Novel diagnostic biomarkers related to necroptosis and immune infiltration landscape in acute myocardial infarction.

Background: Acute myocardial infarction (AMI) can occur suddenly, which may induce deadly outcomes, and the population suffering from AMI presents a younger trend. Necroptosis, the new cell necrosis type, is associated with the pathogenic mechanisms of diverse cardiovascular diseases (CVDs). Its diagnostic value and molecular mechanisms in AMI are still unclear. Objective: This study focused on determining key necroptosis-related genes as well as immune infiltration in AMI. Methods: We first examined the GSE66360 dataset for identifying necroptosis-related differentially expressed genes (NRDEGs). Thereafter, GO and functional annotation were performed, then a PPI network was built. In addition, "CIBERSORT" in R was applied in comparing different immune infiltration degrees in AMI compared with control groups. The receiver operating characteristic (ROC) curve was plotted to evaluate whether hub NRDEGs could be used in AMI diagnosis. Associations of immune cells with candidate NRDEGs biomarkers were examined by Spearman analysis. Finally, hub NRDEGs were validated by cell qPCR assays and another two datasets. Results: A total of 15 NRDEGs were identified and multiple enrichment terms associated with necroptosis were discovered through GO and KEGG analysis. Upon module analysis, 10 hub NRDEGs were filtered out, and the top six hub NRDEGs were identified after ROC analysis. These top six NRDEGs might have a certain effect on modulating immune infiltrating cells, especially for mast cells activated, NK cells activated and neutrophils. Finally, two AMI datasets and qPCR assay came to identical findings. Conclusion: Our results offer the reliable molecular biomarkers and new perspectives for necroptosis in AMI, which lay a certain foundation for developing novel anti-AMI therapeutic targets.

Occurrence of microplastic pollution in rivers globally: Driving factors of distribution and ecological risk assessment.

Microplastics, as global emerging pollutants, have received significant attention worldwide due to their ubiquitous presence in the rivers. However, there is still a lack of clarity on the occurrence, driving factors, and ecological risks of microplastics in rivers worldwide. In this study, a global microplastic dataset based on 862 water samples and 445 sediment samples obtained from 63 articles was constructed, which revealed the temporal and spatial distribution of abundance and morphological characteristics of microplastics in rivers across the globe. In global rivers, the abundance of MPs in both water and sediment spans across 10 and 4 orders of magnitude, respectively. The MP comprehensive diversity index based on the physical morphological characteristics of MPs indicated a significant positive correlation between the pollution sources of MPs in different environmental media. Based on the data was aligned to the full-scale MPs, a novel framework was provided to evaluate the ecological risk of MPs and the interaction effects between the influencing factors driving the distribution characteristics of MPs in rivers around the world. The results obtained demonstrated a wide variation in the key driving factors affecting the distribution of microplastics in different environmental media (water and sediment) in rivers globally. The diversity indices of the morphological characteristics of MPs in densely populated areas of lower-middle income countries in Asia were significantly higher, implying that the sources of microplastics in these regions are more complex and extensive. More than half of the rivers are exposed to potential ecological risks of MPs; however, microplastics may pose only immediate risks to aquatic species in Burigang River, Bangladesh. This can provide valuable insights for formulating more effective scientific strategies for the management of MP pollution in rivers.

Untargeted Plasma Lipidomics Reveal Perturbed Metabolites of Glycerophospholipids, and Sphingolipids in Moderate-to-Severe Acne.

Background: Acne vulgaris (AV) is a common inflammatory disorder involving the pilosebaceous unit. The study aimed to explore the plasma lipidome signatures and identify specific lipid biomarkers in moderate-to-severe acne patients. Patients and Methods: Untargeted plasma lipidomic analysis using ultra-high performance liquid chromatography system (UHPLC) coupled to q-extraction plus was employed on 30 moderate-to-severe acne patients aged between 16-25 years and 30 healthy controls. Multivariate data analyses were used to identify the distinguishing lipid metabolites. Results: All 1449 species of 37 lipid subclasses were identified from the MS data. There were apparent differences in plasma lipid profiles between acne groups and control groups. With variable influence on projection (VIP) > 1.0 and P-value < 0.05, 26 significantly different lipid metabolites were identified. These metabolites consisted mainly of glycerophospholipids (GPs), sphingolipids (SPs), and glycerolipids (GLs). Combining with AUC≥0.800 as the elected criteria, we obtained five differential lipids with good diagnostic performance for acne severity, including 2 sphingomyelins (SM), 1 phosphatidylglycerol (PG), 1 trihexosylceramide (Hex3Cer), and 1 Phosphatidylcholine (PC). Among them, PG (44:0) had the highest AUC values. Conclusion: Our study revealed the plasma lipidome signature of patients with moderate-to-severe acne. The results will provide a novel light into the perturbed lipid metabolism leading to the development of acne.

