High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris

High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZαA with the Saccharomyces cerevisiae α-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additional sequences of the c-myc epitope and (His)₆-tag of the vector were fused to the C-terminus of cutinase, while the other expression vector was constructed without any additional sequence. P. pastoris expressing the non-tagged cutinase exhibited about two- and threefold higher values of protein amount and cutinase activity in the culture supernatant, respectively. After simple purification by diafiltration process, both cutinases were much the same in the specific activity and the biochemical properties such as the substrate specificity and the effects of temperature and pH. In conclusion, the high-level secretion of F. solani cutinase in P. pastoris was demonstrated for the first time and would be a promising alternative to many expression systems previously used for the large-scale production of F. solani cutinase in Saccharomyces cerevisiae as well as Escherichia coli.     

农杆菌介导转化莱茵衣藻体系的优化

[目的]为建立根癌农杆菌介导的莱茵衣藻快速简便高效的遗传转化体系,本研究以模式生物莱茵衣藻为受体材料,从转化方法和转化子快速鉴定两个方面进行了优化.[方法]比较了固体培养基共培养转化方法和液体培养基共培养转化方法对根癌农杆菌LBA 4404介导的莱茵衣藻CC425转化效率的影响;研究并比较了(1)首先经过TE裂解再进行...     

爵床的甲醇提取物对小菜蛾的生物活性作用

为探明爵床(Justicia procumbens)甲醇提取物对小菜蛾的生物活性,采用室内生测法测定了爵床甲醇提取物对小菜蛾的触杀、拒食、胃毒、生长发育抑制和产卵忌避作用。结果表明,爵床甲醇提取物对小菜蛾幼虫具有较强的触杀、拒食、胃毒和生长发育抑制活性,对小菜蛾成虫具有较强的产卵忌避活性。在触杀试验中,药后1、2 d和3 d爵床甲醇提取物对小菜蛾3龄幼虫的致死中浓度(LC50)分别为5.17、4.05和3.06 mg/m L;在拒食试验中,药后1 d和2 d提取物对3龄幼虫的选择性拒食中浓度(AFC50)分别为2.64和3.13 mg/m L,药后1 d和2 d提取物对3龄幼虫的非选择性拒食中浓度(AFC50)分别为3.70、4.54 mg/m L;在胃毒试验中,药后4、5、6 d和7 d提取物对3龄幼虫的致死中浓度(LC50)分别为8.13、3.65、2.88、2.23 mg/m L;在生长发育抑制试验中,药后1 d和2 d提取物对3龄幼虫的抑制中浓度(IC50)分别为2.02、1.40 mg/m L;在产卵忌避试验中,药后1、2 d和3 d提取物对小菜蛾成虫的选择性产卵忌避中浓度(AOC50)分别为2.61、3.66、4.58 mg/m L,药后1、2和3 d提取物对小菜蛾成虫的非选择性产卵忌避中浓度(AOC50)分别为3.19、4.52、5.65 mg/m L。由此证实,爵床提取物对小菜蛾具有显著的毒杀活性,具有开发为新型高效、低毒植物源农药的潜在价值。     

连翘提取物对嗜水气单胞菌群体感应系统的影响

【背景】传统抑菌剂的大量使用导致细菌产生多重耐药性与抗性,而基于细菌群体感应靶点调控的新型抑菌剂可缓解细菌耐药性与抗性,是未来抑菌剂的发展方向之一。【目的】研究连翘(Forsythiasuspensa)提取物对嗜水气单胞菌(Aeromonashydrophila)群体感应系统的影响及可能的作用机制,为新型抑菌剂的开发提供理论依据。【方法】以紫色杆菌(Chromobacterium violaceum)CV026为报告菌株,以嗜水气单胞菌为供试菌株,采用倍比稀释法测定连翘提取物对2种菌的最小抑菌浓度(minimal inhibitory concentration,MIC),通过微量法测定提取物对嗜水气单胞菌生长、群集运动及蛋白酶活性的影响,利用高效液相色谱串联质谱法分析提取物中的主要成分,采用分子对接模拟探究提取物对嗜水气单胞菌群体感应系统的作用机制。【结果】连翘提取物对紫色杆菌CV026和嗜水气单胞菌的MIC均为16.00mg/mL。在亚抑菌浓度下,连翘提取物处理显著抑制了CV026紫色菌素的产生,最大抑制率高达56.30%。经8.00mg/mL连翘提取物处理后,嗜水气单胞菌的群集运...     

Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea

Bacillus licheniformis N1, which has previously exhibited potential as a biological control agent, was investigated to develop a biofungicide to control the gray mold of tomato caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were developed using fermentation cultures of the bacteria in Biji medium, and their ability to control gray mold on tomato plants was evaluated. The results of pot experiments led to the selection of the wettable powder formulation N1E, based on corn starch and olive oil, for evaluation of the disease control activity of this bacterium after both artificial infection of the pathogen and natural disease occurrence under production conditions. In plastic-house artificial infection experiments, a 100-fold diluted N1E treatment was found to be the optimum biofungicide spray formulation. This treatment resulted in the significant reduction of symptom development when N1E was applied before Bo. cinerea infection, but not after the infection. Both artificial infection experiments in a plastic house and natural infection experiments under production conditions revealed that the N1E significantly reduced disease severity on tomato plants and flowers. The disease control value of N1E on tomato plants was 90.5% under production conditions, as compared to the 77% conferred by a chemical fungicide, the mixture of carbendazim and diethofencarb (1:1). The prevention of flower infection by N1E resulted in increased numbers of tomato fruits on each plant. N1E treatment also had growth promotion activity, which showed the increased number of tomato fruits compared to fungicide treatment and non-treated control and the increased fruit size compared the non-treated control under production conditions. This study suggests that the corn starch-based formulation of B. licheniformis developed using liquid fermentation will be an effective tool in the biological control of tomato gray mold.     

Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris

To improve the expression level of recombinant Drosophila melanogaster AChE (R-DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R-DmAChE activity were obtained. The highest production of R-DmAChE in shake-flask culture after 5-day induction by methanol was 718.50 units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R-DmAChE in the medium could be used for production of R-DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.     

拟南芥ERD15基因缺失突变体的分子鉴定和表型分析

为探究ERD15基因功能,利用反向遗传学,通过PCR及半定量PCR筛选鉴定出拟南芥(Arabidopsis thaliana) ERD15基因的T-DNA插入纯合突变体,并对其表型进行观察分析。结果表明,erd15突变体莲座叶数目显著增多,提前3～4 d开花,突变体比野生型更早从营养生长转向生殖生长。拟南芥野生型植株主茎为圆柱体,平均直径1.29 mm,而erd15突变体主茎扁平,平均直径达到2.27mm,具极显著差异。与野生型相比,erd15突变体果实心皮发育受到影响,隔膜上排列有多排种子,果荚顶端膨大,长度缩短37.67%,但角果平均结籽数升高。因此,ERD15基因参与了调控拟南芥植株的生殖生长过程。     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

荧光假单胞菌2P24中opgGH基因影响抗生素2,4-二乙酰基间苯三酚的产生

【目的】抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol,2,4-DAPG)是生防菌株荧光假单胞菌(Pseudomonas fluorescens) 2P24防治植物病害的关键因子,然而对2,4-DAPG生物合成的调控通路并未完全解析。【方法】前期利用Tn5随机突变的方法获得一株对棉花立枯丝核菌(Rhizoctonia solani)拮抗能力完全丧失的突变菌株W3,本研究利用基因互补等方法研究该突变体中被破坏的基因对菌株2P24分泌2,4-DAPG和其他生防相关性状的影响。【结果】Tn5插入位点及其序列分析表明突变菌株W3中Tn5破坏了opgG基因。鉴于opgG和opgH基因组成操纵子,利用同源重组技术构建了opgGH内缺失突变菌株。与野生菌株2P24相比,opgGH突变菌株中2,4-DAPG的产量显著降低。对其他生防相关性状的检测发现,突变opgGH基因并不影响群体感应系统(quorum sensing,QS)信号分子的产生、氢氰酸的产生以及生物膜的形成,但可抑制菌株2P24的游动性。转录融合实验进一步表明opgGH基因并不调控gacA基因及其调控...     

