Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells.

Fluorescent RNAs, aptamers that bind and activate small fluorogenic dyes, have provided a particularly attractive approach to visualizing RNAs in live cells. However, the simultaneous imaging of multiple RNAs remains challenging due to a lack of bright and stable fluorescent RNAs with bio-orthogonality and suitable spectral properties. Here, we develop the Clivias, a series of small, monomeric and stable orange-to-red fluorescent RNAs with large Stokes shifts of up to 108 nm, enabling the simple and robust imaging of RNA with minimal perturbation of the target RNA's localization and functionality. In combination with Pepper fluorescent RNAs, the Clivias enable the single-excitation two-emission dual-color imaging of cellular RNAs and genomic loci. Clivias can also be used to detect RNA-protein interactions by bioluminescent imaging both in live cells and in vivo. We believe that these large Stokes shift fluorescent RNAs will be useful tools for the tracking and quantification of multiple RNAs in diverse biological processes.

Bright and Stable Cyan Fluorescent RNA Enables Multicolor RNA Imaging in Live Escherichia coli.

Fluorescent RNAs (FRs), which are RNA aptamers that bind and activate their cognate small fluorogenic dyes, have provided a particularly useful approach for imaging RNAs in live cells. Although the color palette of FRs is greatly expanded, a bright and stable cyan FR with good biocompatibility and biorthogonality with currently available FRs remains desirable but is not yet developed. Herein, the development of Myosotis is described, an RNA aptamer that emits bright cyan fluorescence upon binding a novel GFP chromophore-like fluorophore called DBT. Myosotis has a nanomolar affinity for DBT and shows a weak dependence on magnesium for folding. Further studies reveal that the Myosotis-DBT complex has a long fluorescence lifetime, good photostability, and enhance cellular brightness. It is further shown that Myosotis-DBT is biorthogonal to Pepper and Clivia FRs, allowing multiplex fluorescence imaging of RNA in live bacteria. Myosotis can also use to image mRNA in live bacteria, revealing potential coupling between mRNA translation and stability. It is believed that this cyan FR will be a useful tool for studying the functionality and mechanism of RNA underlying diverse biological processes.

The construction of recombinant Lactobacillus casei vaccine of PEDV and its immune responses in mice.

BACKGROUND: Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV) characterized by vomiting, diarrhea, anorexia, and dehydration, which have caused huge economic losses around the world. At present, vaccine immunity is still the most effective method to control the spread of PED. In this study, we have constructed a novel recombinant L. casei-OMP16-PEDVS strain expressing PEDVS protein of PEDV and OMP16 protein of Brucella abortus strain. To know the immunogenicity of the recombinant L. casei-OMP16-PEDVS candidate vaccine, it was compared with BL21-OMP16-PEDVS-F, BL21-OMP16-PEDVS, and BL21-PEDVS recombinant protein. RESULTS: The results showed that we could detect higher levels of IgG, neutralizing antibody, IL-4, IL-10, and INF-γ in serum and IgA in feces of L. casei-OMP16-PEDVS immunized mice, which indicated that L. casei-OMP16-PEDVS candidate vaccine could induce higher levels of humoral immunity, cellular immunity, and mucosal immunity. CONCLUSION: Therefore, L. casei-OMP16-PEDVS is a promising candidate vaccine for prophylaxis of PEDV infection.

Development of Acrylamide-Based Rapid and Multicolor Fluorogenic Probes for High Signal-to-Noise Live Cell Imaging.

Protein covalent labeling is dramatically useful for studying protein function in living cells and organisms. In this field, the chemical tag technique combined with fluorogenic probes has emerged as a powerful tool. Herein, a series of TMP tag fluorogenic probes have been developed to span the green to full blue spectral range. These probes feature an acrylamide unit that acts as a linker group to conjugate the fluorophore and the ligand as well as a quencher and a covalent reaction group. After the probes bind to eDHFR:L28C, the acrylamide unit specifically reacts with the thiol group of the L28C residue beside the ligand binding pocket, achieving protein-specific labeling without any liberation of leaving groups. With these probes, multicolor and specific protein labeling with a fast reaction rate ( t1/2 = 33 s) and dramatic fluorescence enhancement (4000-fold) were obtained. Furthermore, no-wash protein labeling in both living cells and zebrafish was successfully achieved. We expect it may provide a general and highly effective chemical tool for the study of protein function in living cells and organisms.