Comparative volatiles profiling in milk-flavored white tea and traditional white tea Shoumei via HS-SPME-GC-TOFMS and OAV analyses.

White tea is a mildly fermented tea processed with withering and drying. Milk-flavored white tea has a unique milk flavor compared to the traditional white tea. Little is known about the aromas that make white tea taste milky. Here we conducted the volatile profiling via headspace solid-phase microextraction (HS-SPME)-gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and chemometrics to explore the key volatiles making milk-flavored white tea taste milky. Sixty-seven volatiles were identified, with 7 volatiles (OAV > 1 and VIP > 1) were characterized as the typical aromas. Green and light fruity scent volatiles, such as methyl salicylate, benzyl alcohol, and phenylethyl alcohol, were richer in TFs than MFs. Strong fruity and cheese aromas, such as dihydro-5-pentyl-2(3H)-furanone, 2-pentyl-furan, (E)-6,10-dimethyl-5,9-undecadien-2-one, and hexanal, were more abundant in MFs than TFs. Dihydro-5-pentyl-2(3H)-furanone, recognized as coconut and creamy aroma, should be the essential volatile for milky flavor. Also, (E)-6,10-dimethyl-5,9-undecadien-2-one and 2-pentyl-furan may contribute to the milk scent formation.

Comparison of a direct aspiration first pass technique vs. stent retriever thrombectomy for the treatment of acute large vessel occlusion stroke in the anterior circulation with atrial fibrillation.

Objectives: The stent retriever thrombectomy (SRT) and a direct aspiration first-pass technique (ADAPT) are the two main mechanical thrombectomy (MT) techniques for acute ischemic stroke. Few data are available for comparing the therapeutic effects associated with the two mechanical thrombectomy techniques in acute ischemic stroke with atrial fibrillation. The purpose of this study was to compare the efficacy and safety of both techniques for the treatment of acute large vessel occlusion stroke in the anterior circulation with atrial fibrillation. Methods: Retrospective analysis was performed in stroke patients with atrial fibrillation admitted to Guangzhou Red Cross Hospital from January 2018 to June 2022 who received mechanical thrombectomy by either SRT or ADAPT. Comparisons were made with regards to the initial traits, course of therapy, effectiveness indicators, and complications of these individuals. The primary outcome is recanalization rate. Results: In this study, after screening 431 patients, 92 eligible patients, with 48 patients received SRT and 44 patients received ADAPT, were included. There was no significant difference in the recanalization rate between the two groups (SRT 87.5% vs. ADAPT 84.1%, P = 0.639). Compared with SRT, patients in ADAPT group had a shorter puncture to recanalization time [33.5 min (27.0-59.5) vs. 50.5 min (31.5-91.5), P = 0.009], a higher first pass success recanalization rate (54.5 vs. 33.3%, p = 0.040), and a higher rate of patients with improvement of NIHSS scores ≥4 at discharge (84.1 vs. 56.3%, P = 0.004). However, distal embolization occurred more frequently in the ADAPT group than that in SRT group (50.0 vs. 22.9%, P = 0.007). There was no significant difference between the two groups in the 3-month mRS score, symptomatic cerebral hemorrhage, or mortality. Conclusions: Compared with SRT, ADAPT has similar recanalization rate for the treatment of acute large vessel occlusion stroke in the anterior circulation with atrial fibrillation. However, ADAPT might be more effective in terms of shorter puncture to recanalization time and higher first pass success recanalization rate. Further studies are needed for confirming our results.

Integrated metabolomic and transcriptomic analysis reveal the effect of mechanical stress on sugar metabolism in tea leaves (Camellia sinensis) post-harvest.

