苦荞二氢黄酮醇4-还原酶的原核表达与多克隆抗体制备

苦荞二氢黄酮醇4-还原酶(DFR)是花青素合成途径的关键酶。该研究以苦荞种子灌浆期cDNA为模板,采用RT-PCR方法克隆苦荞DFR编码基因,并将其连接到表达载体pET47b上,转化获得苦荞DFR编码基因的大肠杆菌BL21(DE3)工程菌,通过IPTG诱导表达,用SDS-PAGE分析表达产物,用亲和层析方法纯化蛋白,制备苦荞DFR多克隆抗体。RT-PCR技术获得了苦荞DFR编码基因的开放阅读框,重组表达载体经PCR和测序鉴定,表明表达载体构建成功,SDS-PAGE分析表达产物分别以可溶和不可溶的形式高效表达,亲和层析纯化得到融合蛋白,Western blotting显示,制备的多克隆抗体能特异识别其对应的抗原,天然的苦荞DFR蛋白在苦荞种子灌浆期中大量表达。苦荞DFR编码基因的原核表达与多克隆抗体的制备,为进一步开展DFR编码基因功能的研究奠定了基础。     

荞麦属植物淀粉酶和甲酸脱氢酶同功酶研究(英文)

以聚丙烯酰胺凝胶电泳方法研究了荞麦属植物8个种42个收集系干种子和发芽种子的淀粉酶和甲酸脱氢酶同功酶。结果表明,荞麦淀粉酶在于种子中缺乏活性,但是在发芽种子中活性很强。在供试材料的发芽种子中共发现23个淀粉酶谱带,其中甜荞和苦荞分别有10条和8条。不同荞麦种间淀粉酶谱带差异很大,但是同种内不同收集系间差异较小。谱带聚类分析表明大野荞和毛野荞分别与甜荞和苦荞较近缘,支持它们分别为甜荞和苦荞祖先种的假说。在干种子和发芽种子中,发现所有荞麦种类均只有1条位置一致的甲酸脱氢酶谱带,暗示该酶在进化中具有高度稳定性。     

自交可育甜荞、金苦荞、米苦荞不同品系总黄酮、粗蛋白及其蛋白组分含量研究北大核心CSCD

为探究荞麦新品种的营养保健价值,该文对自交可育甜荞、金苦荞、米苦荞共56个不同品系荞麦种子的粗蛋白、总黄酮、蛋白组分含量及其果实性状的变异进行了研究。结果表明:(1)甜荞、金苦荞、米苦荞种子中粗蛋白含量平均值分别为13.19%、15.44%、11.75%,总黄酮含量平均值分别为0.14%、2.50%、2.09%,清蛋白含量的平均值分别为5.22%、6.13%、4.56%,球蛋白含量的平均值分别为1.29%、1.15%、0.91%,醇溶蛋白含量的平均值分别为0.42%、0.58%、0.55%,谷蛋白含量的平均值分别为2.66%、3.36%、2.80%,三种荞麦的蛋白组分均符合清蛋白>谷蛋白>球蛋白>醇溶蛋白。(2)果实性状中,甜荞果实千粒重、果实面积、果实直径的变异系数最大,米苦荞果实周长、果实长宽比、果实长、果实宽和50 mL容重的变异系数最大。(3)相关分析表明甜荞种子粗蛋白含量与果实长宽比、果实长,金苦荞种子粗蛋白含量与果实周长、果实长,米苦荞种子粗蛋白含量与果实宽、总黄酮含量与果实面积、果实宽、果实直径、50 mL容重的相关性均达到了显著或极显著水平。(4)该研究筛选出甜荞(1808-166贵甜2号优系)、金苦荞(多苦74、多苦78)、米苦荞(1906-136黑米荞麦、43-2)等高蛋白含量或高黄酮含量的荞麦品系。该研究结果对荞麦优良品种的选育和荞麦新产品的开发具有一定指导意义。     

苦荞TBW16重组表达及靶向结合蛋白的初步分析

苦荞16kD过敏蛋白(Tartary buckwheat 16kD allergen,TBW16)是定位于种子胚中的过敏蛋白,其生物学功能未知。该研究以苦荞种子灌浆期cDNA文库中获得苦荞过敏原TBW16基因的序列为基础,构建TBW16成熟蛋白的原核表达载体pET47b-TBW16,实现了其在大肠杆菌BL21Star(DE3)中的高效表达。结果表明:该过敏原以包涵体的形式表达,经包涵体复性及金属离子螯合层析纯化了目标蛋白;以1,4丁二醇二缩水甘油醚环氧基介导的蛋白偶联技术固定化TBW16于Sepharose CL 6B上,采用亲和层析分离与TBW16靶向结合的蛋白,MALDI-TOF质谱鉴定显示,苦荞TBW16过敏原靶向结合蛋白与细菌膜孔蛋白高度同源,该研究结果为分析苦荞TBW16生物学功能奠定了基础。     