Echinococcus granulosus: In vitro effectiveness of warm water on protoscolices

Hydatid disease is one of the most important helminthic diseases worldwide. Hydatid cysts may be found anywhere in the body. The most effective treatment of hydatid cyst is surgical operation. Spillage of live protoscolices during the operation is the major cause of recurrence. Instillation of scolicidal agent into hydatid cyst is the most commonly employed measure to prevent this complication. To date, many scolicidal agents have been used for inactivation of the hydatid cyst content, however, most common scolicidal agents may cause unacceptable side-effects, limiting their use. In this study the scolicidal effect of warm water (45, 50, 55, and 60 °C) at different exposure times (1, 2, 3, 4, 5, 6, 8, 10, 12, and 15 min) is investigated. Protoscolices were collected aseptically from sheep livers containing hydatid cyst. Viability of protoscolices was determined by 0.1% eosin staining. Even though the highest scolicidal activity of warm water at 45 °C was 40.4% at the end of 15 min, the best scolicidal effect (100%) of warm water at 50, 55, and 60 °C was obtained after 5, 2, and 1 min, respectively. The results of this in vitro study showed that warm water at 50–60 °C can be regarded as an effective scolicidal agent. Warm water is commonly available, easily prepared, and inexpensive. In vivo scolicidal activity of warm water and also the possible side effects need further investigation.     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

干旱胁迫对蒙古黄芪生长及根部次生代谢物含量的影响

以山西道地药材黄芪一年生幼苗为试验材料,设置常规水分条件(CK)、轻度干旱胁迫(A1)、中度干旱胁迫(A2)、重度干旱胁迫(A3)4个不同处理,研究土壤干旱胁迫对黄芪生长及生理的影响。结果表明:黄芪茎叶快速生长集中在出苗后80—120d,以后生长减缓;当茎叶枯萎时,根中生物量短期快速积累。与常规水分条件相比,干旱胁迫处理显著降低了黄芪苗高及茎叶生物量,但对抗氧化能力、根系生长及次生代谢物积累产生了不同的影响。轻度干旱胁迫下SOD、POD、CAT 3种抗氧化酶活性升高,丙二醛(MDA)含量和细胞膜透性降低,同时根长与根生物量增加、多糖与皂苷两种次生代谢物积累增多,黄芪药材的质量得到显著提高(P0.05);胁迫上升到中度、重度时,SOD酶活性逐渐降低,重度胁迫下低于对照,而POD及CAT酶活性、MDA含量、细胞膜透性均随胁迫增强而升高,相反,根长、根生物量、多糖与皂苷含量降低,导致黄芪药材的质量显著降低(P0.01)。综上表明,干旱胁迫下,SOD酶表现较为敏感,可能在清除活性氧中起主要作用;轻度水分胁迫能有效启动黄芪体内抗氧化酶系统和次生代谢,它们相互协作共同对抗胁迫对细胞产生的伤害,通过降低地上部分的生长,将营养物质优先运往根部,促进根产量及药材质量的提高。这一结论,可在黄芪多糖和皂苷次生代谢物定向培育的水分管理中加以利用。     

Suppression of Botrytis cinerea sporulation by Clonostachys rosea on rose debris: a valuable component in Botrytis blight management in commercial greenhouses

Botrytis blight, caused by Botrytis cinerea (Bc), is an important disease on roses grown in plastic greenhouses in Brazil. Biocontrol with Clonostachys rosea (Cr) applied to leaves and crop debris to reduce pathogen sporulation can complement other control measures for disease management. Two experiments, each with a rose cultivar, were conducted in a plastic greenhouse. For ‘Red Success,’ four treatments were compared: (1) control; (2) fortnightly sprays of Cr; (3) weekly sprays of mancozeb; and (4) weekly sprays of either Cr or mancozeb to the lower third of the plants and the debris. For ‘Sonia,’ treatment 4 was not included. Samples were taken from debris (leaves and petals) at ten 15-day intervals and plated on PCA medium. Sporulation of fungi and incidence of Botrytis blight on buds were assessed. For both cultivars, C treatments significantly (P=0.05) reduced Bc sporulation. However, disease incidence was not consistently reduced, probably because the applications of C. rosea started when Botrytis blight epidemic was advanced and no sanitation practices were performed on nontreated plots. From the present and previous studies, continuous application of Cr on debris, associated with sanitation practices, has the potential to reduce Bc sporulation and disease incidence in the buds.     