Sugar metabolites not only act as the key compounds in tea plant response to stress but are also critical for tea quality formation during the post-harvest processing of tea leaves. However, the mechanisms by which sugar metabolites in post-harvest tea leaves respond to mechanical stress are unclear. In this study, we aimed to investigate the effects of mechanical stress on saccharide metabolites and related post-harvest tea genes. Withered (C15) and mechanically-stressed (V15) for 15 min Oolong tea leaves were used for metabolome and transcriptome sequencing analyses. We identified a total of 19 sugar metabolites, most of which increased in C15 and V15. A total of 69 genes related to sugar metabolism were identified using transcriptome analysis, most of which were down-regulated in C15 and V15. To further understand the relationship between the down-regulated genes and sugar metabolites, we analyzed the sucrose and starch, galactose, and glycolysis metabolic pathways, and found that several key genes of invertase (INV), α-amylase (AMY), ß-amylase (BMY), aldose 1-epimerase (AEP), and α-galactosidase (AGAL) were down-regulated. This inhibited the hydrolysis of sugars and might have contributed to the enrichment of galactose and D-mannose in V15. Additionally, galactinol synthase (Gols), raffinose synthase (RS), hexokinase (HXK), 6-phosphofructokinase 1 (PFK-1), and pyruvate kinase (PK) genes were significantly upregulated in V15, promoting the accumulation of D-fructose-6-phosphate (D-Fru-6P), D-glucose-6-phosphate (D-glu-6P), and D-glucose. Transcriptome and metabolome association analysis showed that the glycolysis pathway was enhanced and the hydrolysis rate of sugars related to hemicellulose synthesis slowed in response to mechanical stress. In this study, we explored the role of sugar in the response of post-harvest tea leaves to mechanical stress by analyzing differences in the expression of sugar metabolites and related genes. Our results improve the understanding of post-harvest tea's resistance to mechanical stress and the associated mechanism of sugar metabolism. The resulting treatment may be used to control the quality of Oolong tea.

Curcumin exerts a protective effect on murine knee chondrocytes treated with IL-1ß through blocking the NF-κB/HIF-2α signaling pathway.

BACKGROUND: Osteoarthritis (OA) is characterized by erosion and degradation of articular cartilage. This study assessed the effects of curcumin on mouse knee cartilage chondrocytes. METHODS: Chondrocytes were treated for 24 hours with interleukin IL-1ß (10 ng/mL) alone, or the combination of curcumin (10, 20, and 50 µM) and IL-1ß. The proliferation, viability, and cytotoxicity of the chondrocytes were evaluated by the MTS assay. Expression of SOX9, AGG, Col2α, MMP9, ADAMTS5, COX2, iNOS, pIκB-α, pNF-κB, and hypoxia-inducible factor-2α (HIF-2α) were detected by western blotting or quantitative polymerase chain reaction (q-PCR). Nuclear translocation of NF-κB and HIF-2α were investigated by immunofluorescence and immunohistochemistry. In in vivo experiments, mice were subjected to destabilization of the medial meniscus (DMM) and given curcumin orally for 6 weeks. Cartilage integrity was evaluated by OARSI (Osteoarthritic Research Society International) scores. RESULTS: Curcumin significantly inhibited the IL-1ß-induced reduction of cell viability, degradation of ECM, and the expression of SOX9, Col2α, and AGG (P<0.01). Western blotting, immunofluorescence and immunohistochemistry experiments demonstrated that curcumin dramatically inhibited the activation of NF-κB/HIF-2α in chondrocytes treated with IL-1ß (P<0.01). The articular scores were significantly lower in the DMM-induced OA mice compared to OA mice treated with curcumin (P<0.01). CONCLUSIONS: Curcumin may have the potential to inhibit OA development, partly through suppressing the activation of the NF-κB/HIF-2α pathway.

A genome-wide survey of interaction between rice and Magnaporthe oryzae via microarray analysis.