苦荞全基因组11S种子储藏蛋白基因的鉴定及表达分析

以苦荞（Fagopyrum tataricum（L.）Gaertn）全基因组数据为平台,采用生物信息学方法,挖掘出9个11S种子储藏蛋白基因,并对其定位、蛋白结构、系统发育及表达模式进行了分析。结果表明,苦荞9个11S种子储藏蛋白基因编码的蛋白长度为189~914 aa,等电点位于5.18~9.82之间,分子量为21.27~103.33 kD;定位分析结果显示,这些成员位于苦荞基因组的6条连锁群上（Megascaffold2/5以及scaffold77/344/395/861）;序列比对分析发现,除了1个11S种子储藏蛋白sample1_00009513-RA具有1个cupin保守结构域外,其余8个都含有2个cupin结构域,并且在cupin保守结构域中,苦荞和拟南芥（Arabidopsis thaliana（L.）Heynh）共有14个保守的氨基酸残基;蛋白结构预测表明,苦荞11S种子储藏蛋白的结构具有2种类型;苦荞与其它6个物种[拟南芥、花生（Arachis hypogaea Linn.）、大豆（Glycine max（Linn.）Merr.）、杏仁（Armeniaca vulgaris Lam.）、胡桃（Juglans regia L.）和芝麻（Sesamum indicum Linn.）]11S种子储藏蛋白以及苦荞过敏蛋白（TBb和TBt）系统发育分析结果表明,这些蛋白可以分为3类,共具有4对旁系同源蛋白和3对直系同源蛋白;与已报道的苦荞过敏性储藏蛋白以及其它5个物种（花生、大豆、杏仁、胡桃和芝麻）的11S过敏蛋白比较发现,5个11S种子储藏蛋白（sample1_00013128-RA、sample1_00013130-RA、sample1_00021677-RA、sample1_00021668-RA和sample1_00021674-RA）与苦荞2个过敏蛋白的同源性较高,同时它们与胡桃11S过敏蛋白的同源性最高,但尚需进一步实验来确定这5个成员是否为食物过敏原;RNA-Seq转录组数据显示,4个基因（sample1_00018411-RA、sample1_00026786-RA、sample1_00021674-RA、sample1_00022718-RA）在2种荞麦属植物的灌浆期种子中表达水平较高,且在‘大苦1号’中的表达水平要高于‘大甜1号’。     

不同磁处理对芍药种子萌发的影响

目的:研究磁场对芍药种子萌发的影响.方法:采用均匀电磁场分不同的处理强度和处理时间分别磁处理野生芍药种子和栽培芍药种子,种子磁处理后在培养皿中催芽,2个月后统计种子胚根萌发率和胚芽萌发率.结果:3000Gs的磁场处理1.5h能提高野生芍药种子胚芽萌发率和栽培芍药种子胚根萌发率,对于栽培芍药种子4000 Gs的磁场处理2h能够促进胚根和胚芽萌发.结论:一定强度的磁场能打破芍药种子休眠,促进种子的萌发.     

苦荞黄酮-3-羟化酶截短体的原核表达与多克隆抗体制备

为了制备苦荞黄酮-3-羟化酶的多克隆抗体,该研究以苦荞种子灌浆期cDNA文库中获得的苦荞黄酮-3-羟化酶基因截短体(truncated Flavanone-3-hydroxylase,TrF3 H)序列为基础,采用PCR扩增F3 H的截短序列编码区(TrF3 H),构建了原核表达载体pET47b-TrF3 H,并转化入大肠杆菌Rosetta(DE3)plysS中进行诱导表达,将经钴离子螯合层析柱纯化后的目的蛋白切胶回收后制备了高效价的多克隆抗体。结果表明:pET47b-TrF3 H在大肠杆菌Rosetta(DE3)plysS中以包涵体的形式高效表达。蛋白质印迹显示,制备的多克隆抗体能特异识别其对应的抗原,天然的黄酮-3-羟化酶蛋白在苦荞的未成熟种子中大量表达。原核表达体系的建立和多克隆抗体的制备为进一步探讨F3H在苦荞中功能奠定了基础。     

