花生MAPK13基因的克隆及表达分析研究

为了挖掘花生抗果腐病相关基因,本研究以花生品种花育20号为试验材料,从花生果腐病转录组文库中筛选出促丝裂原活化蛋白激酶13(MAPK13)基因编码区序列设计引物,通过RACE-PCR克隆得到AhMAPK 13基因°结果表明AhMAPK 13基因序列全长1818 bp,含有/非编码区593 bp,5非编码区122 bp,开放阅读框全长1104 bp,编码1条含有368个氨基酸的蛋白序列。预测其分子量为42. 5kDa,含有MAPK家族典型的11个结构域,属于MAPK基因家族B组成员。亚细胞定位显示AhMAPK13定位于细胞质和细胞核中° RT-qPCR分析发现AhMAPK 13基因在茎中表达量高于其他组织,说明该基因具有组织表达特异性;AhMAPK 13基因受干旱、低温、NaCl(ABA诱导时表达量上升,说明该基因可能广泛参与植物非生物胁迫响应过程;AhMAPK 13基因受JA、GA、ET、SA诱导时表达量上调,说明该基因可能参与到这些激素介导的抗逆信号转导途径。本研究结果为花生抗果腐病育种研究提供了新的基因资源。     

花生CAX相互作用蛋白4(CXIP4)基因克隆表达分析及载体构建

钙离子是细胞信号传导中重要的第二信使。CAX interacting protein(CXIP)是一类能够激活Cation exchanger(CAX)的钙离子转运活性蛋白,在离子转运体系中起重要作用。本研究利用RACE方法获得花生CAX相互作用蛋白4(CXIP4)基因的cDNA和DNA全长序列。该基因cDNA序列的全长包括966bp的开放阅读框,编码321个氨基酸。DNA全长为966bp,不含有内含子序列。推测的蛋白质分子量为36706.24Da,等电点为9.62。使用荧光定量技术(qRT-PCR)分析了该基因在花生品种日花1号植株中的组织表达和青枯菌胁迫条件下的表达变化规律,并构建了该基因的过表达载体、反义表达载体以及原核表达载体,为进一步研究花生中该基因的功能及其在花生抗青枯病中的作用奠定基础。     

荔枝快速碱化因子基因的克隆与表达分析

从已构建的荔枝果皮cDNA文库中挑取克隆测序,获得一条荔枝快速碱化因子(LcRALF)基因(GenBank登录号:EU024484).该基因的cDNA全长为666 bp,含1个381 bp的开放读码结构,编码126个氨基酸.生物信息学分析表明,该基因的氨基酸序列与拟南芥、烟草等物种的RALF基因一致性较高,具有RALF基因氨基酸序列的典型特征.RT-PCR分析表明,该基因在荔枝果皮中特异表达,且在新采收的荔枝果皮中表达量最高.随着果皮的衰老该基因表达量逐渐下降.     

滨麦蔗糖:果聚糖6-果糖基转移酶(6-SFT)基因全长cDNA的克隆与生物信息学分析

为了挖掘和利用滨麦(Leymus mollis,2n=4x=28,JJNN)的优良基因,以拓宽小麦非生物胁迫抗性基因资源,以滨麦为材料,利用RT-PCR结合RACE技术从滨麦叶片中克隆到6-SFT基因cDNA的全长序列,并对其进行生物信息学分析。结果表明,其cDNA全长为2 086bp,开放阅读框为1 866bp(命名为Lm-6-SFT),编码621个氨基酸;其推导的蛋白分子量为69.1kDa,理论等电点(pI)为5.18,属于酸性蛋白。保守结构域分析表明,该基因推导的氨基酸序列含有SDPDG、RDP和EC结构域。多序列比对及进化树分析表明,滨麦6-SFT与冰草6-SFT在氨基酸水平具有高度的序列相似性。     