The main aim of the work is to study the regulation of gene expression in the interaction between rice and Magnaporthe oryzae by gene chip technology. In this study, we mainly focused on changes of gene expression at 24, 48, and 72 hours post-inoculation (hpi), through which we could conduct a more comprehensive analysis of rice blast-related genes in the process of infection. The results showed that the experimental groups contained 460, 1227, and 3937 significant differentially expressed genes at 24, 48, and 72 hpi, respectively. Furthermore, 115 significantly differentially expressed genes were identified in response to rice blast infection at all three time points. By annotating these 115 genes, they were divided into three categories: metabolic pathways, proteins or enzymes, and organelle components. As expected, many of these genes were known rice blast-related genes; however, we discovered new genes with high fold changes. Most of them encoded conserved hypothetical proteins, and some were hypothetically conserved genes. Our study may contribute to finding new resistance genes and understanding the mechanism of rice blast development.

Rice OsAAA-ATPase1 is Induced during Blast Infection in a Salicylic Acid-Dependent Manner, and Promotes Blast Fungus Resistance.

Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown (OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1-6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice's defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae; conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.

Correction: Towards a Better Detection of Horizontally Transferred Genes by Combining Unusual Properties Effectively.

[This corrects the article DOI: 10.1371/journal.pone.0043126.].

Survey and analysis for impact factors of psychological distress in HIV-infected pregnant women who continue pregnancy.

Objective: The objective of this study is to explore the psychological distress of HIV-infected pregnant women who continue pregnancy, and analyze the possible influencing factors. Methods: A total of 194 HIV-infected pregnant women who continue pregnancy were enrolled for this study by a convenient sampling method during June 2012-August 2016. Participants completed questionnaires including Hospital Anxiety and Depression Scale (HADS), Berger HIV Stigma Scale (BHSS), Distress Thermometer (DT) and Problem List (PL), and to determine the cut-off value of DT in the group. Results: The positive detection rate of psychological distress in the HIV-infected pregnant women who continue pregnancy was 69.1%, and the highest frequency of PL was the emotional problems. The positive detection rate of anxiety was 60.8%, the positive detection rate of depression was 54.1%, and the discrimination score was 113.16 ± 19.21. Spearman relevant analysis showed that psychological distress score was positively correlated with anxiety, depression and discrimination score (p < .001). Multiple linear regression analysis showed that relationship between husband and wife, family misfortune, Medicaid, chronic disease or high-risk pregnancy, viral load, CD4+T cell count, infection and confidentiality could affect the psychological distress (p < .05). The ideal cut-off value of DT in the group was 5. Conclusion: HIV-infected pregnant women who continue pregnancy have higher incidence of psychological distress, and the psychological distress is not inferior to cancer patients. The influencing factors are mainly related to the infection and pregnancy characteristics, and have nothing to do with the general social demographic characteristics. The DT can be used as a screening tool to quickly identify psychological distress of the group.

Anti-inflammatory mechanisms of IFN-γ studied in experimental autoimmune encephalomyelitis reveal neutrophils as a potential target in multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) mediated by T helper (h)1 and/or Th17 CD4 T cells that drive inflammatory lesion development along with demyelination and neuronal damage. Defects in immune regulatory mechanisms are thought to play a role in the pathogenesis of MS. While an early clinical trial indicated that IFN-γ administration was detrimental to MS, studies in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), indicated that IFN-γ exhibits a number of anti-inflammatory properties within the CNS. These mechanisms include inhibition of IL-17 production, induction of regulatory T cells, T cell apoptosis and regulation of chemokine production. Mice deficient in IFN-γ or its receptor were instrumental in deciphering the anti-inflammatory properties of IFN-γ in the CNS. In particular, they revealed that IFN-γ is a major regulator of neutrophil recruitment into the CNS, which by a variety of mechanisms including disruption of the blood-brain-barrier (BBB) and production of reactive oxygen species are thought to contribute to the onset and progression of EAE. Neutrophils were also shown to be instrumental in EAE relapses. To date neutrophils have not been appreciated as a driver of MS, but more recently based largely on strong EAE data this view is being reevaluated by some investigators in the field.

Molecular characterization and application of a novel cytoplasmic male sterility-associated mitochondrial sequence in rice.

