Ubiquitin Research Articles

Human cytomegalovirus interleukin 10 (cmvIL-10) orchestrates crosstalk between the cellular IL-10 receptor and CXCR4.

Histone deacetylase 6 (HDAC6) helps cells manage misfolded proteins by transporting ubiquitin (UB)-associated structures toward the microtubule organizing center, where they can be sequestered and degraded by lysosomes. Here, we show that when cells are subjected to acute protein-folding stress in the endoplasmic reticulum (ER), HDAC6 depletion results in the appearance of enlarged endosomes that are highly decorated with UB and colocalize with both early and late endosome markers. The C-terminal UB-binding domain and adjacent disordered regions of HDAC6 are necessary and sufficient to rescue this endosomal phenotype in cells lacking endogenous HDAC6. HDAC6 deficiency does not appear to prevent the recruitment of endosomal sorting complexes required for transport (ESCRT), which coordinate endosome maturation. However, overexpression of HDAC6 can reverse endosome phenotypes associated with the depletion of the early ESCRT factor HRS. We speculate that HDAC6 facilitates the packaging and processing of endosomal cargo when the endomembrane system is under stress.

Abstract The development of effective genetic transformation tools is crucial for advancing molecular breeding in peanut (Arachis hypogaea). In this study, we identified and characterized a Ubiquitin (UBQ) promoter from peanut and evaluated its potential utility in transgenic research. Using sequence similarity–based identification and transcriptome analysis, we selected a highly expressed UBQ gene, arahy.E356RC, designated AhUBQ4, from which we cloned a 973-bp fragment of the promoter region. To assess its activity, we used this AhUBQ4 promoter fragment to drive expression of the GUS and Ruby reporter genes in transient and stable expression assays in Nicotiana benthamiana and peanut tissues. Compared to the commonly used CaMV 35S promoter, the AhUBQ4 promoter had stable and high transcriptional activity across multiple tissues. Furthermore, we replaced the traditional 35S promoter with the AhUBQ4 promoter in a CRISPR/Cas9 system, enabling efficient gene editing in peanut. Using a peanut hairy root transformation system, we induced site-specific mutations in HY5-HOMOLOG, confirming stable Cas9 expression from the AhUBQ4 promoter for genome editing applications. Our findings highlight the potential of the AhUBQ4 promoter as a valuable genetic tool for improving transformation efficiency and gene expression stability in peanut, paving the way for enhanced functional genomics studies and molecular breeding efforts.

Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin.

Ubiquitylation is involved in various physiological processes, such as signaling and vesicle trafficking, whereas ubiquitin (UB) is considered an important clinical target. The polymeric addition of UB enables cargo molecules to be recognized specifically by multivalent binding interactions with UB-binding proteins, which results in various downstream processes. Recently, protein condensate formation by ubiquitylated proteins has been reported in many independent UB processes, suggesting its potential role in governing the spatial organization of ubiquitylated cargo proteins. We created modular polymeric UB binding motifs and polymeric UB cargos by synthetic bioconjugation and protein purification. Giant unilamellar vesicles with lipid raft composition were prepared to reconstitute the polymeric UB cargo organization on the membranes. Fluorescence imaging was used to observe the outcome. The polymeric UB cargos clustered on the membranes by forming a phase separation codomain during the interaction with the multivalent UB-binding conjugate. This phase separation was valence-dependent and strongly correlated with its potent ability to form protein condensate droplets in solution. Multivalent UB binding interactions exhibited a general trend toward the formation of phase-separated condensates and the resulting condensates were either in a liquid-like or solid-like state depending on the conditions and interactions. This suggests that the polymeric UB cargos on the plasma and endosomal membranes may use codomain phase separation to assist in the clustering of UB cargos on the membranes for cargo sorting. Our findings also indicate that such phase behavior model systems can be created by a modular synthetic approach that can potentially be used to further engineer biomimetic interactions in vitro.