花生中UDP-glucosyltransferase基因的克隆及在非生物胁迫下的表达研究

通过RACE技术从花生叶片cDNA中克隆到了UDP-glucosyltransferase基因,命名为AhUGT83A1-like （Genbank注册号为KF411463）。该基因全长为1 530bp,ORF为1 380bp,编码460个氨基酸。序列比对与进化分析结果表明,该序列有保守的UDPGT结构域,并且与其它植物的UGT蛋白同源性较高。荧光定量PCR结果表明,AhUGT83A1-like基因在高盐和低温处理的花生根和叶片中均上调表达;在干旱处理时,根中表达也受到显著上调,但叶片中表达量则有明显下降。以上结果表明AhUGT83A1-like可能参与了花生对干旱、低温和高盐的抗性调控。ABA处理结果表明,AhUGT83A1-like在花生根中对ABA响应明显,但在叶片中对ABA没有明显响应,表明该基因在花生根中对非生物胁迫的调控可能是以依赖ABA的方式发挥作用的。?     

甘蔗苏氨酸脱氨酶基因的克隆与表达分析

应用电子克隆技术,以烟草AAG59585序列为探针,获得了甘蔗苏氨酸脱氨酶(threonine deaminase)基因的全长cDNA,命名为ScTD。经RT-PCR扩增和验证,该基因序列与电子克隆结果一致性高达99%。序列分析结果表明:ScTD基因全长2 120 bp,具有完整的开放阅读框(ORF,164~1 681 bp),编码505个氨基酸,该基因编码蛋白定位于微体,为可溶性蛋白,不存在信号肽,二级结构原件多为α-螺旋,含有多个保守功能域,主要在氨基酸合成中发挥作用。电子表达分析结果显示,该基因在甘蔗根尖、根颈、花序、叶片、全株、芽和茎中组成型表达,其中在花序和根中的表达量最高。荧光定量PCR结果分析表明:该基因在甘蔗各组织中均有表达,且在根中的表达量最高。此外,该基因的表达受水杨酸胁迫后表达量最高,约为对照组的43.16倍,其次为聚乙二醇和茉莉酸甲酯,分别为6.90倍和4.40倍,推测该基因的表达与甘蔗抗病虫和抗渗透胁迫有关。此结果为甘蔗ScTD基因的克隆和功能验证以及在甘蔗基因工程中的应用奠定基础。     

小麦细胞色素P450(TaP450)基因的克隆与序列分析

细胞色素P450是一种多功能氧化酶,在植物体内担当着生物合成、代谢解毒以及抗逆等重要功能。本研究采用RACE方法从普通小麦中同源克隆到一个新的P450基因,命名为TaP450(Genbank No.KJ541960)。该基因基因组序列全长为2 643bp,含有2个外显子和1个内含子,cDNA全长为2 033bp,含有一个1 557bp的开放读码框,推导蛋白含518个氨基酸;序列分析发现其具有保守的P450结构域,但该蛋白与已报道的小麦P450蛋白序列有较大差异,表明它是小麦P450家族的新成员;蛋白结构特征分析发现,该蛋白的分子量为57.092kD,等电点为8.63,N端具有一个含29个氨基酸的跨膜结构和一个含22个氨基酸的信号肽,亚细胞定位分析表明该蛋白属于分泌蛋白。其在小麦叶片、茎、根与种子中均有表达,但在叶片和茎中表达量最高;在胁迫处理下,该基因的表达量均上调,且在盐胁迫处理下上调尤为显著,表明该基因与盐胁迫密切相关。     

橡胶树胚胎晚期丰富蛋白基因HbLEA1的克隆和表达分析

胚胎晚期丰富蛋白（LEA）是与植物抗逆反应相关的一类重要蛋白。本研究克隆了橡胶树LEA家族一个新基因的全长cDNA序列,命名为HbLEA1。该cDNA全长1 183 bp,其中5'UTR区60 bp,3'UTR区154 bp,CDS区969 bp,共编码322个氨基酸,分子量大小为36 ku,等电点4.84,含有LEA-2和WHy（Water Stress and Hypersensitive response）结构域,属于LEA_2类LEA蛋白。组织表达分析结果表明,HbLEA1在种子中表达量最高,其次是胶乳和稳定叶,在芽中的表达量最低。在未开割树的胶乳中,机械伤害和割胶处理均明显下调HbLEA1表达。在橡胶树幼苗中,低温胁迫处理后该基因在叶片、树皮和根中的表达呈上升趋势,而在高温和干旱胁迫中其表达变化并不明显。结果显示,HbLEA1基因可能参与橡胶树的胁迫应答和胶乳代谢调控。     