Arabidopsis HSP70-16 is required for flower opening under normal or mild heat stress temperatures.

Sepals play important roles in protecting inner floral organs from various stresses and in guaranteeing timely flower opening. However, the exact role of sepals in coordinating interior and exterior signals remains elusive. In this study, we functionally characterized a heat shock protein gene, Arabidopsis HSP70-16, in flower opening and mild heat stress response, using combined genetics with anatomic, physiological, chemical, and molecular analyses. We showed that HSP70-16 is required for flower opening and mild heat response. Mutation of HSP70-16 led to a significant reduction in seed setting rate under 22°C, which was more severe at 27°C. Mutation of HSP70-16 also caused postgenital fusion at overlapping tips of two lateral sepals, leading to failed flower opening, abnormal floral organ formation, and impaired fertilization and seed setting. Chemical and anatomic analyses confirmed specific chemical and morphological changes of cuticle property in mutant lateral sepals, and qRT-PCR data indicated that expression levels of different sets of cuticle regulatory and biosynthetic genes were altered in mutants grown at both 22°C and 27°C temperatures. This study provides a link between thermal and developmental perception signals and expands the understanding of the roles of sepal in plant development and heat response.

Histological and Cytological Characterization of Anther and Appendage Development in Asian Lotus (Nelumbo nucifera Gaertn.).

The lotus (Nelumbo Adans.) is a perennial aquatic plant with important value in horticulture, medicine, food, religion, and culture. It is rich in germplasm and more than 2000 cultivars have been cultivated through hybridization and natural selection. Microsporogenesis and male gametogenesis in the anther are important for hybridization in flowering plants. However, little is known about the cytological events, especially related to the stamen, during the reproduction of the lotus. To better understand the mechanism controlling the male reproductive development of the lotus, we investigated the flower structure of the Asian lotus (N. nucifera). The cytological analysis of anther morphogenesis showed both the common and specialized cytological events as well as the formation of mature pollen grains via meiosis and mitosis during lotus anther development. Intriguingly, an anatomical difference in anther appendage structures was observed between the Asian lotus and the American lotus (N. lutea). To facilitate future study on lotus male reproduction, we categorized pollen development into 11 stages according to the characterized cytological events. This discovery expands our knowledge on the pollen and appendage development of the lotus as well as improving the understanding of the species differentiation of N. nucifera and N. lutea.

Coumarin Photocaging Groups Modified with an Electron-Rich Styryl Moiety at the 3-Position: Long-Wavelength Excitation, Rapid Photolysis, and Photobleaching.

A new class of coumarin photocaging groups modified with an electron-rich styryl moiety at the 3-position was constructed. The large π-conjugated structure and stabilization of the carbocation intermediates by electron donors endowed the new photocaging groups with excellent long-wavelength absorption, large two-photon absorption cross-sections, and high uncaging quantum yields. Moreover, the new photocaging groups displayed unique photobleaching properties after photocleavage as a result of the intramolecular cyclization rearrangement of a carbocation intermediate to form five-membered ring byproducts and block the styryl conjugation at the 3-position. These superior properties of the new photocaging groups are extremely beneficial for high-concentration samples and thick specimens, thus extending the application of photocaging groups in many fields.

Transcriptome exploration for further understanding of the tropane alkaloids biosynthesis in Anisodus acutangulus.

Tropane alkaloids (TAs) such as anisodamine, anisodine, hyoscyamine and scopolamine are extensively used in clinical practice as anticholinergic agents. Anisodus acutangulus produces TAs in root tissue, and although several genes involved in scopolamine biosynthesis have been cloned, yet the biosynthetic pathway of TAs remains poorly understood. To further understand TAs biosynthesis mechanism, transcriptome analysis with deep RNA sequencing in A. acutangulus roots was performed in this study; 48 unigenes related to tropane, piperidine and pyridine alkaloid biosynthesis, 145 linked to the distribution of arginine to TAs biosynthesis, and 86 categorized to terpenoid backbone biosynthesis have been identified in pathway enrichment analyses with eukaryotic orthologous groups (KOG) and Kyoto encyclopedia of genes and genomes. Additionally, 82 unigenes annotated as cytochrome P450 family members seemed to be involved in secondary metabolism. Genes encoding littorine mutase/monooxygenase (CYP80F1), diamine oxidase (DAO), alcohol dehydrogenase (ADH) and aromatic amino acid aminotransferase (ArAT) may also play roles in TAs biosynthetic pathways. Furthermore, over 1,000 unigenes were identified as potential transcription factors of WRKY, AP2/ERF, MYB and bHLH families, which would be helpful to understand transcriptional regulation of secondary metabolite biosynthesis. These data enable novel insights into A. acutangulus transcriptome, updating the knowledge of TAs biosynthetic mechanism at molecular level.