BACKGROUND: Cytoplasmic male sterility (CMS) is a maternally inherited inability to produce functional pollen found in numerous flowering plant species. CMS is associated with mitochondrial DNA mutation, novel chimeric open reading frames (ORFs), and rearrangement of coding and noncoding regions of the mitochondrial genome. RESULTS: BLAST (Basic Local Alignment Search Tool) analysis indicated that L-sp1, a new sequence-characterized amplified region, is non-homologous to atp6-orfH79 (or atp6-orf79) and WA352 cloned CMS-associated genes. L-sp1 was found in 11 of 102 wild rice accessions belonging to four AA genome species: Oryza rufipogon, Oryza nivara, Oryza glumaepatula, and Oryza meridionalis. Using L-sp1, two new CMS lines were developed, from either low natural fertility plants or sterile plants, by backcrossing BC1F1 with Yuetai B. Northern blot and RT-PCR revealed that L-sp1 was only expressed in the anthers of w1/YTB, w2/YTB, w1/YTB//YTB, and w2/YTB//YTB when in the same cytoplasm background. CONCLUSIONS: L-sp1 is a single-copy chimeric CMS-associated gene found in the mitochondrial genome. It can be expressed in anthers with the same specific cytoplasm background, and will be a useful molecular marker for the development and marker-assisted selection of new CMS lines.

Efficacy and safety of selenium nanoparticles administered intraperitoneally for the prevention of growth of cancer cells in the peritoneal cavity.

Peritoneal implantation of cancer cells, particularly postoperative seeding metastasis, frequently occurs in patients with primary tumors in the stomach, colon, liver, and ovary. Peritoneal carcinomatosis is associated with poor prognosis. In this work, we evaluated the prophylactic effect of intraperitoneal administration of selenium (Se), an essential trace element and a putative chemopreventive agent, on peritoneal implantation of cancer cells. Elemental Se nanoparticles were injected into the abdominal cavity of mice, into which highly malignant H22 hepatocarcinoma cells had previously been inoculated. Se concentrations in the cancer cells and tissues, as well as the efficacy of proliferation inhibition and safety, were evaluated. Se was mainly concentrated in cancer cells compared to Se retention in normal tissues, showing at least an order of magnitude difference between the drug target cells (the H22 cells) and the well-recognized toxicity target of Se (the liver). Such a favorable selective distribution resulted in strong proliferation suppression without perceived host toxicity. The mechanism of action of the Se nanoparticle-triggered cytotoxicity was associated with Se-mediated production of reactive oxygen species, which impaired the glutathione and thioredoxin systems. Our results suggest that intraperitoneal administration of Se is a safe and effective means of preventing growth of cancer cells in the peritoneal cavity for the above-mentioned high-risk populations.

Serum thioredoxin reductase levels increase in response to chemically induced acute liver injury.

BACKGROUND: Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low. METHODS: Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl4). RESULTS: TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT. CONCLUSIONS: Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury. GENERAL SIGNIFICANCE: The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions.

CLAVATA3 dodecapeptide modified CdTe nanoparticles: a biocompatible quantum dot probe for in vivo labeling of plant stem cells.

CLAVATA3 (CLV3) dodecapeptides function in plant stem cell maintenance, but CLV3 function in cell-cell communication remains less clear. Here, we coupled CLV3 dodecapeptides to synthesized CdTe nanoparticles to track their bioactivity on stem cells in the root apical meristem. To achieve this, we first synthesized CdTe quantum dots (QDs) using a one-pot method, and then evaluated the cytotoxicity of the QDs in BY-2 cells. The results showed that QDs in plant cells must be used at low concentrations and for short treatment time. To make biocompatible probes to track stem cell fate, we conjugated CLV3 dodecapeptides to the QDs by the zero-coupling method; this modification greatly reduced the cytotoxicity of the QDs. Furthermore, we detected CLV3-QDs localized on the cell membrane, consistent with the known localization of CLV3. Our results indicate that using surface-modified QDs at low concentrations and for short time treatment can improve their utility for plant cell imaging.

Towards a better detection of horizontally transferred genes by combining unusual properties effectively.