VPS28 (vacuolar protein sorting 28) is a subunit of the endosomal sorting complexes required for transport (ESCRTs) and is involved in ubiquitination. Ubiquitination is a critical system for protein degradation in eukaryotes. Considering the recent findings on the role of ubiquitination in the regulation of lipid metabolism, we hypothesized that VPS28 might affect the expression of genes involved in milk fat synthesis. To test this hypothesis, we modulated VPS28 expression in the bovine mammary epithelial cell line (MAC-T) and measured the effects on triglyceride (TG) synthesis using lentivirus-mediated techniques. The results showed that VPS28 knockdown significantly upregulated the levels of the fatty acid transporter CD36 molecule (CD36) and adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production, along with elevated ubiquitin (UB) levels, while reducing proteasome activity. In contrast, VPS28 overexpression increased CD36 levels while not significantly affecting ADFP or TG levels, with a trend toward reduced lipid droplets and increased UB expression and proteasome activity. In addition, inhibition of the ubiquitin-proteasome system and the endosomal-lysosomal pathway using epoxomicin and chloroquine, respectively, further increased CD36, ADFP, and TG levels, thereby enhancing cell viability. These in vitro findings were validated in vivo in a mouse model, where VPS28 knockdown increased mammary CD36, ADFP, UB expression, TG content, and lipid droplets without pathological changes in mammary tissue or blood TG alterations. These results confirm the pivotal role of VPS28 in regulating TG synthesis via the ubiquitination pathway, offering novel insights into the molecular mechanisms of milk fat production in a bovine cell model.

Deubiquitinases are involved in removing ubiquitin (UBQ) from ubiquitylated substrates to regulate their activity and stability. They are involved in various cellular functions including proteasome- and lysosome-dependent proteolysis, gene expression, cell cycle progression, chromosome segregation, kinase activation, histone modification, cell cycle regulation, cell differentiation, spermatogenesis, apoptosis, endocytosis, autophagy, localization, and DNA damage repair. On the other hand, it is also involved in the processes of carcinogenesis and cancer development. DUBs are related to different aspects of human cancer, including proliferation, cell cycle control, apoptosis, DNA damage response (DDR), tumor suppression, oncogenesis, and metastasis. Multiple processes involved in innate and adaptive immunity, such as antigen presentation, cell differentiation, immune defense, and inflammatory responses, are regulated by ubiquitination/deubiquitination. Dysregulation of DUBs is implicated in several human diseases, highlighting the importance of DUB function. This review also provides basic knowledge of DUBs in the development of cancers and highlights the importance of DUBs in T-cell development, apoptosis, and cancer with a specific emphasis on oral cancers. This review highlights the recent advances in the field of cancer biology with a specific role in DUBs in cellular functions. The aberrant expression and regulation of these enzymes have been shown to contribute to promote tumorigenesis, making them promising therapeutic targets for cancer therapy.

Macadamia is an economically significant crop, with its kernel oil being abundant in monounsaturated fatty acids (MUFA). Analyzing the expression of genes related to MUFA biosynthesis is essential for understanding the complex regulatory networks in Macadamia. However, there are few reports on the identification of suitable reference genes for use as internal controls in this species. Consequently, selecting a reliable reference gene for gene expression studies under various conditions is critical. In this study, we evaluated the expression stability of 11 traditional housekeeping genes: α-tubulin (TUBa), β-tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-elongation factor 1 (EF1a), β-elongation factor 1 (EF1b), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UBC), cyclophilin (CYP), and actin (ACT) under abiotic stresses, hormonal treatments and in variety of plant tissues using the online tool RefFinder, which integrates four commonly used software programs: ΔCt, geNorm (version 3.4), NormFinder (version 0953), and BestKeeper (version 1.0). A comprehensive expression stability ranking was established by integrating results from these four methods based on the geometric mean. The findings indicated that ACT was the most stable gene across all samples, including those subjected to cold stress, NaCl stress, PEG stress, ABA treatment, MeJA treatment, and both stem and leaf tissues. EF1b was identified as the most stable gene in GA treatment and heat stress samples, while UBC and CYP were ranked highest in ethrel treatment and root tissue samples, respectively. Finally, the reliability of these findings was further validated using the target gene SAD through qRT-PCR. In summary, this study evaluated and validated appropriate reference genes for qRT-PCR, which will facilitate future investigations into the molecular mechanisms in Macadamia.

