Specific detection of buckwheat residues in processed foods by polymerase chain reaction

A specific and qualitative detection method for buckwheat in foods using the polymerase chain reaction (PCR) was developed. Trace amounts of buckwheat in commercial food products were qualitatively detected by this method. It should be reliable for detecting buckwheat residues in processed foods and practical for monitoring the labeling system for allergenic food materials.     

苦荞和甜荞查尔酮合成酶基因的克隆及序列比较

以苦荞品种‘西农9920’和甜荞品种‘西农9976’为材料,根据其它植物查尔酮合成酶(chalcone synthase,CHS)基因DNA序列的保守区域设计的一对简并引物,进行PCR扩增,从2种荞麦基因组中克隆出了长度均为860 bp的CHS基因片段,对其进行回收、克隆,挑选阳性克隆测序;序列分析表明这2个片段含有CHS基因的N端和C端的结构域,分别为苦荞和甜荞的CHS基因片段,命名为FtCHS和FeCHS。对获得的2种荞麦CHS基因的DNA序列进行比较分析,发现两者间存在多达43处单碱基多态性,这些单碱基多态性可能是苦荞和甜荞种子中类黄酮含量差异的重要原因之一。苦荞和甜荞CHS与其它植物CHS的氨基酸序列的进化分析表明,其与同为蓼科的掌叶大黄和石竹科的满天星的同源性较近。     

虫媒传粉植物荞麦的生物学特性与研究进展

荞麦是禾本科之外的谷物类作物, 具有较高的营养和药用价值。栽培荞麦有甜荞(Fagopyrum esculentum)和苦荞(F. tartaricum), 这两种一年生草本分别为自交不亲和的二型花柱、自交亲和的同型花柱植物; 前者结实依赖昆虫传粉。根据国内外调查研究, 前人对蓼科荞麦属(Fagopyrum)记录了30个物种名, 已有形态学和遗传多样性的调查表明该属的物种多样性中心位于我国西南地区, 特别是长江上游的三江并流区域; 甜荞和苦荞的起源地和祖先物种也认为在该区域。本文在论述前人研究的基础上, 指出对荞麦属的分类修订、野生种质资源的分布、种间关系的调查、优良品种的选育亟待研究。孢粉学和考古学的证据显示在我国长江流域, 人们在4,500年前就开始种植荞麦。荞麦可能曾经是山区人民的主粮, 为孕育长江流域文明提供了食物资源。加强对荞麦基础生物学特性的研究, 运用现代基因组学的方法有望澄清栽培荞麦的起源并探究产量不高的原因, 挖掘和利用其经济和药用价值的性状, 为荞麦成为一类优良的粮食作物提供参考依据。     

Lymphocyte responses to food antigens in food sensitive patients with allergic tension-fatigue syndrome

Scores of radio allergosorbent test (BAST) for cow's milk or buckwheat flour and proliferative responses of peripheral blood mononuclear cells (PBMCs) to bovine serum albumin and-lactoglobulin or buckwheat flour were measured in cow's milk or buckwheat flour sensitive patients with allergic tension-fatigue syndrome. In all 3 cow's milk sensitive patients with allergic tension-fatigue syndrome, RAST scores for cow's milk were negative or slightly positive, but PBMCs well responded to bovine serum albumin and-lactoglobulin, but not to ovalbumin. In a buckwheat flour sensitive patient with allergic tension-fatigue syndrome, RAST scores for buckwheat flour were negative, but PBMCs well responded to buckwheat flour, but not to ovalbumin, bovine serum albumin and-lactoglobulin. Conversely, in cow's milk or buckwheat flour sensitive patients with immediate allergic symptoms, RAST scores for offending foods were positive although PBMCs did not respond to offending food antigens. These results suggest that proliferative responses of PBMCs to food antigens are very useful for detection of offending foods in allergic tension-fatigue syndrome.     

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.     

