大豆悬浮细胞培养及作为外源基因转化受体的研究

以吉林小粒7号大豆品种为材料,切取无菌幼苗下胚轴诱导愈伤组织,初步建立了大豆悬浮细胞系,测定不同硼酸浓度下悬浮细胞生长变化;采用农杆菌介导法对大豆悬浮细胞进行外源GUS基因的转化,先通过不同程度的超声处理确定最佳超声时长,然后在最佳超声处理时长的基础上,探讨了硼酸浓度对转化效率的影响;最后采用PCR和Southern blot杂交对抗性愈伤做分子鉴定。结果表明:大豆悬浮细胞正常生长硼酸浓度在2.0~10 mg·L-1,过高或过低均会抑制大豆细胞正常的生长,高于100 mg·L-1时抑制作用最显著。但在转化过程中,适当增加硼酸浓度(10~30 mg·L-1)则能够提高悬浮细胞的转化效率,并且有助于转化组织的筛选,借以3 min超声的辅助作用,30 mg·L-1硼酸浓度下转化效率达到1 mg转化细胞团中含28个转化体。     

农杆菌介导法将Bt(cryⅠA)基因导入大豆的研究

构建含有Bt(cry Ⅰ A)基因的植物表达载体pCAMBIA3300-Bt,以大豆子叶节为受体,通过农杆菌介导法将Bt基因导人大豆品种黑农37中,获得转基因植株.并进行人豆的再生和遗传转化系统优化的研究,以获得较高的转化率.结果表明:在6-BA浓度为1.7 mg·L-1时,丛生芽分化率最高,确定该品种大豆在从生芽分化阶段的草铵膦筛选浓度为3.5 mg·L-1获得转化质粒pCAMBIA3300-Bt的转基因植株,其中T1代PCR阳性植株19株.采用real-time PCR的方法对T1代抗性植株进行Bt基因的转录水平的分析,初步证明Bt基因已整合到受体大豆的基因组内.     

转cry 1Iem基因大豆的培育及抗虫性检测

以大豆子叶节为外植体,应用农杆菌介导法,将抗虫(cry1Iem)基因转化大豆.筛选标记为bar基因.经Glufasinate筛选,获得大量抗性植株.对转基因T_0、T_1、T_2代植株进行PCR检测,初步证明cry1Iem基因已经整合到大豆基因组中.对T2代PCR阳性植株幼嫩豆荚,采用圆盘分隔法接人初孵幼虫,进行初步的抗虫性检测,得到1株具有明显抗虫效果和7株抗虫效果较好的转基因植株.     

RNA干扰高油GmPEPc基因转入大豆研究

通过构建GmPEPc基因的植物RNAi双元表达载体,并通过农杆菌介导的大豆子叶节遗传转化方法将控制油脂和蛋白合成途径的相关基因GmPEPc转入受体品种沈农9号中,通过抑制大豆内源GmPEPc基因的表达,增加油脂积累,从而获得高油的转基因大豆新品种。在大豆组织培养过程中,共切取大豆外植体407块,获得T0代转化苗35株,转化率8.9%。通过对转基因后代中目的基因的整合及表达情况进行分子鉴定。获得23株T_1代转基因后代,其中高抗草丁膦除草剂(喷施浓度300 mg·mL~(-1))14株,通过PCR检测结果表明其中12株为PCR阳性;PCR和Southern杂交检测表明,GmPEPc基因已经成功插入到转基因大豆植株基因组DNA中。对T_1代测定结果显示,转基因大豆籽粒的平均含油量比对照高9.51%,平均蛋白质含量下降5.44%。这些研究结果为筛选高油脂含量的转基因大豆新株系提供了依据,为下一步高油新品种的选育提供了种质基础。     

双T-DNA表达载体转化大豆的研究

利用含有筛选标记基因和目的基因△6-脂肪酸脱氢酶基因的双T-DNA共转化表达载体pLIN61,经农杆菌EHA101介导,采用子叶节转化法转化大豆,使用除草剂Glufosinate作为筛选剂,获得了一批携带有玻璃苣△6-脂肪酸脱氢酶基因的转基因大豆,转化效率为2.0%,共转化频率及实际转化率分别为56.25%、1.12%.PCR检测和Southern杂交结果证明,玻璃苣D6D已经整合到大豆的基因组上.RT-PCR检测结果显示,玻璃苣△6-脂肪酸脱氢酶基因在转基因大豆的转录水平上得到了表达.     