花生种子的磁处理对其萌发及幼苗生长的影响

本试验着重研究了用交、直流磁场对花生种子进行处理后,在种子萌发及幼苗生长过程中产生的影响。结果表明,一定强度的交流或直流磁场对种子的萌发及幼苗生长有一定的促进作用。处理过的种子存放一年后,磁场对种子萌发的作用依然存在。     

花生种子的磁处理对其萌发及幼苗生长的影响

本试验着重研究了用交、直流磁场对花生种子进行处理后,在种子萌发及幼苗生长过程中产生的影响。结果表明,一定强度的交流或直流磁场对种子的萌发及幼苗生长有一定的促进作用。处理过的种子存放一年后,磁场对种子萌发的作用依然存在。     

苦荞FtTCPlike1和FtTCPlike2的克隆及表达分析

植物TCP转录因子家族是一类调控植物生长、发育的重要转录因子,主要分为class Ⅰ和class Ⅱ两种类型。在本研究中,我们克隆了苦荞(Fagopyrum tataricum)两个TCP转录因子基因并分别命名为FtTCPlike1和FtTCPlike2。蛋白聚类分析表明FtTCPlike1属于class Ⅱ而FtTCPlike2属于class Ⅰ,并且均含有TCP转录因子典型的保守结构域。亚细胞定位实验也表明FtTCPlike1和FtTCPlike2与报道的其他物种TCP转录因子类似,都定位在细胞核中。本文同时收集了苦荞芽期的子叶和下胚轴、第1~3片长出的真叶以及5个时期由幼嫩到成熟的种子和花共11种样本进行了实时荧光定量PCR,发现FtTCPlike1和FtTCPlike2在苦荞的各个不同时期和不同组织中均有不同的表达,并且在种子的某个特定时期(FtTCPlike1和FtTCPlike2分别在种子发育第3、第4时期和第1、第3时期)有较高的表达量,表明这两个基因可能参与种子发育。从苦荞芽期来看,FtTCPlike1在子叶中表达量较高但在下胚轴表达较低,而FtTCPlike2则相反,这说明FtTCPlike1和FtTCPlike2在苦荞早期形态建成中的调控方式可能有很大区别。     

普通荞麦资源的耐铝性研究

利用小容器溶液培养法对耐铝性鉴定条件和52份普通荞麦栽培品种资源的耐铝性进行了研究。结果发现普通荞麦耐铝性鉴定的适宜条件为发芽种子于500μmol/LAlCl3溶液(pH4.5)处理3d,以发芽种子在这三天内的根伸长量衡量耐铝性程度。在该处理条件下,普通荞麦不同品种间的耐铝性有显著差异。其中,陕西大红花甜荞品种、日本大粒荞、织金红花甜荞的耐铝毒胁迫能力最强,值得在荞麦耐铝性育种和耐铝机制研究中利用。     

Adventitious sprouting enables the invasive annual herb Euphorbia geniculata to regenerate after severe injury

Euphorbia geniculata, an annual weed of arable land native to America and invasive in subtropical and tropical regions, is able to regenerate from seeds and is also able to produce adventitious buds on the hypocotyl. Whether sprouting from adventitious buds represents a mechanism for surviving severe injury, and whether this ability is crucial for species invasion is, however, not known. The significance of such sprouting was investigated with a field survey and a pot experiment. Among 897 plants in 25 field populations surveyed in Indonesia, only a few exhibited marks of injury and sprouting from adventitious buds. When seeds were collected from 12 of the populations and used in a pot experiment, however, the seedlings were able to survive severe injury (removal of all tissue above the hypocotyl) by sprouting from adventitious buds on the hypocotyl and were able to set seed, although they produced less vegetative and generative (flowers and fruits) biomass than control plants. Growth but not fitness of plants in the pot experiment was population specific but neither growth characteristic correlated with disturbance level assessed in the field. Although the pot experiment indicates that E. geniculata can cope with severe injury by adventitious sprouting from the hypocotyl, the survey data are inconsistent with the hypothesis that such adventitious sprouting is important for the plant??s invasion in tropical regions.     

Purification and characterization of a 24 kDa protein from tartary buckwheat seeds

A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.     

Determination of Anomeric Configuration of Furanoside Derivatives by Carbon-13 NMR

A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.     

