Effect of celangulin V on detoxification enzymes in Mythimna separata and Agrotis ypsilon

Celangulin V (CA-V), a β-dihydroagrofuran sesquiterpene polyol ester, is extracted from the root bark of Chinese bittersweet, Celastrus augulatus Maxim. It exhibited selective toxicity against different insects. By CO-difference spectral and biochemical method, the effects of CA-V on two kinds of detoxification enzymes, cytochrome P450 (P450 and NADPH-cytochrome P450 reductase) and glutathione S-transferase, were investigated in oriental armyworm, Mythimna separata and black cutworm, Agrotis ypsilon. CA-V showed higher induction against P450 of M. separata than that of A. ypsilon. Treated by CA-V, the maximum absorption of M. separata increased 1.2 and 0.8 nm than the control, respectively. Meanwhile, compared with the control, the P450 content and NADPH-P450 reductase activity in treated M. separata larvae increased 1.46-, 2.26- and 1.26-, 2.56-fold, respectively. But in treated A. ypsilon larvae, they all increased a little more than those of control. So far as M. separata and A. ypsilon, whether there is exposure of CA-V or not, the P450 content and GST activity in A. ypsilon were obviously higher than those in M. separata. It suggested that the content or activity difference of these two kinds of detoxification enzymes may have important roles in the selective toxicity of CA-V in M. separata and A. ypsilon.     

荚膜红细菌DSM1710中硫化物醌还原酶基因的克隆、表达及活性分析

为获得有活性的硫化物醌还原酶(sulfide-quinone reductase,SQR)并研究其功能,试验利用常规PCR方法从荚膜红细菌(Rhodobacter capsulatus)中克隆了SQR基因全长CDS区1284 bp;成功构建了SQR基因的原核表达载体pRSETA-SQR,并转入大肠杆菌中进行诱导表达。SDS-PAGE和Western blotting结果显示,SQR融合蛋白成功表达,在37 ℃条件下,0.4 mmoL/L IPTG诱导表达6 h,50 ku的SQR融合蛋白表达量最高。可溶性分析结果表明,SQR重组蛋白以包涵体形式存在,经过纯化、复性后得到较高纯度的SQR活性蛋白。蛋白活性分析结果表明,该酶具有明显的消化底物decyl-UQ的能力,测定其酶活Km值约为4。     

紫花苜蓿二氢黄酮醇还原酶基因(MsDFR)的克隆与分析

二氢黄酮醇还原酶(dihydroflavonol reductase,DFR)是缩合单宁生物合成途径中的关键酶,在单宁的合成中起着重要的作用。根据同源克隆的原理,利用RACE技术,从“中苜一号”苜蓿中克隆得到DFR基因(MsDFR),并对其进行了序列分析及不同胁迫条件下的表达模式分析。结果表明,MsDFR基因cDNA全长1 402 bp,包括开放阅读框1 023 bp,编码340个氨基酸,在N端存在1个NADP结合位点“VTGASGFIGSWLVMRLMERGY”,中部存在1个底物特异性结合的氨基酸基序“TLNVTEDQKPLWDESCWSDVEFCRRV”。实时荧光定量PCR结果表明,该基因在荚果中表达量较高,根中较弱;在NaCl和GA₃诱导下,MsDFR基因表达受到抑制;在黑暗条件下,该基因被诱导表达。由此推测“中苜一号”苜蓿中可能存在不依赖于GA₃信号的单宁合成途径。     

Chronic response of Anabaena fertilissima Rao, C.B. on growth, metabolites and enzymatic activities by chlorophenoxy herbicide

Study was carried out to investigate the chronic response of cyanobacteria, Anabaena fertilissima to chlorophenoxy herbicide 2,4-dichlorophynoxyacetic Acid (2,4-D) ethyl ester at different concentrations 15, 30 and 60 ppm. The influence of 2,4-D on growth (pigments), release of metabolites such as carbohydrates, protein, amino acid, phenols and nitrate reductase and glutamine synthetase activities was analyzed. The test concentrations caused a concentration-dependent decrease in pigments. Depletion in carbohydrate and protein content was registered with rise in herbicide concentrations. However, phenols were found to rise with increased herbicide concentrations but amino acids were reported to decline. The inhibition of nitrate reductase and glutamine synthetase activity was also concentration-dependent and showed more sensitivity for substituted phenoxy herbicide. This study therefore suggests that decrease in metabolite content and enzyme activity can be used as a signal of herbicide toxicity in cyanobacteria.     

