Mechanism Of ROS-mediated Difference In Regeneration Potential Of Citrus Protoplasts

Protoplasts is the bare cell of the cell which’s wall is removed. Research showed that reactive oxygen species (ROS) in protoplast regeneration had two sides. The right amount of ROS was promoted to protoplast regeneration, but the excessive of ROS would be inhibited to protoplast regeneration. To date, the physiological and molecular mechanisms of reactive oxygen species in the Citrus protoplast isolation and culture process is unclear. Based on this, this paper used citrus callus and leaves as the starting material, analysised the dynamic changes of reactive oxygen species (ROS) in protoplast isolation and culture of the Citrus reticulata Blanco by fluorescent markers. Using the data from the published orange genome,we identified the members of the family of NADPH oxidase gene and analyzed their expression in the process of ponkan protoplast during isolation and culture, in order to select specific NADPH oxidase genes of ponkan protoplast during isolation and culture, cloned a specific gene and analysised its bioinformatics.We construct the over expression vectors of Crrboh10 and introduced it into Agrobacterium tumefaciens EHA105 and preserved,and completed the transformed callus. We analyzed the transgenic callus of the soluble protein content,and the dynamic changes of the level of H2O2, in order to understand the role of CrrbohlO in Citrus callus.1. This study analysised the dynamic changes of reactive oxygen species (ROS) in protoplast isolation and culture of the Citrus reticulata Blanco by fluorescent markers, and used with active oxygen inhibitors to study the sources of reactive oxygen species. The results showed that ROS distributed in the cytoplasm and cell membrane of protoplasts. Compared with mesophyll protoplasts, the level of ROS in callus protoplasts was significantly lower during isolation, but during culture it became significantly higher.2. A decrease in the level of ROS during isolation and culture of callus protoplasts was observed after DPI or NaN3 treatment. Our findings confirm the crucial role of ROS in protoplast regeneration.3. Using bioinformatics methods in citrus genome database to screen 13 citrus NADPH oxidase gene, and analyze these genes in ponkan expression in the process of somatic embryo induction. Results showed that the expression of 9 genes were detected and the trend of the expression has obvious differences in the stages (Crrbohl、Crrboh2、Crrboh5-6、Crrboh8-9 and Crrboh10-12); No detectable expression of the gene is Crrboh3、Crrboh4、Crrboh7、Crrbohl3.4. We cloned citrus CrrbohlO gene, and analysised its expression. Its sequence length is 2812bp, including a coding 916 amino acid and 2748 bp open reading frame. The protein molecular weight of this gene is 103.51 kD, and isoelectric point is 9.11; It has 99.89 percent of similarity with orange. Analysis of CrrbohlO gene expression in citrus tissue found were expressed in roots, stems, leaves, callus, expression descending order callus, leaves, roots, stems, using real-time quantitative PCR.5. We successfully constructed the gene’s over expression vector for pSN1301-Crrboh10. Then CrrbohlO introduced into Agrobacterium tumefaciens EHA105 and preserved,and completed the transformed callus.6. We researched the physiological and biochemical changes on citrus transgenic callus of soluble protein content and H2O2 content, where in the transgenic callus soluble protein content was up to 0.543 g/L, rather than the non-transgenic callus was 0.457 g/L; transgenic callus H2O2 content was 11.24 mmol/gprot, non-transgenic callus H2O2 content was 4.26 mmol/ gprot.