GFP绿色荧光法辅助筛选玉米转基因植株

利用GFP(Green Fluorescence Protein)绿色荧光蛋白基因作为辅助性筛选标记基因,基因枪法轰击玉米自交系A188和Qi319未成熟胚的胚性愈伤组织。瞬间表达实验结果表明,轰击后GFP表达可持续近20 d,轰击后10 d左右表达量仍保持在较高的水平。采用潮霉素筛选、GFP荧光法鉴定,获得一批玉米转基因植株,转化频率为0.33%~0.47%。分子鉴定和后代分析的结果表明,外源基因已经整合到了玉米基因组中,并能够稳定地表达和遗传。GFP作为辅助性筛选标记基因可以直观、快速和有效地消除玉米遗传转化工作中的假阳性现象。     

大豆Gy1基因种子特异性表达载体的构建及在玉米中的转化

实验构建了2个大豆贮藏蛋白Gy1基因种子特异性表达载体,并将其分别转入玉米愈伤组织中。载体1以潮霉素为筛选标记,将Gy1基因片段插入到含有种子特异启动子的植物表达载体pCAMBIA1301-7αP上,得到种子特异性表达载体pCAMBIA1301-7αP-Gy1;载体2以BADH为筛选标记,将Gy1基因插入到带有耐盐基因BADH的植物表达载体pCAMBIA1301-BADH上,得到安全性无抗生素选择标记的种子特异性表达载体pCAMBI-A1301-sbeⅡb-Gy1。通过农杆菌介导法将其分别导入玉米自交系H99的愈伤组织中,共获得132株再生植株;经过PCR检测,初步证明获得了34株转基因阳性植株。     

刺葡萄DFR基因植物表达载体构建及转化茉莉花愈伤组织的研究

本研究以从刺葡萄克隆获得的DFR基因为目的基因，同时从质粒pCAMBIA1302中扩增获得CaMV35S启动子和NOS终止子，并利用双酶切法构建由CaMV35S启动子驱动DFR基因且带有NOS终止子的表达载体，验证测序结果显示成功构建了pCAMBIA1302-35S-DFR-NOS植物表达载体，并将该载体转化到农杆菌EHA105中。采用农杆菌注射渗透法转化烟草叶片进行瞬时表达，经鉴定，转化后的烟草叶片中能检测到GFP基因的表达。采用农杆菌介导法将该植物表达载体转化受体茉莉花愈伤组织，经多轮抗性筛选获得了转基因抗性愈伤组织，经PCR鉴定，茉莉花转基因抗性愈伤组织中检测到GFP、Hyg和DFR基因等的存在，初步证明目的基因DFR已成功整合到宿主的基因组中。茉莉花愈伤组织遗传转化体系的建立，验证了花色苷合成相关基因转化茉莉花愈伤组织的可行性，为今后通过植物转基因技术丰富茉莉花的花色奠定基础。     

利用种子萌发法快速检测抗草铵膦转基因大豆

转基因后代植株遗传存在不稳定性,能够快速有效地筛选后代阳性植株成为转基因研究工作的重点。通过在培养基中添加不同浓度的草铵膦进行种子萌发试验,结果普通受体大豆品种东农50在大于5.0 mg.L-1草铵膦筛选压力下,种子萌发率显著降低,植株根系生长受到抑制,基本不能完成正常的生活史;携带bar基因的Southern拷贝T2代转基因大豆种子在添加5.0 mg.L-1草铵膦的MS培养基中34.7%能正常萌发生长,利用CaMV35S和bar 2对引物对5.0 mg.L-1草铵膦筛选得到的转基因植株进行检测,能扩增出正确的条带,结果均为阳性。研究表明应用种子萌发法能够快速检测草铵膦转基因大豆。     