当归种子的生态学特性

当归种子在自然条件下,存放一年后,即失去发芽力,这一问题成为推广“立秋直播”新技术的严重障碍。为查明导致发芽力迅速下降的主要原因,探讨保持发芽力的有效措施,特对当归种子在自然条件下,发芽力的年变化,以及种子含水量(4—20%)、贮种温度(-10一33℃)和发芽温度(5—35℃)等对种子发芽力的影响进行了比较系统的试验分析。结果表明,当归种子属于“耐干藏种子类型”,高温和高湿是降低发芽力的主要因素。若将种子含水量降低到5—8%,并配合以较低的温度(15℃以下),发芽力则可长达三年以上。在当归产区,若能改“常温堆藏法”为“低温、干燥、密闭贮存法”,便可达到妥善保存当归种子的目的。     

甜荞果皮开裂类型及其对籽粒早期萌发性状的影响

甜荞是我国传统的药食同源植物,籽粒是甜荞的重要贮藏器官,籽粒果皮的开裂类型及其开裂行为影响种子的萌发性状。该研究以5份甜荞为材料,进行果皮裂口的建模与分型,并通过纸床发芽法,检测种子的发芽率、胚根及下胚轴长度。结果表明:(1)甜荞籽粒果皮开裂主要包括纵向完全开裂型、种孔端部半开裂型、种脊中部半开裂型、种脐端部半开裂型和横裂型等5种类型;(2)开裂型籽粒比完整型萌发时间提早约12 h;(3)开裂型籽粒早期萌发率均高于相应的果皮完整型,但48 h后,完整型籽粒的萌发率超过果皮开裂型;(4)开裂型籽粒胚根及下胚轴的早期增长速度比完整型快,但果皮开裂型籽粒胚根与下胚轴增长比值显著小于果皮完整型;(5)不同开裂类型的萌发率从大到小依次为种孔端部半开裂型、纵向完全开裂型、种脐端部半开裂型、种脊中部半开裂型、横裂型,而种子霉烂率从大到小依次为种脐端部半开裂型、纵向完全开裂型、种脊中部半开裂型、种孔端部半开裂型、横裂型。果皮开裂虽然可以提高甜荞种子早期的萌发率,促进胚根及下胚轴早期的伸长速度,但会降低甜荞整体的萌发率以及胚根/下胚轴增长比值,种子霉烂率显著增加,不利于田间壮苗与全苗的形成。     

Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds.

A homogeneous preparation of metalloproteinase, purified 1000-fold, was obtained from buckwheat (Fagopyrum esculentum) seeds. The Mr of the enzyme, determined by SDS/PAGE, was 34,000 (it was 39,000 by gel chromatography). Its pH optimum was 8.0-8.2 with 13 S globulin, from buckwheat seeds, as substrate. Atomic-absorption spectroscopy revealed the presence of one Zn2+ ion per enzyme molecule. The enzyme was completely inhibited by EDTA (1 mM), zincone (1 mM) and 1, 10-phenanthroline (1 mM). The metalloproteinase performed limited proteolysis of the following seed storage proteins: 13 S globulin from buckwheat seeds and 11 S globulin from soybean (Glycine max) seeds. It hydrolysed three peptide bonds formed by the amino groups of Leu15, Tyr16 and Phe25 in the oxidized B-chain of insulin. In its main properties the enzyme is similar to metalloproteinases of animal and bacterial origin.     

Germination of soybean seeds exposed to the static/alternating magnetic field and algal extract

In the present study, the effect of the static and alternating magnetic field applied individually and in combination with an algal extract on the germination of soybean seeds (Glycine max (L.) Merrill) and chlorophyll content was examined. The exposure time of seeds to the static magnetic field was 3, 6, and 12 min, whereas to the alternating magnetic field was 1, 2.5, and 5 min. The static magnetic field was obtained by means of a permanent magnets system while the alternating magnetic field by means of magnetic coils. Algal extract was produced from a freshwater macroalga—Cladophora glomerata using ultrasound homogenizer. In the germination tests, 10% extract was applied to the paper substrate before sowing. This is the first study that compares the germination of soybean seeds exposed to the static and alternating magnetic field. The best effect on the germination and chlorophyll content in seedlings had synergistic action of the static magnetic field on seeds for 3 min applied together with the extract and alternating magnetic field used for 2.5 min. It is not possible to clearly state which magnetic field better stimulated the germination of seeds, but the chlorophyll content in seedlings was much higher for alternating magnetic field.