HSSP基因植物表达载体构建及其对大豆的遗传转化

大豆中含硫氨基酸匮乏,限制了大豆的营养价值和商业价值。为了提高大豆的含硫氨基酸含量,利用人工设计合成的高含硫氨基酸贮藏蛋白基因(HSSP)转化大豆。以载体p TF101.1为骨架,构建植物表达载体p TFGS,利用农杆菌介导法转化大豆品种东农50,经PCR及试纸条检测,获得转基因大豆16株,基因的转化效率为1.94%。对转基因大豆株系GSDL5进行组织特异性分析,结果表明,HSSP基因在种子中超量表达,而在其它组织部位仅有微量表达。采用接头PCR方法对株系GSDL5基因组插入位点侧翼序列进行克隆,获得与大豆基因组序列匹配的436 bp侧翼序列,HSSP基因插入2号染色体基因的非编码区。经研究,获得了遗传背景明确的,仅在大豆种子中超量表达HSSP的转基因株系GSDL5。     

未成熟种子子叶节转化体系优化及转EPSPS/GAT基因大豆新材料创制

以丛生芽率为指标,将农杆菌介导的大豆未成熟种子子叶节转化与成熟种子转化体系进行比较,明确了诱导过程中草甘膦筛选浓度、未成熟种子生理状态,改进大豆外植体的获得方式并减少了组培环节,建立了未成熟种子子叶节转化体系。利用Jack黄熟后期的种子作为受体材料,在15 mg·L-1的草甘膦筛选浓度下获得了9株PCR检测阳性植株并且生长正常可育,T0代经测序及Southern Blot分析,进一步证明外源基因已整合进大豆基因组中,其中2个株系的T1代植株经PCR检测符合3∶1的分离比,且表型鉴定筛选出抗性转基因植株,为抗草甘膦转基因大豆育种提供了新材料。     

农杆菌介导的大豆子叶节遗传转化技术流程及操作要点

大豆遗传转化效率低,是大豆转基因育种亟待解决的重要问题。以大豆子叶节为外植体采用农杆菌介导法,系统地研究了大豆子叶节遗传转化各技术环节的操作要点,包括工程菌液制备、无菌苗获得及外植体制备、农杆菌的侵染及共培养、不定芽诱导、不定芽伸长、根诱导、驯化与移栽、抗性植株的获得及转基因植株的检测等,建立了一套较稳定、高效的大豆子叶节遗传转化体系。并利用该体系,将野生大豆耐盐碱相关基因A1对大豆品种绥农28、合丰50、合丰55、东农50进行遗传转化,系统地探讨了大豆基因型、影响T-DNA的转移效率的各环节、筛选剂浓度及不同基因型转化效率等几个重要因素。     

我国大豆转基因研究进展

我国大豆转基因发展很快,本文就大豆转基因中常用的方法:农杆菌介导法、电击法、PEG转化法、基因枪法、花粉管通道法、农杆菌传导的原位基因转化等的技术环节、在大豆抗虫、抗病、抗除草剂等方面转化外源基因上的应用状况,大豆再生体系建立以及对国内大豆转基因的展望.     

大豆GmRAV基因与暗形态建成相关

实时定量PCR(RT-PCR)研究表明大豆GmRAV基因表达受黑暗强烈诱导,转基因烟草研究发现该基因在黑暗下促进植株的黄化,抑制子叶的展开和促进下胚轴的伸长,说明该基因可能参与大豆暗形态建成过程.     