玉米光周期敏感基因ZmELF4的克隆及其亚细胞定位

以热带玉米自交系CML288为供试材料,根据拟南芥ELF4基因保守序列设计引物,采用RT-PCR方法克隆了ELF4在玉米上同源基因的全长cDNA序列(GenBank上的登录号为HQ009862)。序列分析表明,该基因cDNA序列全长683 bp,开放阅读框为432 bp,编码了144个氨基酸,与拟南芥、水稻的氨基酸序列同源性分别高达62%和75%,此外它们还共同包含一个高度保守的DUF1313未知功能结构域。利用基因重组技术分别构建了能够高效表达的过量表达载体pBI-ZmELF4和带有GFP标记的融合表达载体pGIT-ZmELF4-GFP,利用融合表达载体进行洋葱表皮瞬时表达实验,结果将ZmELF4定位于细胞核内,说明该基因在细胞核内起作用。     

花生胚胎发育晚期丰富蛋白基因AhLEAL的克隆及其在非生物胁迫下的表达分析

低温、高盐和干旱等非生物胁迫严重影响花生的生长和产量,而花生中有关非生物胁迫的研究及抗性基因的挖掘较少。胚胎发育晚期丰富蛋白(Late embryogenesis abundant,LEA)是一类亲水性蛋白,其功能主要是参与调控植物应答脱水相关的非生物胁迫包括耐盐、抗旱和抗冻等。本研究以花生品种花育33号为试验材料,根据cDNA文库中已知的胚胎发育晚期丰富蛋白基因全长序列设计引物,通过RT-PCR克隆到该基因。该基因全长为555bp,开放阅读框为291bp,编码97个氨基酸。核苷酸序列比对表明,该基因与花生中已注册基因AhARG2(XM_016113504)同源性为100%,与AhLEA6-1(HM543588)同源性为99%。蛋白序列比对分析表明,该基因编码的蛋白与Genbank上已经注册的沙冬青(Ammopiptanthus mongolicus)、牧豆树(Prosopis juliflora)和柠条锦鸡儿(Caragana korshinskii)等植物中的胚胎发育晚期丰富蛋白同源性达到近65%。我们将该基因命名为AhLEAL(Arachis hypogaea Late Embryogenesis Abundant Like)。预测该基因编码的蛋白可能定位于细胞液、线粒体、内质网和叶绿体中。荧光定量PCR结果显示,AhLEAL在花生叶片和根中的表达受低温和高盐胁迫的强烈诱导,在根中受干旱胁迫的强烈诱导,说明该基因可能参与了花生对三种非生物胁迫的适应性调控;此外,AhLEAL在花生叶片和根中的表达也明显受ABA的诱导,说明该基因对花生逆境胁迫的调控可能是以依赖ABA的方式进行的。     

跨入新世纪开创新局面——浙江省茶叶学会第八届理事会工作报告

1　四年工作概况本届理事会是跨世纪的一届理事会。世纪之交的 1 997～ 2 0 0 1年 ,是伟大祖国取得辉煌成就的时期 ;也是浙江茶业深化改革 ,不断开拓新局面的时期。四年来 ,本届理事会在省科协的直接领导、挂靠单位和有关部门的大力支持以及广大会员的积极参与下 ,认真分析了我省茶叶产销形势 ,认真组织并调动各方面积极性 ,克服重重困难 ,有重点、有针对性的开展了一系列活动 ,取得了新的成绩 ,为浙江茶业经济的持续发展作出了重要贡献。为此 ,我会被评为 1 997～ 1 999年度全省先进学会 ,“九五”科普先进单位 ;并再次评为“全国学会之星…     