苯甲酸对黄瓜幼苗叶绿素含量、氮代谢及膜透性的影响

采用不同浓度的苯甲酸对2种两叶一心期的黄瓜幼苗进行处理,研究其对2种黄瓜幼苗叶绿素含量、氮代谢及膜透性的影响。结果表明,在0.25~1.00 mmol.L-1之间,随着苯甲酸处理浓度的增加,‘密刺王黄瓜’和‘津杂2号’幼苗叶绿素和类葫萝卜素的含量、硝酸还原酶活性、硝态氮含量均不断降低,电解质外渗率均不断增加。‘津杂2号’幼苗的变化趋势弱于‘密刺王黄瓜’,这表明‘津杂2号’较‘密刺王黄瓜’耐一定程度的苯甲酸作用。     

二氢黄酮还原酶基因植物表达载体构建及对烟草的遗传转化研究

以pBI121为基础载体,通过分步酶切连接分别构建组成型CaMV35S启动子、光合组织特异型PNZIP启动子驱动的DFR基因植物表达载体pBIDFR和pPNDFR;采用直接转化法将pBIDFR和pPNDFR导入根癌农杆菌菌株,采用pPNDFR/EHA105菌株对普通烟草进行了遗传转化研究.在Kanamycin选择压力下获得了烟草转化不定芽和完整植株,经过PCR鉴定,该DFR基因已成功导入烟草基因组中.     

Three lignans and one coumarinolignoid with quinone reductase activity from Eurycorymbus cavaleriei

Four compounds, including one new lignan cavaol H (1), one new coumarinolignoid 5′-hydroxycleomiscosin B (4) and two known lignans (2–3) were isolated from the 95% ethanol extract of the twigs of Eurycorymbus cavaleriei. Their structures were established on the basis of various spectroscopic analyses including 1D-(¹H, ¹³C, and DEPT) and 2D-NMR (COSY, HMQC, and HMBC). The absolute stereochemistry of the two known lignans was firstly reported in this article by ¹H NMR studies on Mosher's ester derivatives. In the present study, quinone reductase induction activities of compounds 1–4 were assayed, compound 4 showed moderate quinone reductase induction with concentration to double the enzyme activity (CD) of 10.5 ± 0.8 μg/mL. Then we established LC-MS-MS analysis of glutathione (GSH) incubation with compound 4 to explain whether compound 4 induced quinone reductase through alkylating of the sulfhydryl groups of Keap1. Reconstructed selected ion chromatogram (SIC) of m/z 706 after compound 4 incubation with GSH was different from that with Tris–HCl buffer solution, which meant the quinone reductase induction activity of compound 4 attributed to alkylating the sulfhydryl groups of Keap1.     

杨梅硝酸还原酶活性与根瘤固氮活性的研究

离体根瘤的固氮活性的测定采用乙炔还原法,硝酸还原酶的测定采用改进活体法,测试杨梅各器官硝酸还原酶活性的差异和温度、化合态氮对硝酸还原酶活性和固氮活性的影响,研究结果表明:①确定1%的正丙醇为活体法测定杨梅硝酸还原酶时所使用的渗透剂的最佳浓度。②杨梅NO3- 还原的主要部位在叶片,根瘤的硝酸还原酶活性受到宿主植物和内生菌的影响。③温度对植物体内硝酸还原酶和根瘤固氮活性水平影响很大,温度太高抑制酶活性,20℃时杨梅各器官的硝酸还原酶活性均达最高点,根瘤的固氮活性也达最高点。④用含10mmol/LNO3-的Hogland营养液培养的植株各部位的硝酸还原酶活性均高于对照,10mmol/L的NH4+ 对植株的硝酸还原酶活性未见明显影响,但能抑制根瘤的固氮活性,其抑制程度高于NO3-。⑤硝酸还原酶活性和根瘤固氮活性之间没有直接关系。      

Iron-Utilization System in Vibrio vulnificus M2799

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.     

Effect of Cadmium Stress on the Growth,Antioxidative Enzymes and Lipid Peroxidation in Two Kenaf (Hibiscus cannabinus L.) Plant Seedlings

The effects of cadmium stress on the growth, antioxidative enzymes and lipid peroxidation in two kenaf plants, Fuhong 991 and ZM412, were analysed under control (0.5-strength Hoagland's nutrient solution) or five levels of cadmium stress (0.5-strength Hoagland's nutrient solution containing different concentrations of Cd²⁺). The leaves and roots of control and cadmium-stressed plants were harvested after 3 wk. At the same Cd concentration, the Cd tolerance index of Fuhong 991 was higher than that of ZM412, indicating that Fuhong 991 may be more tolerant to Cd than ZM412. The superoxide dismutase (SOD), catalase activity (CAT) and peroxidase (POD) activities fluctuated in the leaves of the Cd-stressed plants compared to the control, whereas the glutathione reductase activity (GR) was much larger than the control for Fuhong 991, ensuring that sufficient quantities of GSH were available to respond to the cadmium stress. In comparison to the control, the dynamic tendency of the SOD, CAT and POD activities in roots of the Cd-stressed plants all increased and then declined, but the POD activity of Fuhong 991 remained nearly unchanged at all of the stress levels. The increase in the enzyme activities demonstrated that Fuhong 991 was more tolerant to cadmium than ZM 412. The lipid peroxidation was enhanced only in the leaves of Cd-stressed ZM 412. These findings indicated that antioxidative activities may play important roles in Cd-stressed Fuhong 991 and ZM 412 and that the leaf and root cell membranes of Fuhong 991 have a greater stability than those of ZM 412. For pollution monitoring purposes, the GR activity in the roots and leaves may serve as a biomarker of Cd for Fuhong 991, whereas lipid peroxidation may serve as biomarker for ZM 412.