BCL2 promotor methylation and miR-15a/16-1 upregulation is associated with sanguinarine-induced apoptotic death in rat HSC-T6 cells.

Previous studies show that several pathways are involved in sanguinarine-induced apoptotic cell death, including AKT downregulation, inhibition of NF-kB activation, mediation of ROS production, downregulation of anti-apoptosis proteins XIAP and cIAP-1, upregulation of BAX, and downregulation of BCL2. In this study, we found out that the quenching of ROS generation by N-acetyl-l-cysteine (NAC), a scavenger of ROS, reversed sanguinarine-induced apoptosis effects, also we found out that sanguinarine-induced rat hepatic stellate T6 cells (HSC-T6 cells) apoptosis was correlated with the generation of increased ROS, which was followed by the activation of caspase-8 (-3, -6, and -9), and the decreasing in the miltochondrial membrane potential (MMP) and the down-regulation of anti-apoptotic protein Bcl-2. It is not clear whether BCL2's downregulation relates to its promoter methylation and miR-15a/16-1 expression which can bind to BCL2 3'-UTR (un-translation reagon). We showed that sanguinarine-induced down regulation of BCL2 was associated with the increased methylation rate of BCL2 promotor district and the increased expression of miR-15a/16-1. HSC-T6 cells treatment with 5-Aza-2'-deoxycytidine (5'-Aza-CdR) impeded sanguinarine-induced BCL2 promotor district methylation and recovered BCL2's expression. Over expression of BCL2 using pEGFP-N1 vector decreased sanguinarine-induced HSC-T6 cells apoptotic death significantly but not completely. These observations clearly showed that BCL2 down regulation was associated with its promoter methylation and miR-15a/16-1 upregulation in sanguinarine-induced Rat HSC-T6 cells.

Angiopoietin-like protein 2 may mediate the inflammation in murine mastitis through the activation of interleukin-6 and tumour necrosis factor-α.

Mastitis is the inflammation of the mammary gland. Recent research has shown that Angiopoietin-like protein 2 (ANGPTL2) is a key inflammatory mediator. In the present study, we tested whether there is a correlation between increased ANGPTL2 expression and inflammation in response to Staphylococcus aureus in murine mastitis and the mechanisms involved. Thirty mice were divided into two groups: blank control group, challenged group. The entire infused mammary glands were removed to observe the changes of histopathology, myeloperoxidase (MPO) activity, production of tumour necrosis factor-α (TNF-α) and interleukin (IL)-6, and genes expression of ANGPTL2, TNF-α and IL-6. In challenged group, the structure of mammary glands was damaged and the large areas of cell fragments were observed. The MPO activity, IL-6 and TNF-α concentrations, ANGPTL2, IL-6, and TNF-α mRNA levels were significantly elevated in challenged group compared with blank control group. The present findings indicate ANGPTL2 may mediate the inflammation in murine mastitis through the activation of IL-6 and TNF-α.

miRNA-940 reduction contributes to human Tetralogy of Fallot development.

Tetralogy of Fallot (TOF) is a complex congenital heart defect and the microRNAs regulation in TOF development is largely unknown. Herein, we explored the role of miRNAs in TOF. Among 75 dysregulated miRNAs identified from human heart tissues, miRNA-940 was the most down-regulated one. Interestingly, miRNA-940 was most highly expressed in normal human right ventricular out-flow tract comparing to other heart chambers. As TOF is caused by altered proliferation, migration and/or differentiation of the progenitor cells of the secondary heart field, we isolated Sca-1(+) human cardiomyocyte progenitor cells (hCMPC) for miRNA-940 function analysis. miRNA-940 reduction significantly promoted hCMPCs proliferation and inhibited hCMPCs migration. We found that JARID2 is an endogenous target regulated by miRNA-940. Functional analyses showed that JARID2 also affected hCMPCs proliferation and migration. Thus, decreased miRNA-940 affects the proliferation and migration of the progenitor cells of the secondary heart field by targeting JARID2 and potentially leads to TOF development.