水稻种子特异性谷蛋白GluB1启动子在水稻愈伤组织中驱动外源基因表达

【目的】水稻谷蛋白启动子GluB1(GluB1promoter,pGluB1)常用于外源基因在种子中特异性高效表达的研究,也是研究种子储藏蛋白基因表达调控机制的模型。前人研究表明,pGluB1只在水稻胚乳中表达,而在根、茎、叶片、叶鞘、颖壳等组织中均无表达活性。研究的目的是为了克服种子特异表达启动子筛选周期长的缺点。【方法】将由pGluB1驱动的霍乱毒素B亚单位和重组胰岛素原组成的融合基因(a fusion gene of the cholera toxin B subunit and human proinsulin, CTBIN)表达载体pCAMBIA1302-pGluB1sig-CTBIN-NOS经农杆菌介导法转化水稻成熟胚愈伤组织。通过RT-PCR和蛋白质印迹杂交试验检测融合基因CTBIN在水稻愈伤组织中的转录和翻译表达。【结果】获得的7个转基因愈伤克隆中,有6个克隆的融合基因CTBIN在转录水平上表达。选取其中4个克隆进一步进行蛋白质印迹杂交试验检测证实融合基因CTBIN均在翻译水平上表达,而且从分子量大小推断融合蛋白包含的谷蛋白GluB1的N-端信号肽序列(24个氨基酸残基)在所测的愈伤组织细胞中均被成功切除。【结论】水稻种子特异性启动子pGluB1在愈伤组织中具有驱动外源基因表达活性,种子蛋白体亚细胞定位信号肽序列可在愈伤组织细胞中被切除。这为在愈伤组织细胞中快速检测种子特异表达启动子活性和探索愈伤组织中蛋白质的亚细胞分拣机制奠定了基础。     

可去除选择标记的转CpTI基因抗虫玉米研究

将组成型表达的玉米泛素启动子与豇豆胰蛋白酶抑制剂基因CpTI连接,插入根癌农杆菌双T-DNA质粒,构建一个T-DNA结构域含有抗潮霉素选择标记基因hyg;另一个T-DNA结构域含有抗虫基因的双T-DNA单子叶植物表达载体,用以转化农杆菌菌株,再通过共培养转化玉米胚性愈伤组织。通过潮霉素培养基抗性筛选,用特异PCR扩增和Southern杂交检测,从分化再生的T0代植株中,鉴定出7个转化CpTI基因的阳性植株。目前,正结合进行田间分离纯合和DNA分子鉴定,培育去除选择标记基因的转基因抗虫玉米自交系。     

甘蔗花青素转录因子基因ScRS克隆与基因枪转化研究

花青素不仅是天然色素,而且具有保健功能。花青素转录因子基因对于调控花青素的合成具有关键作用。本研究根据玉米花青素转录因子基因RS序列设计引物,利用同源克隆技术,得到甘蔗花青素转录因子基因ScRS的全长序列;在ScRS序列两端分别添加KpnⅠ、SalⅠ酶切位点,构建植物表达载体pCAMBIA1301-35SN-ScRS,包被微粒载体通过基因枪对甘蔗愈伤组织进行轰击,转化的愈伤组织明显呈紫红色;将紫红色愈伤组织分化培养获得再生甘蔗植株,经PCR检测,获得转ScRS基因阳性植株,初步证实利用克隆的基因转化甘蔗可以使愈伤组织显色。研究结果对于建立新型的遗传转化可视化示踪体系和培育高花青素甘蔗品种具有重要意义。     

GNA基因表达载体的构建及遗传转化甘蔗

利用经人工改造的gna基因分别与Ubi、Rbcs启动子组合,构建抗虫植物表达载体pUNG和pRNG,通过农杆菌介导法导入甘蔗愈伤组织,经PPT筛选和PCR检测,获得Ubi-gna转基因植株124株,Rbcs-gna转基因植株35株。对部分转基因植株进行RT-PCR检测,初步证明外源基因已经整合到甘蔗基因组中,并得到有效表达;对RT-PCR阳性植株进行GNA(植物外源凝集素)活性测验,结果表明,甘蔗叶片表达的凝集素可以使健康公鸡的红细胞凝集,初步证明表达的GNA具有正常的生物学活性。     

源于玉米幼苗的幼嫩叶段愈伤诱导及其植株再生的研究

以6个不同基因型的玉米自交系幼嫩叶段为外植体,研究其愈伤诱导、继代和分化过程。结果表明,3mg/L 2,4-D是诱导叶段愈伤组织较为适宜的,玉米愈伤组织的诱导及分化与基因型有密切关系。从供试的6个基因型中筛选出出愈率较高、愈伤组织形态较好的3个基因型齐319、Mo17、鲁原92,对它们的愈伤组织进行GUS瞬时表达检测表明,来源于幼苗叶段的愈伤组织GUS+转化率较高。     

转基因水稻中重组植酸酶的表达

为通过在转基因植株中表达植酸酶降解植酸来提高水稻中无机磷利用率,构建了由玉米泛素基因Ubi启动子控制的植酸酶基因植物表达载体,并以来源于水稻未成熟胚的愈伤组织作为转化受体,经农杆菌介导法将植酸酶基因导入水稻中,共获得15个独立的转基因株系。对转基因水稻总DNA的PCR和Southern 杂交分析证明目的基因已整合入转基因水稻植株基因组中,并能稳定遗传。对部分转基因水稻未成熟种子总RNA进行RT PCR分析,表明导入的植酸酶基因能够在转基因水稻种子中正常表达。无机磷含量分析表明含目的基因的转基因水稻种子及其后代叶片中的无机磷含量较未转化植株均有了明显的提高。     

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.     

