Summary Heterosis utilization is the most effective way to improve rice yields. The cytoplasmic male‐sterility (CMS) and photoperiod/thermosensitive genic male‐sterility (PTGMS) systems have been widely used in rice production. However, the rate of resource utilization for the CMS system hybrid rice is low, and the hybrid seed production for the PTGMS system is affected by the environment. The technical limitations of these two breeding methods restrict the rapid development of hybrid rice. The advantages of the genic male‐sterility (GMS) rice, such as stable sterility and free combination, can fill the gaps of the first two generations of hybrid rice technology. At present, the third‐generation hybrid rice breeding technology is being used to realize the application of GMS materials in hybrid rice. This study aimed to use an artificial CMS gene as a pollen killer to create a smart sterile line for hybrid rice production. The clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) technology was used to successfully obtain a CYP703A3‐deficient male‐sterile mutant containing no genetically modified component in the genetic background of indica 9311. Through young ear callus transformation, this mutant was transformed with three sets of element‐linked expression vectors, including pollen fertility restoration gene CYP703A3, pollen‐lethality gene orfH79 and selection marker gene DsRed2. The maintainer 9311‐3B with stable inheritance was obtained, which could realize the batch breeding of GMS materials. Further, the sterile line 9311‐3A and restorer lines were used for hybridization, and a batch of superior combinations of hybrid rice was obtained.
OsNramp5 is a key gene involved in the control of the uptake of Cd, Mn, and other metal ions by rice root cells. The functional deficiency of this gene can significantly reduce the accumulation of Cd in rice grains, but the effects of its mutation on agronomic traits such as yield and quality have not been investigated comprehensively yet. In the present study, three Huanghuazhan-based OsNramp5 mutants [LCH1 (Low Cadmium Huanghuazhan 1), LCH2 (Low Cadmium Huanghuazhan 2), and LCH3 (Low Cadmium Huanghuazhan 3)] were obtained using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. The mutation-type analysis showed that LCH1, LCH2, and LCH3 encoded defective OsNramp5 protein sequences containing only 76aa, 176aa, and 266aa, respectively. The determination of metal content and the statistics of related agronomic traits revealed that the functionally deficient OsNramp5 not only significantly reduced the accumulation of Cd in the grains of the mutants but also affected rice yield and quality. However, with the decrease of OsNramp5 mutation degree, its effects on chlorenchyma Mn accumulation, yield, and quality were also diminished. Additionally, we also found that the increase in the concentration of Mn in the soil restored the phenotype of the declined yield and quality due to the functional deficiency of OsNramp5. Our findings provide novel insights into and new materials for breeding rice varieties with low Cd accumulation and excellent agronomic traits under severe Cd pollution environment.
Microelectrodes were fabricated to study impedance labelless detection of DNA hybridization. The probe molecule was attached onto the platinum microelectrode surface by electrochemically copolymerizing pyrrole and the probe oligonucleotides. Measured impedance complexes showed that an electrochemical redox-reaction occurred and the electron-transfer resistance increased after DNA hybridization. It was proposed that the hybridization of DNA in the conductive polymer matrix slowed down the anionic doping/undoping process, resulting impedance changes for the target DNA detection. Impedance measurements were conducted at the complementarily hybridized probe oilgomer-attached polypyrrole film electrodes in different anionic solutions to exam the anionic effects. Results showed that higher concentration and smaller size of anions had the lower electron-transfer resistance. The results not only provide further evidence to support the detection mechanism proposed, but also offer a method to improve the signal to noise ratio for the DNA biosensor. The research also tested the specificity of the methods and experimental results, indicating good specificity of the method. A concept array chip was fabricated and used to demonstrate the capability of the labelless detection method. Nano-Molar concentrations were detected and showed fairly linear responses versus the target molecule concentrations. The method is simple and inexpensive. The technique based genosensors could have potential applications in clinical diagnosis, drug discovery, environmental and food analysis.
In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway.
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