Studies on natural repellents against potato tuber moth (Phthorimaea operculella Zeller) in country stores

Summary The effects of the leaves of five plant species, one from each of the generaAmbrosia, Anemone, Eupatorium, Eucalyptus andLantana, on potato tuber moth were investigated under indigenous storage conditions at the Central Potato Research Station, Shillong (1800 m above sea level). Their action was compared with that of a biological insecticide (spores ofBacillus thuringiensis), a chemical insecticide (carbaryl), and an untreated control. The data collected after six months storage on tuber damage, sprout damage and the rotting indicated that the leaves ofLantana aculeata provided most protection to the tubers, reducing damage from over 70% in the check to below 5%, and sprout damage from over 45% to below 3%. Next best wasEucalyptus globulus followed byB. thuringiensis. They may be used on tubers stored for table use or for seed as they had no adverse effect on germination or on the yield of a subsequent crop.     

Comparative Assessment of the Efficacy of Various Nitrogen Fertilizers

Summary

The efficiency of N fertilizers is usually poor; often less than 50% of the applied N is taken up by the crop. This review focuses on various N fertilizers with respect to the significance of different N loss pathways, namely (i) ammonia volatilization, (ii) dinitrogen and nitrogen oxide emissions, and (iii) nitrate leaching. Further, the significance of biological N immobilization, ammonium fixation and, finally, the impact of nitrate vs. ammonium uptake on crop yield are also discussed. The reviewed literature shows that N fertilizers may differ markedly in their susceptibility to losses. There is, however, considerable scope to improve N efficiency of each N source by proper N management practices.     

In vitro antimicrobial activity of different accessions ofSolanum commersonii Dun. from Uruguay

Summary The in vitro antimicrobial activity of extracts from accessions ofSolanum commersonii Dun. collected in the south of Uruguay was investigated against five microorganisms including the pathogenRalstonia solanacearum. A total of 30 extracts corresponding to organic and aqueous extracts were studied. Interestingly, most of the positive results for growth inhibition were againstR. solanacearum. The extracts were also analyzed for the presence of glycoalkaloids and lectins. Six of the ten aqueous extracts showed lectin presence and a wide variation in the type and amounts of glycoalkaloids, was found. Results indicate that there is no clear relationship between the antimicrobial activity against the five microorganisms screened and the presence or amounts of lectins and glycoalkaloids, traditionally regarded as possible antimicrobial metabolites in theSolanum genus, which indicates the presence of as yet unidentified antimicrobial compounds.     

Pharmacokinetics of Marine-Derived Drugs

Marine organisms represent an excellent source of innovative compounds that have the potential for the development of new drugs. The pharmacokinetics of marine drugs has attracted increasing interest in recent decades due to its effective and potential contribution to the selection of rational dosage recommendations and the optimal use of the therapeutic arsenal. In general, pharmacokinetics studies how drugs change after administration via the processes of absorption, distribution, metabolism, and excretion (ADME). This review provides a summary of the pharmacokinetics studies of marine-derived active compounds, with a particular focus on their ADME. The pharmacokinetics of compounds derived from algae, crustaceans, sea cucumber, fungus, sea urchins, sponges, mollusks, tunicate, and bryozoan is discussed, and the pharmacokinetics data in human experiments are analyzed. In-depth characterization using pharmacokinetics is useful for obtaining information for understanding the molecular basis of pharmacological activity, for correct doses and treatment schemes selection, and for more effective drug application. Thus, an increase in pharmacokinetic research on marine-derived compounds is expected in the near future.     

