Creating a novel herbicide-tolerance OsALS allele using CRISPR/Cas9-mediated gene editing

Weeds and weedy rice plague commercial rice fields in many countries. Developingherbicide-tolerance rice is the most efficient strategy to control weed proliferation. CRISPR/Cas9-mediated gene editing, which generates small InDels and nucleotide substitutions atand around target sites using error-prone non-homologous end joining DNA repairing, hasbeen widely adopted for generation of novel crop germplasm with a wide range of geneticvariation in important agronomic traits. We created a novel herbicide-tolerance allele inrice by targeting the acetolactate synthase (OsALS) gene using CRISPR/Cas9-mediated geneediting. The novel allele (G628W) arose from a G-to-T transversion at position 1882 of OsALSand conferred a high level of herbicide tolerance. Transgene-free progeny carryinghomozygous G628W allele were identified and showed agronomic performance similar tothat of wild-type plants, suggesting that the G628W allele is a valuable resource fordeveloping elite rice varieties with strong herbicide tolerance. To promote use of the G628Wallele and to accelerate introgression and/or pyramiding of the G628W allele with other elitealleles, we developed a DNA marker for the G628W allele that accurately and robustlydistinguished homozygous from heterozygous segregants. Our result further demonstratesthe feasibility of CRISPR/Cas9-mediated gene editing in creating novel genetic variation forcrop breeding.     

一种用于灵活配电系统故障程度预测的分散解决方案

随着各种分布式发电设备并入电网,传统的配电网已经发展为灵活配电网。分散发电设备的并入所带来的重大挑战之一是故障电流可能超过相关断路器的允许容量。介绍一种分散解决方案,可以依附于配电馈线继电保护装置,仅利用就地测量的电压和电流即可在线计算出预计故障程度。首先,通过所测的电流和电压变化计算等量电阻以及等量源电压,然后,可以通过计算出的电阻和背侧源电压得出预计故障程度。试验结果证明了该方案的有效性。     

Cellular mechanisms underlying Hyperin-induced relaxation of rat basilar artery

Background and aim

Hyperin, a flavonol compound extracted from the Chinese herb Abelmoschus manihot L. Medic, is reported to exert protective actions in cerebral ischemic injury. The specific aim of the present study was to study the relaxation of Hyperin in rat isolated basilar artery and identify the underlying cellular mechanisms.

Methods

Rat isolated basilar artery segments were cannulated and perfused while being superfused with PSS solution. Vessel images were recorded by video microscopy and diameters measured. Membrane potential was recorded using glass microelectrodes to evaluate the basilar artery smooth muscle cell hyperpolarization.

Results

Perfusion of Hyperin (1 ~ 100 μM) elicited a concentration-dependent relaxation of basilar artery segments preconstricted with 0.1 μM U46619. The response was significantly inhibited by the removal of the endothelium. Hyperin also elicited marked and concentration-dependent hyperpolarization of smooth muscle cells. 30 μM nitro-L-arginine (an inhibitor of nitric oxide synthase) and indomethacin (an inhibitor of cyclooxygenase), partially inhibited Hyperin-induced relaxation and hyperpolarization leaving an attenuated, but significant, endothelium-dependent relaxation and hyperpolarization. This remaining effect was almost completely blocked by 1 mM tetraethylammonium (an inhibitor of Ca²⁺-activated K⁺ channels), or by 100 μM DL-propargylglycine, an inhibitor of cystathionine-γ-lyase (a synthase of the endogenous H₂S).

Conclusion

These findings show that Hyperin produces significant hyperpolarization in rat basilar artery smooth muscle cells and relaxation through both endothelium-dependent and endothelium-independent mechanisms. The underlying mechanisms appeared to be multi-factorial involving nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). Our data further suggest that endogenous H₂S is a component of the EDHF-mediated hyperpolarization and relaxation to Hyperin.     

Intestinal absorption of forsythoside A in different compositions of Shuang-Huang-Lian

Shuang-Huang-Lian (SHL), a traditional Chinese formula containing Lonicerae japonicae flos (LJF), Scutellariae radix (SR) and Forsythiae fructus (FF), is commonly used to treat acute upper respiratory tract infection, acute bronchitis and light pneumonia. Forsythoside A is one of the main active ingredients in Forsythiae fructus, a key herb in SHL. In the present study, effects of different compositions in SHL on the intestinal absorption of forsythoside A were investigated. The observations from in situ intestinal circulation model showed that A/%(h^− 1) of forsythoside A in FF + LSF, FF + SR and SHL were all reduced greatly compared with that in FF. However, in pharmacokinetics study, C_max and AUC_0 → 1440 of forsythoside A all increased and T_1/2 prolonged in SHL, FF + LJF and FF + SR compared with FF. The results indicated that the different compositions of SHL decreased absorption but increased bioavailability of forsythoside A, which may be related to its metabolism inhibited in intestine or liver.     