Spatial heterogeneity: Necessary and feasible for revealing soil trace elements pollution, sources, risks, and their links.

The source diversity and health risk of trace elements (TEs) in soil make it necessary to reveal the relationship between pollution, source, and risk. However, neglect of spatial heterogeneity restricts the reliability of existing identification methods. In this study, spatial heterogeneity is proposed as a necessary and feasible factor for accurately dissecting the pollution-source-risk link of soil TEs. A comprehensive framework is developed by integrating positive matrix factorization, Geodetector, and risk evaluation tools, and successfully applied in a mining-intensive city in northern China. Overall, the TEs are derived from natural background (28.5 %), atmospheric deposition (25.6 %), coal mining (21.8 %), and metal industry (24.1 %). The formation mechanism of heterogeneity for high-variance TEs (Se, Hg, Cd) is first systematically deciphered by revealing the heterogeneous source-sink relationship. Specifically, Se is dominated (76.5 %) by heterogeneous coal mining (q=0.187), Hg is determined (92.6 %) by the heterogeneity of metal mining (q=0.183) and smelting (q=0.363), and Cd is caused (50.9 %) by heterogeneous atmospheric deposition (q>0.254) co-influenced by the terrains and soil properties. Highly heterogeneous sources are also noteworthy for their potential to pose extreme risks (THI=1.122) in local areas. This study highlights the necessity of integrating spatial heterogeneity in pollution and risk assessment of soil TEs.

Aberrant dynamin 2-dependent Na(+) /H(+) exchanger-1 trafficking contributes to cardiomyocyte apoptosis.

Sarcolemmal Na(+) /H(+) exchanger 1 (NHE1) activity is essential for the intracellular pH (pHi ) homeostasis in cardiac myocytes. Emerging evidence indicates that sarcolemmal NHE1 dysfunction was closely related to cardiomyocyte death, but it remains unclear whether defective trafficking of NHE1 plays a role in the vital cellular signalling processes. Dynamin (DNM), a large guanosine triphosphatase (GTPase), is best known for its roles in membrane trafficking events. Herein, using co-immunoprecipitation, cell surface biotinylation and confocal microscopy techniques, we investigated the potential regulation on cardiac NHE1 activity by DNM. We identified that DNM2, a cardiac isoform of DNM, directly binds to NHE1. Overexpression of a wild-type DNM2 or a dominant-negative DNM2 mutant with defective GTPase activity in adult rat ventricular myocytes (ARVMs) facilitated or retarded the internalization of sarcolemmal NHE1, whereby reducing or increasing its activity respectively. Importantly, the increased NHE1 activity associated with DNM2 deficiency led to ARVMs apoptosis, as demonstrated by cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, Bcl-1/Bax expression and caspase-3 activity, which were effectively rescued by pharmacological inhibition of NHE1 with zoniporide. Thus, our results demonstrate that disruption of the DNM2-dependent retrograde trafficking of NHE1 contributes to cardiomyocyte apoptosis.

The ABORTED MICROSPORES regulatory network is required for postmeiotic male reproductive development in Arabidopsis thaliana.

The Arabidopsis thaliana ABORTED MICROSPORES (AMS) gene encodes a basic helix-loop-helix (bHLH) transcription factor that is required for tapetal cell development and postmeiotic microspore formation. However, the regulatory role of AMS in anther and pollen development has not been fully defined. Here, we show by microarray analysis that the expression of 549 anther-expressed genes was altered in ams buds and that these genes are associated with tapetal function and pollen wall formation. We demonstrate that AMS has the ability to bind in vitro to DNA containing a 6-bp consensus motif, CANNTG. Moreover, 13 genes involved in transportation of lipids, oligopeptides, and ions, fatty acid synthesis and metabolism, flavonol accumulation, substrate oxidation, methyl-modification, and pectin dynamics were identified as direct targets of AMS by chromatin immunoprecipitation. The functional importance of the AMS regulatory pathway was further demonstrated by analysis of an insertional mutant of one of these downstream AMS targets, an ABC transporter, White-Brown Complex homolog, which fails to undergo pollen development and is male sterile. Yeast two-hybrid screens and pull-down assays revealed that AMS has the ability to interact with two bHLH proteins (AtbHLH089 and AtbHLH091) and the ATA20 protein. These results provide insight into the regulatory role of the AMS network during anther development.