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.

     

Marine Pharmacology in 2016–2017: 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 review of the 2016–2017 marine pharmacology literature was prepared in a manner similar as the 10 prior reviews of this series. Preclinical marine pharmacology research during 2016–2017 assessed 313 marine compounds with novel pharmacology reported by a growing number of investigators from 54 countries. The peer-reviewed literature reported antibacterial, antifungal, antiprotozoal, antituberculosis, and antiviral activities for 123 marine natural products, 111 marine compounds with antidiabetic and anti-inflammatory activities as well as affecting the immune and nervous system, while in contrast 79 marine compounds displayed miscellaneous mechanisms of action which upon further investigation may contribute to several pharmacological classes. Therefore, in 2016–2017, the preclinical marine natural product pharmacology pipeline generated both novel pharmacology as well as potentially new lead compounds for the growing clinical marine pharmaceutical pipeline, and thus sustained with its contributions the global research for novel and effective therapeutic strategies for multiple disease categories.     

Effects of Micronutrient Deficiencies on Human Health: Its Status in South Asia

SUMMARY

Vitamin A, iodine, and iron deficiencies affect large numbers of people worldwide. A deficiency of one or more of these micronutrients adversely affects the physical and mental abilities of humans. South Asia contains a high percentage of these individuals, not just because of its large population, but also the prevalence of deficiency is higher there than in many other parts of the world. The reasons for the high rates of deficiency in South Asia are many, but they include inadequate availability of micronutrient-rich foods, sub-optimal dietary habits, and high rates of infection. Across the region, national plans vary from (a) increasing availability of appropriate foods, (b) introducing new dietary behaviors, (c) improving health status, and (d) directly addressing deficiency through intervention programs. The extent to which each of the countries is making progress toward the goals of these plans can help in understanding the priorities for future efforts to address micronutrient malnutrition. This article discusses the adverse effects of micronutrient deficiencies in humans with special reference to South Asia.     

Management of Soil Micronutrient Deficiencies in the Rice-Wheat Cropping System

Summary

Within the last three decades, the rice-wheat cropping system has triggered, and with time, aggravated soil micronutrient deficiencies in the Indo-Gangetic Plains (IGP). This has largely been due to the shift from an earlier rice and wheat monoculture with low yielding, long duration indigenous varieties, to an intensive rice-wheat rotation cropping system with short duration modern high yielding varieties on the same piece of land. The problems related to micronutrient deficiency in the IGP are more due to the size of its available pools in the soil rather than its total contents and are greatly influenced by crop management, or rather its mismanagement. Deficiency of zinc is widespread in the IGP, but with the extensive use of zinc sulfate, zinc deficiency has reduced in some areas of the region. Meanwhile, the deficiency of Fe, Mn and B has increased in the IGP. Deficiency of Cu and Mo is location specific and can limit rice and wheat yields. The adoption and spread of the rice-wheat system in permeable coarse textured soils, particularly in the western IGP, not only caused iron deficiency in rice but also resulted in the emergence of manganese deficiency in wheat. In highly calcareous and acidic soils, boron is the next limiting micronutrient in crop production after zinc. Bumper rice and wheat harvests in the past decade, the declining use of organic manures in the region and except for the widespread use of zinc sulfate, a general lack of awareness amongst farmers on micronutrient deficiency problems has contributed to micronutrients limiting rice and wheat yields in the IGP. Approaches to alleviating micronutrient deficiencies include matching the crop removals of the micronutrients with its replenishments through their respective external carriers, supplementation through organic sources and mobilization/utilization through cultivation of micronutrient efficient crop cultivars. Identification of efficient micronutrient carriers and finding the optimum rate, mode and time of its application is important in ameliorating the micronutrient deficiencies. This article reviews the extent of micronutrient deficiency and discusses various management options available to reduce micronutrient deficiency induced crop yield reduction for rice and wheat in the Indo-Gangetic Plains.     

