| Rice is one of the most important grain crops in the world and it is often suffered from a number of abiotic stresses such as drought. Drought has become one of the major adverse factors for rice yield production. With the completion of genome sequencing of rice and the arrival of the post genome era, looking for and functioning the genes which are related to drought stress have the important meaning in breeding practice. Previously, we found that the expression of Os04g0117100 gene was obviously decreased in an OsNOX2-knockout mutant. As an important gene for reactive oxygen species(ROS) production in plants, OsNOX2 plays a crucial role in drought tolerance of rice. Therefore, Os04g0117100 gene might participate in the OsNOX2-mediated pathway in drought stress response of rice. Based on the characteristic of sequence of Os04g0117100, we found out that the protein coded by Os04g0117100 has homologous sequence with the subunit of the ATP synthase located in mitochondria of animals. However, the functions of Os04g0117100 gene, which is named as OsmtATPS1 here, are greatly unknown. In the present study, the fullength cDNA sequence of OsmtATPS1 gene was cloned and the biological roles of the gene were preliminarily investigated. The main research results are as followed:(1)The fullength cDNA sequence of OsmtATPS1 gene was cloned by RT-PCR. The results based on bioinformatics analysis showed that the protein coded by OsmtATPS1 gene has only 52 amino acids. The 11~52 section of amino acids contains the E subunit of F0 part which is the component of mitochondrial ATP synthase. The senior structure of the protein has both hydrophilic and hydrophobic part in the end, and an alpha helix in the middle. Results from the phylogenetic analysis showed that similar sequences as OsmtATPS1 are universally existed in many important crops including rice, maize, sorghum and millet.(2)According to the results of semi-quantitative RT-PCR, we found out that OsmtATPS1 gene had different degree of expression during different growth period, which is including 30 days seedling, two weeks seedling stage and the mature stage. We found out that OsmtATPS1 gene expressed in all organizations in addition to young panicles outside. The expression levels of OsmtATPS1 gene was higher in the root part than that in leaves in two weeks stage. However, the expression level of OsmtATPS1 gene in leaves is the highest in 30 days seedling stage. The highest expression level of the gene of OsmtATPS1 is in stem while the minimum expression level of the gene of OsmtATPS1 is in leaves in the mature period. The induced expression spectrum shown that OsmtATPS1 gene had different responses to different biological or hormone stress treatment, including high temperature, low temperature, PEG, NaCl, MeJA, SA, ABA, IAA, MV and so on. We can figure out that the expression level of OsmtATPS1 gene from overground tissue is higher in MeJA treated group while which was restrained after high temperature, IAA and SA treatment. The expression of OsmtATPS1 gene was strongly induced by high temperature and low temperature, but which was inhibited by the MV in overground tissue of rice.(3)We constructed the fusion expression plasmid of OsmtATPS1-GFP gene with the vectors of PTF486 and p35s-eGFP, and transformed those into the protoplast from rice and tobacco. After observation of the laser confocal, we found out that fusion protein of OsmtATPS1-GFP was granular distribution in cell, some of that in the chloroplasts, but surplus were unknown distribution position in rice protoplast. After transient transfections in tobacco leaves, the localization of the fusion protein of OsmtATPS1–GFP was consensus with the results of rice protoplast as above shown. The fusion expression plasmid of OsmtATPS1-GFP gene was transformed into rice callus by agrobacterium mediated. We harvested the protoplast from the callus having OsmtATPS1-GFP, and then dyed the protoplast through the mitochondrial dye. The results showed that fusion protein of OsmtATPS1-GFP was expressed in the mitochondria. We came to the conclusion that the OsmtATPS1 is localization in both mitochondria and chloroplasts in rice cell.(4) We respectively constructed the expression vectors OsmtATPS1 gene, and transformed those vectors into rice callus from mature embryo. We got 8 strains of overexpression transgenic plants, 16 strains of silencing(antisense RNA) transgenic plants and 72 strains of silencing transgenic plants(interference RNA). The results shown that the highest of expression level of OsmtATPS1 gene is 8 times in overexpression transgenic plants while that is close to 0.1 times in silencing(interference RNA) transgenic plants. We found out that the number of tiller was decreased in RNAi transgenic plants, but the overexpression and silencing(antisense RNA) transgenic plants did not have abnormal phenotype. In addition, this study also transformed the vector of overexpression into Arabidopsis thaliana by agrobacterium mediated, and we have harvested the seeds of the T2 now.(5)The results shown the expression levels of NOX2 genes was increased in overexpression transgenic plants while which was significant changed in silent expression transgenic plants. Analyzing the expression level of the gene of other subunit of ATP synthase in transgenic plants, we found out that expression level of MATP6 gene become slightly decrease, level of RMATPδ is obviously lower and the expression level of RMATP9 and RMATPα were dramatic increased in overexpression transgenic plants while the expression levels of other subunits of ATP synthase did not significantly change in silent expression transgenic plants.(6) Analyzing the promoter by the online software of PLANTCARE, we found out that the promoter sequences of OsmtATPS1 gene are consist of the promoter core components-TATA box, drought induced MYB transcription factor binding sites of MBS, the related to MeJA original TGACG motif and CGTCA motif, and the function of the ABA response related original ABRE. This study found the promoter sequences of OsmtATPS1 gene in rice genome database IRGSP, and we successfully cloned it by the PCR. The vector of promoter driving the GUS reporter have been constructed, which was transformed into the rice callus. Furthermore, we have received the positive transgenic callus.In summary, this study shows that OsmtATPS1 protein can express both in mitochondria and chloroplasts. It responds to the high and low temperature, MeJA and MV treatment. We found out that there are existed the co-expression between OsmtATPS1 gene and NOX2 genes by analyzing T0 transgenic plants. The expression of this gene does affect the expression level of other subunit of ATP synthase in mitochondria. While we got the transgenic plants including Osmt ATPS1-eGFP, overexpression and silencing, it displays the foundation for further studying the function of OsmtATPS1 gene. |
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