Perfluorooctanoic acid (PFOA) has been widely studied due to its environmental persistence and bioaccumulation potential, raising concerns about its effects on aquatic life. This research evaluates the impact of PFOA on the antioxidant defenses and stress response systems of the mussel Mytilus galloprovincialis. Mussels were exposed to three concentrations of PFOA (1, 10, and 100 µg·L−1) over 28 days. Several biomarkers, including glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), lipid peroxidation (LPO), total antioxidant capacity (TAC), vitellogenin (VTG), ubiquitin (UBI), and caspase-3 (CASP) were analyzed. The results suggest stress responses, particularly in animals exposed to higher concentrations, as shown by GST and SOD activities which increased according to PFOA concentrations. Additionally, oxidative stress markers such as MDA and CAT showed variable responses depending on the exposure concentration tested. This study underscores the need for further investigation into the effects of PFOA on mollusks but also the need to unveil gender-specific responses in aquatic organisms exposed to this contaminant. The concentrations of PFOA used in our research are lower than those examined in previous studies, providing crucial insights into the impacts of even minimal exposure levels. It highlights the potential of M. galloprovincialis as a bioindicator in environmental monitoring programs, providing crucial insights for environmental management and policymaking regarding regulating and monitoring PFOA in marine settings. Consequently, in a country where seafood consumption is the second largest in Europe, implementing environmental policies and regulatory measures to manage and monitor PFOA levels in marine environments is crucial.

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa 'Doulv', notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from 'Doulv', nine candidate reference genes-ACT (Actin), TUB (β-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)-were selected. Their expression stability was assessed across various tissues and developmental stages of 'Doulv' flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of 'Doulv', whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from 'Doulv' diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for 'Doulv' peony. These results provide a solid experimental foundation for further research on 'Doulv' peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in 'Doulv', identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies.

In contrast to intracellular gene transfer, the direct delivery of expressed proteins is a significantly challenging yet essential technique for elucidating cellular functions, including protein complex structure, liquid-liquid phase separation, therapeutic applications, and reprogramming. In this study, we developed a hybrid nanotube (HyNT) stamp system that physically inserts the HyNTs into adhesive cells, enabling the injection of target molecules through HyNT ducts. This system demonstrates the capability to deliver multiple proteins, such as lactate oxidase (LOx) and ubiquitin (UQ), to approximately 1.8 × 107 adhesive cells with a delivery efficiency of 89.9% and a viability of 97.1%. The delivery of LOx enzyme into HeLa cancer cells induced cell death, while enzyme-delivered healthy cells remained viable. Furthermore, our stamp system can deliver an isotope-labeled UQ into adhesive cells for detection by nuclear magnetic resonance (NMR).

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (β-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. β-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (β-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. β-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

Identification of the mechanisms underlying the genetic control of spatial structure formation is among the relevant tasks of developmental biology. Both experimental and theoretical approaches and methods are used for this purpose, including gene network methodology, as well as mathematical and computer modeling. Reconstruction and analysis of the gene networks that provide the formation of traits allow us to integrate the existing experimental data and to identify the key links and intra-network connections that ensure the function of networks. Mathematical and computer modeling is used to obtain the dynamic characteristics of the studied systems and to predict their state and behavior. An example of the spatial morphological structure is the Drosophila bristle pattern with a strictly defined arrangement of its components – mechanoreceptors (external sensory organs) – on the head and body. The mechanoreceptor develops from a single sensory organ parental cell (SOPC), which is isolated from the ectoderm cells of the imaginal disk. It is distinguished from its surroundings by the highest content of proneural proteins (ASC), the products of the achaete-scute proneural gene complex (AS-C). The SOPC status is determined by the gene network we previously reconstructed and the AS-C is the key component of this network. AS-C activity is controlled by its subnetwork – the central regulatory circuit (CRC) comprising seven genes: AS-C, hairy, senseless (sens), charlatan (chn), scratch (scrt), phyllopod (phyl), and extramacrochaete (emc), as well as their respective proteins. In addition, the CRC includes the accessory proteins Daughterless (DA), Groucho (GRO), Ubiquitin (UB), and Seven-in-absentia (SINA). The paper describes the results of computer modeling of different CRC operation modes. As is shown, a cell is determined as an SOPC when the ASC content increases approximately 2.5-fold relative to the level in the surrounding cells. The hierarchy of the effects of mutations in the CRC genes on the dynamics of ASC protein accumulation is clarified. AS-C as the main CRC component is the most significant. The mutations that decrease the ASC content by more than 40 % lead to the prohibition of SOPC segregation.