Plitidepsin has a safe cardiac profile: a comprehensive analysis

Plitidepsin is a cyclic depsipeptide of marine origin in clinical development in cancer patients. Previously, some depsipeptides have been linked to increased cardiac toxicity. Clinical databases were searched for cardiac adverse events (CAEs) that occurred in clinical trials with the single-agent plitidepsin. Demographic, clinical and pharmacological variables were explored by univariate and multivariate logistic regression analysis. Forty-six of 578 treated patients (8.0%) had at least one CAE (11 patients (1.9%) with plitidepsin-related CAEs), none with fatal outcome as a direct consequence. The more frequent CAEs were rhythm abnormalities (n = 31; 5.4%), mostly atrial fibrillation/flutter (n = 15; 2.6%). Of note, life-threatening ventricular arrhythmias did not occur. Myocardial injury events (n = 17; 3.0%) included possible ischemic-related and non-ischemic events. Other events (miscellaneous, n = 6; 1.0%) were not related to plitidepsin. Significant associations were found with prostate or pancreas cancer primary diagnosis (p = 0.0017), known baseline cardiac risk factors (p = 0.0072), myalgia present at baseline (p = 0.0140), hemoglobin levels lower than 10 g/dL (p = 0.0208) and grade ≥2 hypokalemia (p = 0.0095). Treatment-related variables (plitidepsin dose, number of cycles, schedule and/or total cumulative dose) were not associated. Electrocardiograms performed before and after plitidepsin administration (n = 136) detected no relevant effect on QTc interval. None of the pharmacokinetic parameters analyzed had a significant impact on the probability of developing a CAE. In conclusion, the most frequent CAE type was atrial fibrillation/atrial flutter, although its frequency was not different to that reported in the age-matched healthy population, while other CAEs types were rare. No dose-cumulative pattern was observed, and no treatment-related variables were associated with CAEs. Relevant risk factors identified were related to the patient's condition and/or to disease-related characteristics rather than to drug exposure. Therefore, the current analysis supports a safe cardiac risk profile for single-agent plitidepsin in cancer patients.     

Super-races are not likely to dominate a fungal population within a life time of a perennial crop plantation of cultivar mixtures: a simulation study

ABSTRACT: BACKGROUND: Deployment of cultivars with different resistance in mixtures is one means to manage plant diseases and prolong the life of resistance genes. One major concern in adopting mixtures is the development of 'super-races' that can overcome many resistance genes present in the mixture. A stochastic simulation model was developed to study the dynamics of virulence alleles in two-cultivar mixtures of perennial crops, focusing on the effects of cost of virulence and pathogen reproduction mechanism. The simulated mechanism of virulence has characteristics of both major and minor genes. RESULTS: Random genetic drift due to repeated population crashes during the overwintering phase led to fixation of a single fungal genotype (in terms of its virulence), often within 100 seasons. Overall, cost of virulence is most important in determining the virulence dynamics under the present model formulation. With cost of virulence incorporated, nearly all simulation runs ended up with a single fungal genotype that can infect only one of the two cultivars. In absence of cost of virulence, most of the simulation runs ended up with fungal genotypes that can infect both host cultivars but in many cases do not contain the maximum possible number of virulence alleles due to random drift. A minimum of 20% sexual reproduction between strains from different cultivars is necessary to ensure that the final fixed strains are able to infect both cultivars. Although the number of virulence alleles in the final genotype and the time to fixation are affected by simulation factors, most of the variability was among replicate simulation runs (i.e. stochastic in nature). The time to fixation is generally long relative to cropping cycles. CONCLUSIONS: A single fungal genotype will dominate a population due to the bottleneck in overwintering with cost of virulence primarily determining whether the dominant genotype can infect both cultivars. However, the dominate genotype is unlikely to accumulate all the virulence alleles due to genetic drift. The risk of emergence and spread of super-races is insufficiently great to prevent the use of cultivar mixtures of perennial crops as a means to reduce disease development provided that host resistance structure in mixtures is altered every cropping cycle.     