Antifungal activity of tea tree oil from Melaleuca alternifolia against Trichophyton equinum: An in vivo assay

Dermatophytes are a group of keratinophilic and keratinolytic molds, some of which are responsible for ringworm. Among them Trichophyton equinum, which mostly infects equids, can cause extensive outbreaks in stud farms. The conventional treatment of equine trichophytosis is topic, based upon medicated shampoos to reduce the spread of infection among the animals. Nevertheless the popularity of phytotherapy is at an all-time peak, and the interest for natural alternatives or complements to conventional drug therapy is challenging both in human and veterinary field. Among herbal remedia Tea Tree Oil (TTO) shows a wide range of antimicrobial activities. A randomized open clinical trial was carried out on 60 thoroughbred breeding horses affected by equine ringworm. The animals were randomly divided into 2 groups of 30 subjects. Diagnostic criteria were the presence of clinical signs and positive T. equinum culture. Specificity control using TTO mixture in 5 not dermatophyte affected animals was achieved also. The antimycotic activity against T. equinum of a mixture containing 25% TTO in sweet almond oil, was evaluated in vivo treating 30 subjects, the others were administered enilconazole 2% solution. The animals of both groups were topically treated twice a day for 15 days with a 25% mixture of TTO diluted in sweet almond oil and every 3 days, four times with enilconazole rinses, respectively. The clinical and mycological outcome were evaluated at day 30 from the start of the treatments. Data analysis was performed by chi square test. All the treated animals showed complete clinical and aetiological healing. Part of control subjects also, showed an improvement and none of them exacerbate the lesions. This therapeutic protocol appears to be effective and versatile, being applicable immediately after physical examination, prior to have the laboratory response. It could be an alternative for practitioners interested in herbal medicines, contributing to fulfill the gap existing between in vitro and clinical studies.     

红平菇产漆酶条件优化及对孔雀绿染料脱色的研究

采用LNAS(低氮天冬酰胺-琥珀酸)培养基添加方式,对红平菇Pleurotus djamor HP1进行培养,检测不同时间培养液对不同底物的氧化作用,进而得到光密度值的变化情况,作为漆酶的产生及活性测定的主要依据。结果表明:在含Cu2+的培养液中漆酶最大酶活为235.4 U/L。含Cu2+的培养液添加底物木屑后漆酶最大酶活为458.8 U/L。提取经优化筛选后的培养基培养出的漆酶粗酶液,对4种具有不同化学结构的染料进行了脱色试验。结果表明:三苯基甲烷类的孔雀绿在6 h时脱色率为87.5%,蒽醌类的SN4R在24 h时脱色率为49.4%,偶氮类的甲基橙在24 h时脱色率为45%,杂环类的中性红在24 h时脱色率为23.6%。因此,显示出红平菇漆酶对孔雀绿染料脱色具有较大的应用潜力,进而对废水处理具有更好的应用前景。     

