Functional Analysis And Cloning Of OsPRX2 For Rice (Oryzae Sativa)

In normal circumstances,right amount of reactive oxygen is the signal molecules to regulate and control the cell metabolism,but excessive reactive oxygen will resulting in oxidative damage through destroy the dynamic balance.There is a complex antioxidant network system in plant cells and the PRX in plants is considered to be associated with stress conditions;however,to date,there is no relevant research focusing on 2-CysPrx in rice.In this study,candidate OsPRX2 gene was successfully cloned and identified as BAS1 gene in rice through a series of bioinformatics analysis.Subsequently,OsPRX2 was associated with K-deficiency by analyzing the transgenic plants and through the yeast two-hybrid technique 8 potential interactors has been found.During the experiment process,it was found that the calli of indica rice is more recalcitrant to subculture than that of japonica rice and the immature embryos is more efficient in calli subculture than mature seeds.However,the underlying biochemical mechanisms remain obscure,RNA-Seq technology was used to perform the comparative transcriptome profiling to analyze the responsible mechanism in this study.The main conclusions are as follows:1.The full-length cDNA sequences of OsPRX2 were cloned using smarter race,and through a series of bioinformatics the candidate gene was identified as BAS1 gene in rice.2.The results of qRT-PCR and GUS staining showed that OsPRX2 was mainly expressed in the leaves;Subcellular localization indicated that OsPRX2 was located in the chloroplast.3.The anatomical analysis showed there are significant differences in the degree of stomatal opening,the size of outer cells of vascular bundle,the size of the bundle sheath extension,the number of xylem cells and the number of bubbles between the leaves of the OsPRX2-CRISPR knockdown plants and the OsPRX2 overexpression plants.4.Observed the phenotypic of the transgenic plants after a series of stress treatment,and combined with qRT-PCR and Western blot analysis,the OsPRX2 was found to be associated with K-deficiency.By analyzed the expression level of nine antioxidant related-genes and their enzyme activity system in the transgenic plants after K-deficiency stress,OsPRX2 have significant effect on the antioxidant network system.5.Yeast two-hybrid assay found 8 potential interactors,which laid the foundation for subsequent research.6.Through the statistical analysis of the callus of mature embryos induced by 93-11 and Nipponbare in 60 kinds of culture medium,MS plus 3.0 mg/L 2,4-D and 3%sucrose was identified as the optimal basic medium for simultaneous inducing the calli of 93-11 and Nipponbare,the subculture callus of the immature embryos and mature seeds of 93-11 and Nipponbare from this medium showed remarkable morphological differences through scanning electron microscopy and paraffin sections.7.Twelve RNA-Seq libraries from three biological duplicates of the callus of mature embryos induced by 93-11 and Nipponbare,were sequenced with the Illumina Hiseq2000 technology and shown that plant hormone signal transduction and hormone biosynthetic pathways are key factors explaining why indica rice is more insensitive to in vitro culture conditions than japonica rice,and why the quantity and quality of calli induced from immature embryos is higher than those of calli induced from mature seeds.Meanwhile,analyzed the expression levels of 67 hormone-specific genes based on FPKM values,and combined with the endogenous hormones determination,found that the molecular mechanism is completely different,like SA and JA play a major role in the subculture difference between the mature embryos of Nipponbare and 93-11,ABA、IAA、ZA and JA-Il are the main reason result in the subculture difference between immature embryos of Nipponbare and 93-11.