Novel food and non-food uses for sorghum and millets

Sorghum and millets have considerable potential in foods and beverages. As they are gluten-free they are suitable for coeliacs. Sorghum is also a potentially important source of nutraceuticals such antioxidant phenolics and cholesterol-lowering waxes. Cakes, cookies, pasta, a parboiled rice-like product and snack foods have been successfully produced from sorghum and, in some cases, millets. Wheat-free sorghum or millet bread remains the main challenge. Additives such as native and pre-gelatinised starches, hydrocolloids, fat, egg and rye pentosans improve bread quality. However, specific volumes are lower than those for wheat bread or gluten-free breads based on pure starches, and in many cases, breads tend to stale faster. Lager and stout beers with sorghum are brewed commercially. Sorghum's high-starch gelatinisation temperature and low beta-amylase activity remain problems with regard to complete substitution of barley malt with sorghum malt . The role of the sorghum endosperm matrix protein and cell wall components in limiting extract is a research focus. Brewing with millets is still at an experimental stage. Sorghum could be important for bioethanol and other bio-industrial products. Bioethanol research has focused on improving the economics of the process through cultivar selection, method development for low-quality grain and pre-processing to recover valuable by-products. Potential by-products such as the kafirin prolamin proteins and the pericarp wax have potential as bioplastic films and coatings for foods, primarily due to their hydrophobicity.     

La pourriture fongique des tubercules de pomme de terre en atmosphère contrôlée

Résumé Fusarium culmorum et, dans une moindre mesure,F. avenaceum, F. sambucinum, Colletotrichum coccodes etCylindrocarpon sp. ont été isolés de tubercules entreposés en atmosphère contr?lée (AC) et atteints de pourriture grave. Des expériences d'inoculation de tubercules avec 10 espèces ou variétés fongiques et conservation en AC à 8 et 10°C ont montré que: 1) les champignons non pathogènes en atmosphère normale (ex.:Cylindrocarpon sp.) le devenaient en AC, 2) ceux qui étaient faiblement pathogènes en atmosphère normale étaient nettement plus virulents en AC (ex.:F. culmorum), 3) les espèces ou variétés virulentes en conditions d'air normales (Fusarium sulphureum etPhoma exigua var.foveata) ne provoquaient pas de pourriture plus grave en AC et leur activité pouvait même être légérement freinée.

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Summary The extensive rotting of tubers which occurred during storage trials in controlled atmospheres has led to a mycological and pathological study of the rots. The most frequently isolated species wasFusarium culmorum althoughF. avenaceum, F. Sambucinum, Coletotrichum coccodes and aCylindrocarpon sp. were also present. Tubers of cvs Bintje and Ulla were inoculated with 19 isolates of 10 fungal species or varieties associated with rotting of stored tubers (Table 1). The tubers were incubated at 8 or 10°C in normal or controlled (2% O₂:1% CO₂, 2% O₂:6% CO₂, 2% O₂:12% CO₂, 6% O₂:6% CO₂) atmospheres and the extent of rotting was determined a month later (Tables 2 and 3 and Fig. 2). Species which were weakly virulent in a normal atmosphere (variousFusarium spp. andPhoma exigua var.exigua) were clearly more aggressive in the controlled atmosphere and this was especially evident in the case ofF. culmorum. Species ot pathogenic in a normal atmosphere under experimental conditions (Colletotrichum coccodes, Cylindrocarpon sp.,Phoma eupyrena) caused rotting in the controlled atmosphere. The two species which were virulent in a normal atmosphere (F. sulphureum andP. exigua var.foveata) were also virulent in the controlled atmosphere and in some cases the extent of rotting was even less. As a general rule, rotting caused by weakly virulent fungi was greater in both cultivars in the controlled than in a normal atmosphere.

