-scale functional genomics and systems for expression profiling of growth period of rice, research on the expression and regulation of the plastid genome is lagging behind.In this study, we collected 19 organs/tissues cover the whole growth period of japonica rice cultivar ZH11. According to previous reports of RNA editing sites, by RT-PCR and cDNA sequencing, we analyzed the dynamic characteristics of rice plastid RNA editing. This work will provide enormous data for understanding the regulation of rice chloroplast gene expression, thus will help to identify the RNA editing cis-acting elements and trans-acting factors, as well as the rice plastid RNA editing regulatory mechanism basis. The main results were obtained as follows:1、We compared the rice plastid RNA editing sites with maize and tobacco, the results showed that, the nearer the phylogenetic relationships of species, the more are the same editing sites.2、We analyzed 19 materials cover the whole growth period, resulting a total of 456 RNA editing sites, and found RNA editing showed site-specificity.3、Using direct sequencing to calculated RNA editing efficiency in all tissues, the results showed, in different tissues editing efficiency showed dynamic changes, and RNA editing efficiency is not only corresponds to the development stages and, also varied at different editing sites. For example, we found radicle was an interesting organs, in which the editing efficiency of the most editing sites were lower than in other organs, especially in ndhB, ndhD and ndhF.4、The response of RNA editing efficiency to environmental factors (hormones, light) were analyzed. Results showed, effects of hormones on RNA editing efficiency is not obvious, while dark stress can affect editing efficiency:in different tissues the editing efficiency is not the same, and for different genes and different locis in gene, the efficiency of editing is varied5、We analyzed the editing efficiency of photosynthesis-related genes (atpA, ndhA, ndhB, ndhD, ndhF, ndhG, ycf3). In various tissues, ycf3 gene is fully edited in various tissues; the editing efficiency of atpA gene is relatively high (more than 80%), and the variation was slight. For NADH dehydrogenase genes, their editing efficiency in various tissues were dynamic changed. However, The relationship between photosynthesis and editing efficiency still need further verification.