Ce^3+和La^3+对人工老化沙葱种子活力及生理特性的影响

以未老化和人工老化后的沙葱(Allium mongolicum Regel.)种子为材料,采用氯化铈(Ce3+)和氯化镧(La3+)浸种,测定种子萌发和生理指标,探讨Ce3+和La3+浸种对种子萌发、老化种子活力和生理特性的影响。结果显示:(1)在老化0~5 h时,Ce3+和La3+处理可显著促进沙葱种子萌发,提高种子活力;在老化5 h后,Ce3+和La3+处理对种子萌发无明显促进作用。(2)在老化0~15 h时,Ce3+和La3+处理的沙葱种子中抗氧化酶活性和抗坏血酸(AsA)含量提高,其超氧阴离子自由基(O2-·)产生速率、过氧化氢(H2O2)含量和丙二醛(MDA)含量显著降低;在老化15 h后,Ce3+和La3+处理的种子抗氧化酶活性提高、AsA含量降低,O2-·产生速率和MDA含量提高。(3)在老化5 h时,沙葱种子呼吸速率发生跃变达到最大,Ce3+和La3+处理显著降低了种子呼吸速率。(4)Ce3+和La3+处理在老化0~5 h时提高了沙葱种子超弱发光(UWL)强度,但在老化5 h后沙葱种子的UWL强度降低。研究认为,在沙葱种子人工老化初期,Ce3+和La3+浸种处理可以诱导增强种子抗氧化酶活性和提高AsA含量,有效清除因老化产生积累的过量活性氧(ROS),减轻过氧化伤害,提高种子活力;种子老化中后期,其内部ROS产生与清除系统发生紊乱,加剧了ROS对种子结构的损伤,Ce3+和La3+浸种处理的缓解效应丧失。     

乌鳢源杀鱼爱德华菌的分离鉴定及药敏特性研究

【背景】江苏省扬州市某乌鳢养殖场发生疾病,给养殖户造成了严重的经济损失。【目的】确定病因并筛选出敏感药物,为乌鳢相关疾病的防治提供参考。【方法】从患病乌鳢体内分离致病菌,并从形态、生理生化特征、16S rRNA和gyrB基因序列及特异性PCR检测等方面对分离菌株进行鉴定,同时开展人工感染试验分析其致病性,通过纸片扩散法进行药敏特性分析。【结果】从患病乌鳢体内分离获得优势菌株SHL,经形态特征、理化特性、16SrRNA和gyrB基因序列及特异性PCR检测鉴定为杀鱼爱德华菌。进一步人工感染试验证实其对乌鳢有较强的致病性,LD50为1.6×105 CFU/g,发病症状与自然发病症状相似。药敏试验结果显示,该菌株对青霉素、氯霉素、四环素等28种抗菌药物高度敏感,对红霉素中度敏感,对苯唑西啉、克拉霉素、万古霉素等6种药物耐药。【结论】引起江苏省扬州市某养殖场的乌鳢体表溃烂及死亡的病原菌为杀鱼爱德华菌,这是我国首次从淡水鱼类中检出致病性杀鱼爱德华菌,表明该菌的感染谱在扩大,需引起水产养殖领域的重视,在养殖过程中可根据药敏实验结果选用合适的国标渔药进行防治。     

Labdane diterpenoids from Aframomum sceptrum: NMR study and antiparasitic activities

Two novel labdane diterpenoids, 15ξ-methoxy-labdan-8(17),11(E),13(14)-trien-15,16-olide (1) and 12(S)-hydroxy-15ξ-methoxy-labdan-8(17),13(14)-dien-15,16-olide (2) were isolated from the rhizomes of Aframomum sceptrum K. Schum (Zingiberaceae). Their structures were established on the basis of their spectroscopic data. Stigmast-4-en-6β-ol-3-one and caryophylene oxide were also obtained. In vitro trypanocidal and leishmanicidal activities of labdanes 1 and 2 were evaluated. Compound 2 exhibited activity similar to that of reference drugs against Leishmania donovani.     

旱麦草EtAP2基因的克隆及其对干旱胁迫的响应表达

以旱麦草(Eremopyrum triticeum)为实验材料,利用RT-PCR技术从旱麦草叶片中克隆了1个AP2/ERF家族基因,命名为EtAP2(GenBank登录号KX622583)。EtAP2基因含有1 128bp开放阅读框,编码375个氨基酸,相对分子质量40.87kD,等电点为5.36。多序列比对和进化树分析表明,该基因编码蛋白具有2个AP2保守结构域,与小麦AP2/ERF家族蛋白具有较近的亲缘关系。实时荧光定量PCR分析表明,15%PEG 6000模拟干旱胁迫可诱导EtAP2基因在根和叶中表达,且在根中对干旱胁迫的响应大于叶片。研究表明,EtAP2可能参与旱麦草对干旱逆境胁迫应答的调节。