Verrucosamide,a Cytotoxic 1,4-Thiazepane-Containing Thiodepsipeptide from a Marine-Derived Actinomycete

A new cytotoxic thiodepsipeptide, verrucosamide (1), was isolated along with the known, related cyclic peptide thiocoraline, from the extract of a marine-derived actinomycete, a Verrucosispora sp., our strain CNX-026. The new peptide, which is composed of two rare seven-membered 1,4-thiazepane rings, was elucidated by a combination of spectral methods and the absolute configuration was determined by a single X-ray diffraction study. Verrucosamide (1) showed moderate cytotoxicity and selectivity in the NCI 60 cell line bioassay. The most susceptible cell lines were MDA-MB-468 breast carcinoma with an LD₅₀ of 1.26 µM, and COLO 205 colon adenocarcinoma with an LD₅₀ of 1.4 µM. Also isolated along with verrucosamide were three small 3-hydroxy(alkoxy)-quinaldic acid derivatives that appear to be products of the same biosynthetic pathway.     

Rode Eersteling: a false name for a well-known tuber colour mutant

Summary It is shown that the monectochimeraRote Holl?ndische Erstling used in Klopfer's chimera experiments does not correspond with the present homohistic Dutch potato varietyRode Eersteling. The latter seems to be identical with Klopfer's homohisticErstling Rouge.⁴ This misapprehension is caused mainly by replacing the original monectochimerical varietyRode Eersteling in 1942 by a homohistic red sport, derived from it, without changing the variety name.     

Pollution control via a dredged pool at the inlet of a reservoir

We investigated the applicability of a dredged pool formed at the inlet of a reservoir for pollution control. To quantify the effect of a dredged pool on the water quality of a reservoir, we monitored the water quality of the Masan Reservoir, located in the city of Asan, Choongnam Province, Korea, before and after dredging. In addition, a completely mixed box model was applied to simulate the water quality of the dredged pool and reservoir. The model included a water balance equation and chemical mass balance equation, into which several interactions among water quality components, such as phytoplankton, total nitrogen (T-N), total phosphorus (T-P), dissolved oxygen, and chemical oxygen demand (COD), were incorporated. The water quality monitoring and modeling results indicated that reservoir water quality was greatly affected by the dredged pool. The loads of T-N and T-P into the reservoir were reduced by the dredged pool, which may have induced the removal of nutrients by settling. However, the dredged pool may have had a negative effect on the reservoir in terms of COD and chlorophyll-a because of the internal production of organic matter and/or algae with water detention. Therefore, a dredged pool may be used for pollution control in a reservoir, as long as it is combined with measures to reduce concentrations of organic matter and/or algae.     

Characterization of a novel serine protease inhibitor gene from a marine metagenome

A novel serine protease inhibitor (serpin) gene designated as Spi1C was cloned via the sequenced-based screening of a metagenomic library from uncultured marine microorganisms. The gene had an open reading frame of 642 base pairs, and encoded a 214-amino acid polypeptide with a predicted molecular mass of about 28.7 kDa. The deduced amino acid sequence comparison and phylogenetic analysis indicated that Spi1C and some partial proteinase inhibitor I4 serpins were closely related. Functional characterization demonstrated that the recombinant Spi1C protein could inhibit a series of serine proteases. The Spi1C protein exhibited inhibitory activity against α-chymotrypsin and trypsin with K(i) values of around 1.79 × 10(-8) and 1.52 × 10(-8) M, respectively. No inhibition activity was exhibited against elastase. Using H-d-Phe-Pip-Arg-pNA as the chromogenic substrate, the optimum pH and temperature of the inhibition activity against trypsin were 7.0-8.0 and 25 °C, respectively. The identification of a novel serpin gene underscores the potential of marine metagenome screening for novel biomolecules.     