BACKGROUND: Horizontal gene transfer (HGT) is one of the major mechanisms contributing to microbial genome diversification. A number of computational methods for finding horizontally transferred genes have been proposed in the past decades; however none of them has provided a reliable detector yet. In existing parametric approaches, only one single compositional property can participate in the detection process, or the results obtained through each single property are just simply combined. It's known that different properties may mean different information, so the single property can't sufficiently contain the information encoded by gene sequences. In addition, the class imbalance problem in the datasets, which also results in great errors for the gene detection, hasn't been considered by the published methods. Here we developed an effective classifier system (Hgtident) that used support vector machine (SVM) by combining unusual properties effectively for HGT detection. RESULTS: Our approach Hgtident includes the introduction of more representative datasets, optimization of SVM model, feature selection, handling of imbalance problem in the datasets and extensive performance evaluation via systematic cross-validation methods. Through feature selection, we found that JS-DN and JS-CB have higher discriminating power for HGT detection, while GC1-GC3 and k-mer (k = 1, 2, …, 7) make the least contribution. Extensive experiments indicated the new classifier could reduce Mean error dramatically, and also improve Recall by a certain level. For the testing genomes, compared with the existing popular multiple-threshold approach, on average, our Recall and Mean error was respectively improved by 2.81% and reduced by 26.32%, which means that numerous false positives were identified correctly. CONCLUSIONS: Hgtident introduced here is an effective approach for better detecting HGT. Combining multiple features of HGT is also essential for a wider range of HGT events detection.

Genetical and molecular analysis reveals a cooperating relationship between cytoplasmic male sterility- and fertility restoration-related genes in Oryza species.

Although the characterization of genes associated with cytoplasmic male sterility (CMS) and fertility restoration (Rf) has been well documented, the evolutionary relationship between nuclear Rf and CMS factors in mitochondria in Oryza species is still less understood. Here, 41 accessions from 7 Oryza species with AA genome were employed for analyzing the evolutionary relationships between the CMS factors and Rf candidates on chromosome 10. The phylogenetic tree based on restriction fragment length polymorphism patterns of CMS-associated mitochondrial genes showed that these 41 Oryza accessions fell into 3 distinct groups. Another phylogenetic tree based on PCR profiles of the nuclear Rf candidates on chromosome 10 was also established, and three groups were distinctively grouped. The accessions in each subgroup/group of the two phylogenetic trees are well parallel to each other. Furthermore, the 41 investigated accessions were test-crossed with Honglian (gametophytic type) and Wild-abortive (sporophytic type) CMS, and 5 groups were classified according to their restoring ability. The accessions in the same subgroup of the two phylogenetic trees shared similar fertility restoring pattern. Therefore, we conclude that the CMS-associated mitotypes are compatible to the Rf candidate-related nucleotypes, CMS and Rf have a parallel evolutionary relation in the Oryza species.

Gametophytically alloplasmic CMS line of rice (Oryza sativa L.) with variant orfH79 haplotype corresponds to specific fertility restorer.

For years discovery and identification of the cytoplasmic male sterility (CMS) resource in wild rice is the most intriguing events in breeding field. orfH79, a chimeric gene in mitochondria, has been suggested being the determinant for Honglian CMS in rice. In this report orfH79 gene as molecular marker to screen the wild rice, we found eight accessions with orfH79 gene in the total 42 investigated objects. Sequence analysis revealed that there were a total of nine nucleotide substitutions resulting in the change of nine amino acids in the newly identified orfH79 in wild rice, which further fell into seven haplotypes. In order to investigate the underlying relationship between orfH79 haplotypes and the corresponding fertility restorers, four accessions were selected with different orfH79 haplotype as female parents to hybridize the Honglian maintainer line, Yuetai B. After eight consecutive recurrent backcrosses, four alloplasmic CMS lines with different orfH79 haplotype were developed. Microscopic observation exhibited that their pollen grains were spherical and clear in 1% I(2)-KI solution same as that of Honglian CMS line. Moreover, these four CMS lines displayed various fertility restoring model through test cross, suggesting that each orfH79 haplotye represents a new CMS type and corresponds to their specific Rf allele.

[Construction of T7 phage display library from the anther of Honglian hybrid line of rice].

Phage display is a powerful method to study protein-protein interactions. In order to study the molecular mechanism of cytoplasmic male sterility and fertility restoration in Honglian rice, the mRNA was isolated with PolyA Tract mRNA Isolation Kit from the anther of F1 hybrid rice and the double strand (ds) cDNA was synthesized by reverse transcription. Then the directional EcoRI /Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoR I and Hind, which resulted in ds cDNA with EcoR I and Hind III ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated with Mini Column, then ligated into the T7 Select 10-3b vertor with EcoR I and Hind III ends. After packaging in vitro, the T7 Select 10-3b vertor was transformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03 x 106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14 x 1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.