Irrigation but not N fertilization enhances seedhead density in plains rough fescue ( F estuca hallii)

Plains rough fescue ( F estuca hallii) is an important forage grass species in western Canada. Seed for use in pastures and ecological restoration is in high demand but supply is limited because F. hallii is an erratic seed producer. Seed producers require an understanding of the factors that influence flowering and seed set in this species. The aim of this study was to assess the effect of irrigation and nitrogen fertilization on the flowering and growth of F. hallii in a native rough fescue prairie in Alberta, Canada. Irrigation had a strong positive effect on seedhead density, whereas fertilization had limited effects on growth and reproduction of F. hallii. These results demonstrate that under field conditions, available moisture is likely to be a key factor driving flowering and seed production in F. hallii.     

Marine Drugs Regulating Apoptosis Induced by Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)

Marine biomass diversity is a tremendous source of potential anticancer compounds. Several natural marine products have been described to restore tumor cell sensitivity to TNF-related apoptosis inducing ligand (TRAIL)-induced cell death. TRAIL is involved during tumor immune surveillance. Its selectivity for cancer cells has attracted much attention in oncology. This review aims at discussing the main mechanisms by which TRAIL signaling is regulated and presenting how marine bioactive compounds have been found, so far, to overcome TRAIL resistance in tumor cells.     

Biochemical and morphological characterization of potato clones differing in their resistance to late blight

Summary Morphological and biochemical parameters that could be involved in resistance to late blight were studied in non-infected and in infected potato hybrids resulting from a cross betweenSolanum phureja, resistant toPhytophthora infestans, and a susceptibleSolanum tuberosum. Some morphological differences between resistant and susceptible hybrids, indicating a positive correlation between stem diameter and phloem thickness in the stem and resistance toP. infestans, were observed. The lignin content in the leaves of the resistant hybrid rose upon infection byP. infestans. In the leaves of the susceptible hybrid, a diminution of the lignin content could be observed upon infection byP. infestans. In the same context, peroxidasic activity raised upon infection byP. infestans in both resistant and susceptible hybrids. Further characterization of the hybrid clones based on the polymorphism of peroxidases was attempted using isoelectric focusing.     

Applied Biotechnology to Combat Late Blight in Potato Caused by <Emphasis Type="Italic">Phytophthora Infestans</Emphasis>

Potato is an important crop, grown worldwide. It suffers from many pests and diseases among which late blight, caused by the oomycete Phytophthora infestans, is the worst. The disease is still causing major damage in many potato production areas and control is only possible by applying fungicides frequently. The knowledge on the molecular biology and genetics of the interaction between the plant and the oomycete is developing rapidly. These are relevant fields of study, currently dominated by the discovery of many resistance genes and numerous effector proteins and the analysis of their specific mode of action. These studies may yield essential information needed for the development of durable resistance. The long-term and worldwide effort to breed for resistance so far has had little effect. A novel breeding approach may change this. It is based on cisgenic modification (CM) consisting of marker-free pyramiding of several resistance genes and their spatial and temporal deployment yielding dynamic varieties that contain potato genes only. It is envisioned that this CM approach with potato’s own genes will not only prove societally acceptable but may also result in simplifications in the legislation on use of the CM approach. Various parties in the potato research arena intend to cooperate in this novel approach in a number of developing countries where potato substantially contributes to food security. The use of resources such as land, water and energy improves when the effect of late blight is markedly reduced.     

Resistance to <Emphasis Type="Italic">Potato virus Y</Emphasis> in a Multitrait Potato Breeding Scheme without Direct Selection in Each Generation

In an experimental breeding scheme to improve late blight (Phytophthora infestans) and white potato cyst nematode (Globodera pallida) resistance of tetraploid potato over three generations of crossing and selection, 15 clones survived the final selection, and these were derived from 15 great-grandparents. There was no direct selection for resistance to Potato virus Y (PVY), but 14 out of the 15 great-grandparents were resistant to PVY and three had extreme resistance. Thirteen of the 15 descendants had PVY resistance and one extreme resistance. This was within the range expected for a random (unselected) sample from the genotypes of the great-grandparents. Hence, we found no evidence for any positive or negative association between PVY resistance and the attributes selected. The conclusion is that laborious selection is not required in every generation when many parents have PVY resistance, including some with more than one copy of a PVY resistance gene or resistance at more than one locus. However, in the future, determining the major virus resistance genes present in potential parents in each generation using diagnostic molecular markers would prevent susceptible × susceptible crosses being made and maximise the number of resistant × resistant ones.