Integrating a simple shading algorithm into CERES-wheat and CERES-maize with particular regard to a changing microclimate within a relay-intercropping system

Wheat/maize related multi-cropping systems are the dominant cropping systems in North China. To improve and adjust those systems, and to study competition effects within intercropping, extended field experiments are necessary. As field experiments are time consuming, laborious and expensive, a viable alternative is the use of crop growth models that can quantify the effects of management practices on crop growth and productivity. Field experiments showed that intercropped maize yielded as high as monocropped maize, and grain yield of intercropped wheat increased by up to 32%. Based on a process-oriented modeling approach, this study focuses on analyzing and modeling competitive relationships in a wheat/maize relay intercropping system with regard to yield, solar radiation and microclimate effects. A simple shading algorithm was applied and integrated into the CERES models, which are part of the DSSAT software shell vs. 4.5. The algorithm developed estimates the proportion of shading as affected by neighbouring plant height. The model was tested to investigate the applicability of this shading algorithm within the CERES models in the simulation of grain yield and dry matter yield of wheat and maize. Model error of grain and dry matter yield for both species was below 10%. There was a tendency for grain yield to be simulated adequately but for dry matter yield to be slightly underestimated. Increased top soil temperature in intercropped wheat increased the mineralization of nitrogen and improved N supply. The wheat/maize system was N efficient. Thus, N dynamics were taken into account for simulation as well as CO₂ dynamics based upon modified wind speed. Wheat border rows were exposed to a higher wind speed until mid-June and to reduced wind speed thereafter. As a result, solar radiation, soil temperature and wind speed differed between monocropping and intercropping and could provide a starting point for simulating intercropping. Microclimate effects are often small, subtle or non-existent, while spatial and climate variability and the heterogeneity of plant populations can be considerable. Quantifying microclimatic effects may prove difficult but should not be neglected when simulating intercropping systems.     

Optimal gate operation of a main drainage canal in a flat,low-lying,agricultural area using a tank model

Chiyoda basin is located in Saga Prefecture in Kyushu Island, Japan, and lies next to the tidal compartment of the Chikugo River to which the excess water in the basin is drained away. Chiyoda basin has a total area of about 1,100 ha and is a typical flat and low-lying paddy-cultivated area. The main problem in this basin is the appropriate operation of drainage structures during and after flood events in order to minimize the inundation damages for crop yield and to fulfill the irrigation requirement in the irrigation period. This paper presents a mathematical model of a drainage system in Chiyoda basin for calculating the flood inundation and optimizing the operation of gates in a main drainage canal. First, the algorithm of gate operation was simulated and the drainage model was then evaluated by comparing the simulated water levels with those observed during an actual rainfall event. The results show that the observed and simulated water levels are in good agreement, indicating that the proposed model is applicable for drainage and inundation analyses in flat, low-lying paddy-cultivated areas. Second, the optimization of gate operation was investigated by trial and error method using a stochastic rainfall time series with a return period of 30 years and the tidal conditions of spring and neap tides in the Chikugo River. Comparing a total inundation time, a total inundation area and maximum inundation depth in the paddy tanks at the upstream and the downstream ends, it was concluded that the present operation based on the gate operators’ experience was almost the optimal one, and the sooner start of opening operation and the later start of closing operation within the operationality of check gates were recommended to minimize the inundation damage. The present operation could be able to minimize the total inundation time, the total inundation area and the maximum inundation depths in paddy tanks and to meet fully both the drainage and the irrigation requirements.     

对一起违法经营大麦种子案件处罚的探讨

20 0 4年 8月 31日 ,接群众举报 ,反映射阳县某种业经营部经营的大麦种涉嫌假冒。射阳县农业行政执法大队随即派员前往调查。经查 ,该经营部经营的“新啤 2号”大麦种 ,包装上标注的生产单位为 :盐城市某农场 ,经销商为 :盐城某种业有限公司 ,品种说明中介绍为 ,品种来源为 :江苏某农场开发有限公司和盐城市某种业有限公司对俗名“趴地虎”大麦进行穗选改良、提纯复壮后选育而成。当事人于 2 0 0 4年 8月 30日从盐城市某种业有限公司购进该批种子 9375kg ,至检查时尚未销售。执法人员进行了询问笔录 ,随机抽取了样品 ,并当场扣押了该批种子…