PCR method for detecting trace amounts of buckwheat (Fagopyrum spp.) in food

Buckwheat often causes severe allergic reactions, even when its ingestion level is extremely low. Therefore, buckwheat is listed in several countries as a common food allergen. In addition to common buckwheat and Tartarian buckwheat that are cultivated and consumed widely, wild buckwheat may be potentially allergenic. Food containing undeclared buckwheat poses a risk to patients with the buckwheat allergy. We describe in this report a PCR method to detect buckwheat DNA by using primers corresponding to the internal transcribed spacer region and the 5.8S rRNA gene. The method is buckwheat-specific and compatible with both cultivated and wild buckwheat of the Fagopyrum spp. Its sensitivity was sufficient to detect 1 ppm (w/w) of buckwheat DNA spiked in wheat DNA. This method should benefit food manufacturers, clinical doctors, and allergic patients by providing information on the presence of buckwheat contamination in food.     

日光紫外线B辐射对甜荞苯丙烷代谢的影响

对甜荞(Fagopyrum esculentum Moench)苯丙烷次生代谢受紫外线B辐射的响应进行了研究.结果表明:不仅卢丁、槲皮素等黄酮类化合物含量在紫外线B辐射下显著升高,而且叶片苯丙烷单环酚类化合物,如阿魏酸含量等也大幅度升高,两类化合物的应激提高对甜荞的UV-B胁迫提供了重要的保护作用.     

荞麦属种间13S球蛋白基因序列比较研究

荞麦13S球蛋白是荞麦种子中的一类主要贮藏蛋白。本研究选用荞麦属植物甜荞栽培种及其野生类型、苦荞栽培种及其野生类型、毛野荞、左贡野荞、细柄野荞和硬枝万年荞6个种共44份材料,进行PCR特异性扩增、测序得到荞麦13S球蛋白基因的保守片段序列。对序列进行差异分析,结果发现44份供试材料13S球蛋白基因片段的285个排列位点中不变位点为24个,多态性位点S为261个(含简约信息位点数198个和单型可变位点63个),序列总突变位点Eta为503个。野生甜荞种内13S球蛋白基因序列差异明显高于栽培甜荞,但野生苦荞种内13S球蛋白基因序列差异仅稍高于栽培苦荞。推测其一方面可能与繁殖方式有关,另一方面可能与荞麦驯化过程中通常只有少数野生型群体被驯化有关。系统聚类分析发现栽培甜荞与野甜荞亲缘关系近,与左贡野荞亲缘关系次之;栽培苦荞与野苦荞亲缘关系近,与毛野荞亲缘关系次之;细柄野荞和硬枝万年荞的13S蛋白基因片段序列差异较小,说明其亲缘关系较近。上述结果为荞麦属种间遗传多样性与进化关系研究提供了理论依据。     

Functional compounds in fermented buckwheat sprouts

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.     

一种苦荞主要过敏原基因cDNA的克隆及序列分析

为了获得苦荞中主要过敏原的cDNA和由此推导的蛋白质序列 ,分析其结构特点 ,以苦荞幼根根尖为材料 ,提取总RNA并反转录mRNA为cDNA第一链 .通过RT PCR、3′RACE、基因克隆及序列测定 ,获得一种苦荞主要过敏蛋白基因的cDNA片段 (GenBank登录号为AY0 4 4918) .该cDNA片段由 76 8bp组成 ,包括 3′端非编码区 180个bp ,开放阅读框 5 88bp .可编码一个由 195个氨基酸残基组成的功能蛋白及一个终止密码 .苦荞主要过敏原基因与甜荞 2 2kD过敏蛋白、豆球类蛋白的核苷酸序列分别有 95 %和 93%的同源性 .其推导的氨基酸序列与甜荞球蛋白、刀豆蛋白、甜橙柠檬素分别有 93%、83%和 5 7%的同源性 .该过敏蛋白 183～ 188位氨基酸残基KEEEKE在多数不同过敏原中均存在 ,推测可能为其中的抗原决定簇序列     

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’。     

Progress and prospects for interspecific hybridization in buckwheat and the genus Fagopyrum