Aluminum-induced decrease in CO2 assimilation in citrus seedlings is unaccompanied by decreased activities of key enzymes involved in CO2 assimilation

'Cleopatra' tangerine (Citrus reshni Hort. ex Tanaka) seedlings were irrigated daily for 8 weeks with 1/4 strength Hoagland's nutrient solution containing 0 (control) or 2 mM aluminum (Al). Leaves from Al-treated plants had decreased CO2 assimilation and stomatal conductance, but increased intercellular CO2 concentrations compared with control leaves. On a leaf area basis, 2 mM Al increased activities of key enzymes in the Calvin cycle, including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), stromal fructose-1,6-bisphosphatase (FBPase), and a key enzyme in starch synthesis, ADP-glucose pyrophosphorylase (AGPase), compared with control leaves. Aluminum had no effect on cytosolic FBPase activity, but it decreased sucrose phosphate synthase (SPS) activity. Aluminum had no effect on area-based concentrations of carbohydrates, glucose-6-phosphate (G6P) and fructose 6-phosphate (F6P) or the G6P:F6P ratio, but it decreased the area-based concentration of 3-phosphoglycerate (PGA). Photochemical quenching coefficient (qP) and electron transport rate through PSII were greatly reduced by Al. Non-photochemical quenching coefficient (NPQ) was less affected by Al than qP and electron transport rate through PSII. We conclude that the reduced rate of CO2 assimilation in Al-treated leaves was probably caused by a combination of factors such as reduced electron transport rate through PSII, increased closure of PSII reaction centers and increased photorespiration.     

Three new water-soluble alkaloids from the leaves of Suregada glomerulata (Blume) Baill

Three new water-soluble alkaloids (1-3) together with twelve known compounds (4-15) have been isolated from the water extract of leaves of Suregada glomerulata. Their structures were determined by spectroscopic analysis and chemical method. Compounds 1-3 were evaluated for their in vitro inhibitory activity against α-glucosidase and HIV-1 replication. However, no significant activities were found.     

Anti-inflammatory, antiviral and quantitative study of quercetin-3-O-β-D-glucuronide in Polygonum perfoliatum L

Quercetin-3-O-β-D-glucuronide, isolated from Polygonum perfoliatum L., was evaluated by antiviral efficacy against influenza A virus and anti-inflammatory activity in vivo in mouse, and it was used for quality evaluation of P. perfoliatum L.. In vivo study, oral administration of quercetin-3-O-β-D-glucuronide significantly suppressed ear edema induced by dimethyl benzene and peritoneal permeability induced by acetic acid in mice, and quercetin-3-O-β-D-glucuronide also showed to possess inhibitory activity against influenza A virus (FLUAV). In the present study, additionally, a rapid, simple and sensitive method for quantitative analysis of quercetin-3-O-β-D-glucuronide in P. perfoliatum L. was developed using high performance liquid chromatography (HPLC) coupled with photodiode array detection. The separation was carried out on a Lichrosher-C18 column (250 mm × 4.6 mm, 5 μm) together with a C18 guard column at isocratic elution systems of methanol (A) and 0.05% aqueous phosphoric acid (B) (43:57, v/v) with detection wavelength at 258 nm and column temperature at 30 °C. The method was validated for linearity, repeatability, limit of quantification (LOQ), precision and robustness. The contents of quercetin-3-O-β-D-glucuronide in 28 samples from different regions of China were between 0.06% and 2.09%. The developed analytical method was applied to investigate P. perfoliatum L. and for quality control of the herb.     

Effects of the exopolysaccharide fraction (EPSF) from a cultivated Cordyceps sinensis on immunocytes of H22 tumor bearing mice

In order to explore the effects of exopolysaccharide fraction (EPSF) from one of the anamorph strains of Cordyceps sinensis on immunocyte activity of H22 tumor bearing mice, ICR mice were treated with EPSF for 7 days by intraperitoneal injection at doses of 15 mg/kg (low-dose), 30 mg/kg (mid-dose) and 60 mg/kg (high-dose) after H22 tumor cells were implanted. At the end of the experiments, the tumor weight of each mouse was measured. Phagocytosis of mouse peritoneal macrophages was tested by neutral red uptake. The TNF-alpha expression of macrophages was assayed by ELISA. Proliferation ability and cytotoxicity of spleen lymphocytes were determined by MTT methods. The mRNA levels of cytokine TNF-alpha and IFN-gamma mRNA of spleen lymphocytes were detected by RT-PCR. The results indicated that EPSF not only significantly inhibited the H22 tumor growth, but also significantly elevated immunocytes' activity. It significantly enhanced the phagocytosis capacity of peritoneal macrophages and proliferation ability of spleen lymphocytes at all the three doses; it significantly promoted macrophages' TNF-alpha expression and spleen lymphocytes' cytotoxicity. EPSF also significantly elevated TNF-alpha and IFN-gamma mRNA expression of splenic lymphocytes. This experimental finding suggests that EPSF could elevate the immunocytes' activity in H22 tumor bearing mice.