During the Advanced Plant Habitat experiment 2, radish plants were grown in two successive grow-outs on the International Space Station (ISS) for 27 days each. On days 10, 18, and 24, leaf punch (LP) samples were collected and frozen. At harvest, bulb tissue was sampled with oligo-dT functionalized Solid Phase Gene Extraction (SPGE) probes. The space samples were compared with samples from ground controls (GC) grown at the Kennedy Space Center (KSC) under the same conditions as on the ISS, with notably elevated CO2 (about 2500 ppm), and from lab plants grown under atmospheric CO2 but with light and temperature conditions similar to the KSC control. Genes corresponding to peroxidase (RPP), glucosinolate biosynthesis (GIS), protein binding (CBP), myrosinase (RMA), napin (RSN), and ubiquitin (UBQ) were measured by qPCR. LP from day 24 and bulb samples collected at harvest were compared with RNA-seq data from material that was harvested, frozen, and analyzed after return to Earth. The results showed stable transcription in LP samples in GC but decreasing values in ISS samples during both grow-outs, possibly indicative of stress. SPGE results were similar between GC and ISS samples. However, the RNA-seq analyses showed different transcription profiles than SPGE or LP results, possibly related to localized sampling. RNA-seq of leaf samples showed greater variety than LP data, possibly because of different sampling times. RSN and RPP showed the lowest transcription regardless of method. Temporal analyses showed relatively small changes during plant development in space and in ground controls. This is the first study that compares developmental changes in space-grown plants with ground controls based on a comparison between RNA-seq and qPCR analyses.

White lupin (Lupinus albus L.) is an important crop with high phosphorus (P) use efficiency; however, technologies for its functional genomic and molecular analyses are limited. Cluster regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been applied to gene editing and function genomics in many crops, but its application in white lupin has not been well documented. Here, we adapted the CRISPR/Cas9-based multiplex genome editing system by using the native U3/U6 and ubiquitin (UBQ) promoters to drive sgRNAs and Cas9. Two target sites (T1 and T2) within the Lalb_Chr05g0223881 gene, encoding a putative trehalase, were selected to validate its efficacy in white lupin based on the Agrobacterium rhizogenes-mediated transformation. We found that the T0 hairy roots were efficiently mutated at T1 and T2 with a frequency of 6.25%-35% and 50%-92.31%, respectively. The mutation types include nucleotide insertion, deletion, substitution, and complicated variant. Simultaneous mutations of the two targets were also observed with a range of 6.25%-35%. The combination of LaU6.6 promoter for sgRNA and LaUBQ12 promoter for Cas9 generated the highest frequency of homozygous/biallelic mutations at 38.46%. In addition, the target-sgRNA sequence also contributes to the editing efficiency of the CRISPR/Cas9 system in white lupin. In conclusion, our results expand the toolbox of the CRISPR/Cas9 system and benefit the basic research in white lupin.