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Zusammenfassung Eine starke F?ulnis von in Kontrotrollierter Atmosph?re gelagerten Kartoffelknollen veranlasste eine mykologische und pathologische Untersuchung. Der am h?ufigsten isolierte Pilz warFusarium culmorum. F. avenaceum, F. sambucinum, Colletotrichum coccodes undCylindrocarpon sp. wurden ebenfalls bestimmt. Neunzehn St?mme von 10 Pilzsorten oder-Arten welche bei Lagerf?ulnis vorkommen (Tab. 1) wurden auf Knollen der Sorten Bintje und Ulla inokuliert. Die Lagerung wurde bei 8 oder 10°C under Normalbedingungen oder kontrollierter Atmosph?re durchgeführt (2% O₂:1% CO₂, 2% O₂:6% CO₂, 2% O₂:12% CO₂, 6% O₂:6% CO₂). Der F?ulnisgrad (Abb. 1) wurde nach einem Monat bonitiert. Die Ergebnisse sind in den Tabellen 2 und 3 dargestellt und in der Abb. 2 illustriert. Unter in Normalbedingungen nur schwach virulente Arten (verschiedeneFusarien undPhoma exigua var. exigua) waren deutlich aktiver in kontrollierter Atmosph?re. Dies was besonders der Fall fürF. culmorum. Pilze, die im Versuch unter Normalbedingungen nicht pathogen waren (Colletotrichum coccodes, Cylindrocarpon sp., Phoma eupyrena), haben unter kontrollierter Atmosph?re Sch?den verursacht. Hingegen werden die beiden unter Normalbedingungen sehr pathogenen Arten (F. sulphureum undP., exigua var.foveata), durch kontrollierte Atmosph?re wenig beeinflusst. Es wurde sogar in einigen F?llen eine gewisse Verringerung des F?ulnisbefalls beobachtet. Ganz allgemein haben die getesteten kontrollierten Bedingungen die Entwicklung der Pilzf?ulnis an beiden Kartoffelsorten gef?rdert, speziell durch Pilzarten, welche unter Normal-bedingungen wenig Sch?den verursachen.

     

Extreme resistance to infection by potato virus X in genotypes of wild tuber-bearingSolanum species

Summary Clones derived from thirty-one different accessions (nineteen of Argentine origin) belonging to eightSolanum species were screened for resistance to infection by potato virus X strain cp (PVX cp) by mechanical inoculation of plantlets that had been micropropagated in vitro. Estimates of PVX multiplication obtained by enzyme linked immunosorbent assay and slot blot nucleic acid hybridization allowed the identification of resistant clones derived from five accessions belonging toS. commersonii S. oplocense, S. sparsipilum andS. tuberosum andigena. Resistant genotypes supported PVX concentrations 5 to 15 times smaller than did the susceptible control cultivar Spunta. Graft inoculation test confirmed the presence of extreme resistance similar to that conferred by the ‘immunity’ gene X¹ (also called RX_act).     

Marine Pharmacology in 2009–2011: Marine Compounds with Antibacterial,Antidiabetic, Antifungal,Anti-Inflammatory,Antiprotozoal, Antituberculosis,and Antiviral Activities; Affecting the Immune and Nervous Systems,and other Miscellaneous Mechanisms of Action ?

The peer-reviewed marine pharmacology literature from 2009 to 2011 is presented in this review, following the format used in the 1998–2008 reviews of this series. The pharmacology of structurally-characterized compounds isolated from marine animals, algae, fungi and bacteria is discussed in a comprehensive manner. Antibacterial, antifungal, antiprotozoal, antituberculosis, and antiviral pharmacological activities were reported for 102 marine natural products. Additionally, 60 marine compounds were observed to affect the immune and nervous system as well as possess antidiabetic and anti-inflammatory effects. Finally, 68 marine metabolites were shown to interact with a variety of receptors and molecular targets, and thus will probably contribute to multiple pharmacological classes upon further mechanism of action studies. Marine pharmacology during 2009–2011 remained a global enterprise, with researchers from 35 countries, and the United States, contributing to the preclinical pharmacology of 262 marine compounds which are part of the preclinical pharmaceutical pipeline. Continued pharmacological research with marine natural products will contribute to enhance the marine pharmaceutical clinical pipeline, which in 2013 consisted of 17 marine natural products, analogs or derivatives targeting a limited number of disease categories.