西瓜第一染色体显微分离及其特异文库的构建

采用玻璃针分离法,通过显微操作系统成功地分离到西瓜(2n=22)的第一单染色体,将分离到的西瓜染色体放入0.2 mL Eppendorf管中,经去蛋白,Sau3A酶切,并在染色体DNA片段两端加上Sau3A人工接头后,进行两轮PCR扩增,得到0.4～2.0 kb之间的DNA片段.用西瓜的基因组标记成探针,与单染色体扩增产物进行southem杂交,表明这些西瓜单染色体扩增片段与西瓜基因组DNA之间有同源性,从而证明单染色体DNA确实已被成功地扩增.将第2轮PCR产物构建质粒文库,得到约20 000个重组子.这是染色体微分离、微克隆技术首次在瓜类作物中的应用,该方法为直接从西瓜单条染色体DNA文库中筛选分子标记奠定了基础,说明利用染色体显微分离、克隆技术对西瓜等瓜类作物基因组进行研究是可行的.     

柚(Citrus grandis Osbeck)单染色体LA-PCR-AFLP分子标记体系的建立

通过微分离和微克隆技术,对柚单染色体LA-PCR-AFLP技术体系中反应程序、模板浓度等关键参数进行了优化和引物筛选。结果表明:退火温度较高的程序TD3[退火梯度:72℃→65℃30 s(-0.5℃/cyc)]扩增效果较好;稀释100倍(10 ng/μL)为最适模板量;10个引物对组合扩增多态性较好。对22条单染色体的AFLP多态性分析和dot-blot杂交验证,表明较好地构建了柚单染色体LA-PCR-AFLP体系。这为柚单染色体的识别和文库构建提供了试验基础。     

漳州水仙随体染色体的分离及DNA文库的构建

以漳州水仙为材料,采用微细玻璃针分离了水仙第7号同源组的3条随体染色体,进行了LA-PCR体外扩增,Southern杂交证明水仙单染色体DNA被成功扩增.将扩增产物插入PMD18-T载体构建了5.0×105个克隆的单染色体DNA文库,为进一步研究中国水仙的起源与进化提供了有益的细胞遗传学和分子生物学依据.     

基于TRAP的长穗偃麦草SCAR标记的开发及应用

为了开发长穗偃麦草的特异标记,依据TRAP技术,利用56对固定引物与随机引物的组合对中国春-长穗偃麦草附加系等材料进行PCR扩增,共筛选出160条分布于长穗偃麦草1E-7E染色体的特异扩增片段。通过对104个片段的序列进行同源比对,选择与小麦无同源序列的区段设计138对引物,分别对中国春、长穗偃麦草、中国春-长穗偃麦草附加系进行PCR扩增,最终获得30个长穗偃麦草特异SCAR标记,发展标记的效率为53.6%。利用这些特异SCAR标记对硬粒小麦(AABB)与异源六倍体小麦(AABBEE)杂交产生的F2中38个单株进行扩增鉴定,9个单株仅附加了1条相同的E染色体,其余为多条E染色体或者无E染色体附加。这些SCAR标记在不同材料和世代表现出优越的特异性和稳定性,可用于检测小麦背景中附加的相关长穗偃麦草染色体。     

利用流式细胞法鉴定马铃薯倍性方法的优化

为了建立适于马铃薯倍性鉴定的流式细胞法试验体系,以二倍体马铃薯‘E’和四倍体马铃薯‘青薯9号’为材料,分别利用LB01,Galbraith's和Tris·MgCl_2 3种细胞核分离缓冲液制备马铃薯组培苗幼叶单细胞核悬浮液,比较离心和不离心及不同核酸染料碘化丙啶(Propidium iodide,PI)和4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)对流式细胞仪检测结果的影响。结果表明,3种核分离缓冲液对制备马铃薯组培苗幼叶单细胞核悬浮液存在差异,其中核分离缓冲液LB01的效果最好;不离心的效果和离心相比,离心的悬浮液中目的细胞少于不离心的;染料DAPI与PI相比,染色效果无明显差异。从而初步建立起一套利用流式细胞术鉴定马铃薯倍性的方法,该方法样品需求量小,且制备简单,测量快速,准确度高,背景碎片和杂峰较少,主峰清晰,是鉴定马铃薯染色体倍性的理想方法。     

