Functional Analysis Of Rice Chloroplast-Located SOD

Rice (Oryza sativa L.) is an important food crops, play an important role in food production and security. Due to the frequency of extreme weather in recent years (the growing problem of frequent drought, temperature extremes), the yield and quality of rice under threat by abiotic stress factors. Coupled with the pressure of population growth and the ecological environment, traditional rice breeding met hods are difficult to meet people’s needs. Therefore, the modern biological technology to improve rice resistance to environmental stress is an important method to ensure food security. A large number of crops will produce reactive oxygen species subjected to various abiotic stress. Thus rice widespread secondary oxidative stress injury. If it not timely be cleared,the reactive oxygen species will directly damage cells, inhibition of growth.The yield loss mainly due to the environmental stress.SOD is the first line to defense against oxidative damage and directly determines the efficiency of reactive oxygen species scavenging.This attracts widespread attention on improved crops resistant molecules. There are a lot of successful examples using overexpression of SOD transgenic plants respond to temperature, salt, drought, resistance to oxidative stress. Chloroplast is the most vigorous aerobic metabolism of plant cells and also the main source of O2-’. The O2-’might have both damage and signal transduction dual role. Therefore,it plays a crucial role for plants to regulate the concentration of chloroplast O2-’ quickly and accurately. So, researching SOD located in the rice chloroplast, exploring SOD rice chloroplast active oxygen scavenging and signal transduction control mode, elucidating SOD molecular mechanism, ultimately this provides a theoretical basis to improve the resilience of rice molecular improvement.In this study, we cloned full-length cDNA sequence of seven rice SOD (OsFSD1,OsFSD2,OsCSD1,OsCSD2,OsCSD3,OsCSD4,OsMSD1)using Nippo -nbare as material, analyzed the subcellular localization of the seven kinds of rice SOD with rice leave protoplast transient expression, while analyzed the biochemical properties and function of SOD located in rice chloroplast, and explored the molecular mechanism with the mutants and interference vector of OsFSDs.Ultimately it can provide a theoretical basis for rice molecular improvement. The results are as follows:(1) We searched the rice SOD gene sequences (LOC_Os07g46990, LOC_Os03g22810, LOC Os03g11960, LOC_Os08g44770, LOC_Os06g05110, LOC_Os06g02500, LOC_Os05g25850) from the NCBI database,and designed respective specific primers. The full-length cDNAs of OsFSD1、OsFSD2、 OsCSD1、OsCSD2、OsCSD3、OsCSD4and OsMSDl were cloned from Japonica, and the sequences were confirmed by DNA sequencing. The OsFSD2has not been reported, so we first cloned a rice iron SOD gene.(2) Fragments of the cDNAs containing the open reading frame (ORF) without the stop codon were fused to the5’terminal of the GFP gene driven by the35S promoter and were inserted into a PUC18backbone. The plasmids were isolated and purified. The plasmids were transformed into rice leave protoplasts, observing the green fluorescent by confocal microscopy. The results show that OsFSD1, OsFSD2, OsCSD4three genes may be located in the rice chloroplast, OsFSD2, OsCSD4distribute throughout the chloroplasts and OsFSDl spottily distributes in the chloroplasts and we speculate that it may be positioned in the chloroplast nucleoid. OsMSDl may be located in the mitochondria, the other three Cu/Zn-SOD may be located in the cytoplasm.(3) To explored the interaction in vitro between the three SOD located in the rice chloroplast,we did the yeast two-hybrid and GST-pull-down experiments.The results show that the interaction occurs between OsFSDl and OsFSDl, OsFSD2and OsFSD1,but there is no interaction between OsFSD2and OsFSD2.At the same time, we use the BIFC experimental methods to verify this result in vivo.The results of BIFC show that OsFSD2and OsFSD1may interact in the chloroplast nucleoid.(4) The literature of Arabidopsis reported that FSDs have enzymatic activity and play a role to protect chloroplast nucleoids against oxidative stress. SOD activity signals on the native PAGE gels were detected with the recombinant proteins, indicating that OsFSD1protein has enzymatic activity, but OsFSD2has not. Moreover, the enzymatic activity of the recombinant proteins is about twice OsFSD1.SOD activity was also measured using the superoxide activity assay kit. Then two experimental results are basically consistent. We speculate that OsFSD1expression can not be increase immediatly when plants suffered sudden abiotic stress, but OsFSD1can recruit OsFSD2into the chloroplasts nucleoid and increases its activity by forming a heterodimer.(5) To further analyze the functions of OsFSD1and OsFSD2, we generated transgenic lines overexpressing OsFSD1, OsFSD2,OsCSD4individually and constructed RNAi vectors and buyed the mutants of OsFSD1and OsFSD2from South Korea T-DNA insertion mutant library. MV causes an increase of superoxide radicals in chloroplasts. We investigated the effect of MV on the transgenic plants by measuring the MV-dependent decrease in activity of the PSII reaction center under normal light conditions at30umol.m-2.s-1. The leaves of transgenic plants overexpressing OsFSD1, OsFSD2and OsCSD4individually showed less inactivation of PSII, than did wild-type plants as indicated by Fv/Fm. Under this level of exposure, transgenic plants expressing OsFSD2transgenes showed less inactivation of PSII than did wild-type plants and other transgenic plants. Taken together, these data indicate that overexpression of OsFSD1, OsFSD2and OsCSD4individually improves tolerance to MV and protects chloroplasts against light-dep endent oxidative stress.We obtain the homozygous mutant of OsFSD2identified by PCR.The homozygous mutant plants had a severe albino phenotype when grown on agar plates. At the same time, the98%transgenic seedlings of OsFSD2RNAi had a severe albino phenotype, but50%Of OsFSD1RNAi had the phenotype. RT-PCR confirmed that both the OsFSD2RNAi and the homozygous mutant transcripts of OsFSD2were absent in the double plants. Therefore OsFSD2plays an important role in the process of growth and development of rice seedlings, but the specific mechanism of action also requires further in-depth study.