Uncontrolled inflammation is a typical feature of sepsis-related lung injury. The key event in the progression of lung injury is Caspase-1-dependent alveolar macrophage (AM) pyroptosis. Similarly, neutrophils are stimulated to release neutrophil extracellular traps (NETs) to participate in the innate immune response. This study aims to illustrate the specific mechanisms by which NETs activate AM at the post-translational level and maintain lung inflammation. We established a septic lung injury model by caecal ligation and puncture. We found elevated NETs and interleukin-1b (IL-1β) levels in the lung tissues of septic mice. Western blot and immunofluorescence analyses was utilized to determine whether NETs promote AM pyroptosis and whether degrading NETs or targeting the NLRP3 inflammasome had protective effects on AM pyroptosis and lung injury. Flow cytometric and co-immunoprecipitation analysesverified intracellular reactive oxygen species (ROS) levels and the binding of NLRP3 and ubiquitin (UB) molecules, respectively. Increased NETs production and IL-1β release in septic mice were correlated with the degree of lung injury. NETs upregulated the level of NLRP3, followed by NLRP3 inflammasome assembly and caspase-1 activation, leading to AM pyroptosis executed by the activated fragment of full-length gasdermin D (FH-GSDMD). However, the opposite effect was observed in the context of NETs degradation. Furthermore, NETs markedly elicited an increase in ROS, which facilitated the activation of NLRP3 deubiquitination and the subsequent pyroptosis pathway in AM. Removal of ROS could promote the binding of NLRP3 and ubiquitin, inhibit NLRP3 binding to apoptosis-associated spotted proteins (ASC) and further alleviate the inflammatory changes in the lungs. In summary, these findings indicate that NETs prime ROS generation, which promotes NLRP3 inflammasome activation at the post-translational level to mediate AM pyroptosis and sustain lung injury in septic mice.

Post-ischemic cardioprotective potential of exogenous ubiquitin in myocardial remodeling late after ischemia/reperfusion injury

IntroductionMorchella importuna (M. importuna) is a rare fungus with high nutrition value and distinct flavor. Despite the successful artificial cultivation, its genetic characteristics and biological processes such as life cycle, reproductive system, and trophic mode remain poorly understood.MethodsConsidering this, we constructed pEH2B and pMH2B vectors by fusing M. importuna endogenous histone protein H2B with fluorescent proteins eGFP or mCherry, respectively. Based on the constructed pEH2B and pMH2B vectors, nuclear fluorescence localization was performed via Agrobacterium tumefaciens-mediated transformation (ATMT). These two vectors were both driven by two endogenous promoters glyceraldehyde 3-phosphate dehydrogenase (GPD) and ubiquitin (UBI). The vector-based reporter systems were tested by the paired culture of two genetically modified strains pEH2B-labeled M04M24 (24e, MAT1-1-1) and pMH2B-abeled M04M26 (26m, MAT1-2-1).ResultsThe fluorescence observation and molecular identification results indicated the successful hyphal fusion and heterokaryon formation. We found that the expression of the reporter genes was stable, and it did not interfere with the growth of the fungus.DiscussionOur constructed nucleus-directed fluorescent systems in M. importuna can be used for monitoring the dynamic development and reproductive processes in living cells and also for monitoring the interaction between morels and plant roots. Therefore, morels exhibit the potential to be a candidate organism used for the research on basic biology and genetics of ascomycetes.

In transgenic experiments, we often face fundamental requirements such as overexpressing a certain gene, developing organelle markers, testing promoter activities, introducing large genomic fragments, and combinations of them. To fulfill these multiple requirements in rice, we developed simple binary vectors with or without maize ubiquitin (UBQ) promoter, Gateway cassette and fluorescent proteins. First, we compared stabilities of cauliflower mosaic virus 35S and maize UBQ promoters for constitutive gene expression in transgenic rice. We show that the 35S promoter was frequently silenced after shoot regeneration, whereas maize UBQ promoter achieved stable expression in various young tissues. Binary vectors with Gateway cassettes under the control of the UBQ promoter allowed us to develop stable organelle markers for nuclei, microtubules and P-bodies in rice. The maize UBQ promoter can be easily replaced with any promoters of interest as exemplified by reporters of mitotic cells and provascular bundles. Finally, by introducing two genomic fluorescent reporters, we showed utilities of the Gateway cassette and two selection markers in large DNA fragment transfer and sequential transformations, respectively. Thus, these binary vectors provide useful choices of transgenic experiments in rice.