玉米株高主效QTL qPH2.4的定位分析

课题组前期以玉米自交系郑58为轮回亲本,以昌7-2为供体亲本,通过分子标记辅助选择获得染色体单片段代换系Z12和W16。Z12和W16在bin2.07区域均含有1个来源于昌7-2的染色体片段,株高均显著高于轮回亲本郑58。利用郑58和Z12为材料构建F2分离群体,基于重测序和混合分离分析(BSA)策略,将株高主效QTL qPH2.4定位于第2染色体13.95 Mb(201 457 953~215 022 157 bp)的区域内。利用在目标区间内筛选出的20对多态性分子标记对包含743个单株的郑58×Z12 F2分离群体和包含1 720个单株的郑58×W16 F2分离群体进行基因型分析,结合田间株高数据进行QTL定位,将株高主效QTL qPH2.4定位在InDel分子标记ph-18和ph-19之间,区间的遗传距离为0.57 cM,物理距离为626 kb。参考B73基因组(RefGen_v4)注释信息,该区间内存在17个注释基因,其中,包含可以调控油菜素内酯信号的基因Zm00001d006677。     

基于甘蔗AP85-441和R570基因组参考序列的微卫星位点鉴定和SSR标记开发

分子标记缺乏是制约甘蔗分子标记技术发展的重要因素。本研究利用甘蔗AP85-441和R570基因组参考序列,使用MISA软件分别鉴定出512 835和97 839个微卫星位点,分别占割手密基因组（AP85-441）和甘蔗基因组（R570）高质量参考序列的0.32%和0.35%。在2个基因组序列中,优势重复单元均为单核苷酸、二核苷酸和三核苷酸,各重复单元均以AT富集的基元为主。割手密和甘蔗基因组序列中分别有472 117和89 748个位点可以开发SSR标记。对割手密、甘蔗与高粱基因进行同源性分析,分别鉴定出16 691个和13 271个对应到高粱1~10号染色体上的同源基因,利用基因序列中的SSR位点,开发出13 224和7624对SSR引物。对开发的引物分别以割手密和甘蔗基因组为模板进行e-PCR检测,这些引物在割手密基因组中以多位点扩增为主,在2个基因组中的有效标记比例分别为79.35%和36.13%、79.01%和93.36%。部分SSR引物在基因组中表现出特异性扩增,有1368对仅在AP85-441中单扩增,有1420对仅在R570序列中单扩增,共有752对SSR引物可在2个基因组中单扩增,且这些SSR的扩增位点和来源基因均分布于所在基因组的全部染色体上。在禾本科作物基因组中e-PCR检测表明,开发的单扩增SSR标记具有较好的特异性。本研究鉴定的SSR位点,有助于进一步丰富甘蔗的分子标记;开发的3540对SSR引物对于栽培种甘蔗遗传图谱构建中遗传来源区分和同源连锁群确定具有重要的参考意义。     

花生微卫星DNA分离方法的研究

比较了2种从AFLP扩增片段中分离花生微卫星DNA(SSR)的方法，结果表明，从预扩增的AFLP片段中富集SSR的方法简便，获得较多的含简单重复序列的片段；而从选择性扩增的AFLP片段中分离SSR后直接从电泳胶上切取差异片段进行测序的方法效率低，不适用于分离花生的微卫星DNA。     

水稻着丝粒特异组蛋白CENH3抗体的制备与应用

【目的】着丝粒是真核生物染色体的基本功能元件之一,其功能是在细胞有丝分裂和减数分裂时期精确地调控染色体配对和分离并维持染色体的稳定。着丝粒结构是由DNA和蛋白质形成的一种复合体。着丝粒特异组蛋白(centromere-specific histone H3,CENH3)是功能着丝粒是否具有活性的最基本特征。所以制备CENH3的相关抗体是进行着丝粒结构与功能研究的前提条件之一。【方法】通过设计短肽进行兔免疫实验,制备了水稻着丝粒特异组蛋白CENH3兔源抗体,利用ELISA和蛋白免疫荧光(immunofluorescence,IF)等检测方法对抗体有效性进行了鉴定。【结果】ELISA检测显示制备的CENH3抗体有效稀释度为1:40万,并且蛋白免疫荧光信号在水稻体细胞每条染色体的着丝粒区域均能检测到。同时,该抗体也可以应用于玉米等其他物种。通过染色质免疫沉淀(ChIP)技术获得与CENH3相结合的DNA分子,并进行PCR扩增和FISH定位分析,结果显示相应的Ch IP-DNA位于水稻功能着丝粒区域。【结论】本研究制备的水稻CENH3兔源抗体能满足着丝粒研究中相关实验的要求,可进一步应用于着丝粒的结构与功能研究。     

转Bt-pta-bar基因玉米侧翼序列分析及特异性检测方法研究

采用染色体步移技术(Genome Walking)分离转Bt-pta-bar基因玉米JL937的插入位点侧翼序列,获得了615 bp的5’端边界序列。据此序列设计特异性引物,建立了JL937玉米的特异性定性PCR检测方法,可特异性地从JL937玉米中扩增出221 bp的产物。此方法具有特异性强、稳定性好的特点,适用于JL937玉米的定性检测。     

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.     

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.     

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.     

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.