Imaging intracellular metabolite and protein changes in live mammalian cells with bright fluorescent RNA-based genetically encoded sensors.

Fluorescent RNA (FR)-based genetically encoded sensors have been engineered to detect various essential metabolites in living systems. However, the unfavorable characteristics of FR impede sensor applications. Here, we describe a strategy for converting Pepper fluorescent RNA into a series of fluorescent sensors to detect their cognate targets both in vitro and in live cells. Compared to previously developed FR-based sensors, Pepper-based sensors exhibited expanded emission of up to 620 nm and markedly improved cellular brightness, allowing robust and real-time monitoring of the pharmacologic-triggered dynamics changes in the intracellular level of S-adenosylmethionine (SAM) and the optogenetic manipulated protein translocation in live mammalian cells. Furthermore, signal amplification in fluorescence imaging of the target was achieved using the CRISPR-display strategy by incorporating a Pepper-based sensor into the sgRNA scaffold. Together, these results demonstrate that Pepper can be readily developed into high-performance FR-based sensors to detect various cellular targets.

Design of a palette of SNAP-tag mimics of fluorescent proteins and their use as cell reporters.

Naturally occurring fluorescent proteins (FPs) are the most widely used tools for tracking cellular proteins and sensing cellular events. Here, we chemically evolved the self-labeling SNAP-tag into a palette of SNAP-tag mimics of fluorescent proteins (SmFPs) that possess bright, rapidly inducible fluorescence ranging from cyan to infrared. SmFPs are integral chemical-genetic entities based on the same fluorogenic principle as FPs, i.e., induction of fluorescence of non-emitting molecular rotors by conformational locking. We demonstrate the usefulness of these SmFPs in real-time tracking of protein expression, degradation, binding interactions, trafficking, and assembly, and show that these optimally designed SmFPs outperform FPs like GFP in many important ways. We further show that the fluorescence of circularly permuted SmFPs is sensitive to the conformational changes of their fusion partners, and that these fusion partners can be used for the development of single SmFP-based genetically encoded calcium sensors for live cell imaging.

MicroRNA-204 is required for differentiation of human-derived cardiomyocyte progenitor cells.

Human cardiomyocyte progenitor cells (hCMPCs) are cardiac progenitor cells that are unique for their efficient differentiation into beating cardiomyocytes without requiring co-culture with neonatal cardiomyocytes. hCMPCs have shown great potential in preserving the function of infarcted mouse myocardium. MiRNA-204 has been reported to be up-regulated in differentiated hCMPCs, however, its biological significance is unclear. In this study, hCMPC proliferation, viability, apoptosis and necrosis were determined using the ELISA Kit (colorimetric BrdU detection), Cell Counting Kit-8, and Annexin V and propidium iodide staining, respectively. MiRNA-204 inhibition promoted hCMPC proliferation without affecting cell viability and the level of apoptosis and necrosis, indicating that miRNA-204 might be required for hCMPC differentiation. Quantitative reverse transcriptase-polymerase chain reactions were used to detect the expression profile of cardiac genes, including MEF2C, GATA-4, Nkx-2.5, TropT, ßMHC, and cActin. Cardiac α-actin staining was used to quantify the degree of differentiation. MiRNA-204 inhibition significantly down-regulated TropT, ßMHC, and cActin and reduced differentiation by 47.81% after 2 weeks of differentiation induction. Interestingly, miRNA-204 mimics (30 nM) did not promote hCMPC proliferation and differentiation. The bioinformatic tool GOmir identified the activating transcription factor 2 (ATF-2) as a potential target, which was confirmed by Western blot and a luciferase reporter assay. ATF-2 overexpression promoted hCMPC proliferation, further demonstrating the role played by ATF-2 as a target gene of miRNA-204. Therefore, miRNA-204 is required for hCMPC differentiation and ATF-2 is a target gene of miRNA-204 in hCMPCs. This study indicates that miRNA-204 is among the regulators that drive hCMPC proliferation and differentiation, and miRNA-204 might be used to influence cell fate.

Impact of P-Chloroaniline on Oxidative Stress and Biomacromolecules Damage in the Clam Ruditapes philippinarums: A Simulate Toxicity Test of Spill Incident.

As a hazardous chemical, p-chloroaniline (PCA) shows intensive adsorption and accumulation after entering the aquatic ecosystem, which can be enriched in organisms and cause damage. With the objective of achieving an integrated and mechanistic view of the toxic effects of PCA in the marine sentinel organism Ruditapes philippinarum, Manila clams were exposed to different concentration of PCA (0.5, 2 and 5 mg/L) for 15 days. Focusing on the gills, first targeting the toxic and digestive gland, the metabolic detoxification organ, we detected dose- and time-related changes inantioxidase activities and biomacromolecular damages in treated clams. Glutathione S-transferase (GST) activity and glutathione (GSH) contents were significantly induced, and superoxide dismutase (SOD) activity increased at the beginning of exposure and then decreased. The malondialdehyde (MDA) and protein methylation (PC) contents which represent lipid peroxidation and carbonylation of proteins, increased first with exposure time and then decreased in the digestive gland. DNA strand break levels were consistently higher than those in the control group. The digestive gland showed more sensitivity to the stress of PCA than the gills. GST and MDA in the gill and GST, GSH, SOD, DNA strand break level in the digestive gland showed significant correlation with PCA exposure, which indicated that these parameters can be used as sensitive biomarkers to indicate toxic effects from chloraniline leakage.

OsC6, encoding a lipid transfer protein, is required for postmeiotic anther development in rice.

Synthesis of lipidic components in anthers, including of the pollen exine, is essential for plant male reproductive development. Plant lipid transfer proteins (LTPs) are small, abundant lipid-binding proteins that have the ability to exchange lipids between membranes in vitro. However, their biological role in male reproductive development remains less understood. Here, we report the crucial role of OsC6 in regulating postmeiotic anther development in rice (Oryza sativa). Found in monocots, OsC6 belongs to a distinct clade from previously identified LTP1 and LTP2 family members found in both dicots and monocots. OsC6 expression is mainly detectable in tapetal cells and weakly in microspores from stage 9 to stage 11 of anther development. Immunological assays indicated that OsC6 is widely distributed in anther tissues such as the tapetal cytoplasm, the extracellular space between the tapetum and middle layer, and the anther locule and anther cuticle. Biochemical assays indicated that recombinant OsC6 has lipid binding activity. Moreover, plants in which OsC6 was silenced had defective development of orbicules (i.e. Ubisch bodies) and pollen exine and had reduced pollen fertility. Furthermore, additional evidence is provided that the expression of OsC6 is positively regulated by a basic helix-loop-helix transcription factor, Tapetum Degeneration Retardation (TDR). Extra granule-like structures were observed on the inner surface of the tdr tapetal layer when the expression of OsC6 was driven by the TDR promoter compared with the tdr mutant. These data suggest that OsC6 plays a crucial role in the development of lipidic orbicules and pollen exine during anther development in rice.

Molecular Cloning and Expression Profile of Class E Genes Related to Sepal Development in Nelumbo nucifera.

The lotus (Nelumbo Adans.) is an important aquatic plant with ornamental, medicinal and edible values and cultural connotations. It has single-, semi-double-, double- and thousand-petalled types of flower shape and is an ideal material for developmental research of flower doubling. The lotus is a basal eudicot species without a morphological difference between the sepals and petals and occupies a critical phylogenetic position in flowering plants. In order to investigate the genetic relationship between the sepals and petals in the lotus, the class E genes which affect sepal formation were focused on and analyzed. Here, SEPALLATA 1(NnSEP1) and its homologous genes AGAMOUS-LIKE MADS-BOXAGL9 (NnAGL9) and MADS-BOX TRANSCRIPTION FACTOR 6-like (NnMADS6-like) of the class E gene family were isolated from the flower buds of the Asian lotus (Nelumbo nucifera Gaertn.). The protein structure, subcellular localization and expression patterns of these three genes were investigated. All three genes were verified to locate in the nucleus and had typical MADS-box characteristics. NnSEP1 and NnMADS6-like were specifically expressed in the sepals, while NnAGL9 was highly expressed in the petals, suggesting that different developmental mechanisms exist in the formation of the sepals and petals in the lotus. The significant functional differences between NnSEP1, NnMADS6-like and NnAGL9 were also confirmed by a yeast two-hybrid assay. These results expand our knowledge on the class E gene family in sepal formation and will benefit fundamental research on the development of floral organs in Nelumbo.