Cultivated buckwheat, such as common (Fagopyrum esculentum Moench.) and tartary (Fagopyrum tataricum (L.) Gaertn.) buckwheat, is one of the most versatile crops for forage and food and has several benefits for human health. Interspecific hybridization between Fagopyrum species is of great importance to improvement of buckwheat. Hybridization would allow the transfer of agronomical beneficial characteristics from wild Fagopyrum species, including self-pollination and increased fertility, frost tolerance, and higher content of beneficial compounds. However, conventional breeding methods are only partially applicable because of the self-incompatibility and incompatibility barriers between different species. Present review summarizes the morphology of self-incompatibility, the genetic and cellular basis of incompatibility between different Fagopyrum species. In many interspecific crosses hybrid embryos are aborted after successful pollination due to post-zygotic incompatibility. The use of in vitro embryo rescue after interspecific hybridization has been successful in circumventing breeding barriers between Fagopyrum species. Methods applied successfully for the construction of interspecific hybrids are discussed in detail.     

Functional Compounds in Fermented Buckwheat Sprouts

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.     

荞麦种子萌发期多种抗氧化酶活性的研究

通过研究荞麦种子萌发期(0～7 d)超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(ASP)四种抗氧化酶的酶活性变化,以及对自由基等的清除效果,分析荞麦种子内在抗氧化酶系统在萌发期对细胞膜结构的修复及保护作用。实验结果表明:荞麦种子在萌发期产生的活性氧、自由基等对四种抗氧化酶均有激活效应,其中超氧化物歧化酶(SOD)活性最先上升,其他三种酶随其后被激活,四种酶的氧化反应存在一定关联性和协同作用。苦荞的抗氧化酶活性高于甜荞。     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na /H 反向转运蛋白基因AtNHX1转入荞麦中,在2·0mg/L6-BA、0·1mg/LIAA、1mg/LKT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株(转化频率为4·17%)。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na 及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K 的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

Effects of buckwheat flowers on leafroller (Lepidoptera: Tortricidae) parasitoids in a New Zealand vineyard

Abstract 1 The provision of floral resources in agricultural ecosystems can potentially enhance biological control of pests by providing nutrients to parasitoids. To test this, the effect of buckwheat Fagopyrum esculentum Moench flowers on leafroller parasitoids was investigated in a New Zealand vineyard. 2 Relative abundance of parasitoids was assessed with yellow sticky traps in buckwheat and control plots. Male Dolichogenidea tasmanica (Cameron) (Hymenoptera: Braconidae) were significantly more abundant in the presence of buckwheat. No significant result was found for female D. tasmanica or either sex of Glyptapanteles demeter (Wilkinson) (Hymenoptera: Braconidae), the other leafroller parasitoid caught. 3 The relative parasitism rate of leafrollers by D. tasmanica was assessed with a leafroller release and recover technique. No difference in parasitism was found between buckwheat and control plots. 4 The sex ratio (% males) of D. tasmanica emerging from recovered leafroller larvae was significantly lower in the presence of buckwheat than in control plots on one of the two release dates. Possible reasons for this increase in female production are discussed.     

Proteolysis of the main storage protein of buckwheat seeds at the early stage of germination

The changes in the main storage protein of seeds of buckwheat ( Fagopyrum esculentum Moench cv. Shatilovskaya 5), 13S globulin, were studied during seed germination. During the first three days of germination the 13S globulin is subjected to a limited proteolysis, which consists in the splitting of some of its subunits into large polypeptide fragments. Insignificant changes in the sedimentation coefficient of the 13S globulin during the first days of germination as well as immunochemical data, indicate that the limited proteolysis of the 13S globulin does not cause any major changes in its structure.
Dormant buckwheat seeds contain a proteolytic enzyme (a metalloproteinase), which can cause limited proteolysis of 13S globulin. The proteinase hydrolyzed some subunits of the 13S globulin to high molecular weight fragments. In the presence of sodium dodecylsulphate the electrophoretic pattern of 13S globulin, isolated from 3-day-old buckwheat seedlings, was almost identical to that of 13S globulin from dormant seeds hydrolyzed with metalloproteinase. It is suggested that the proteolysis of 13S globulin observed in vitro may also take place in vivo in the course of seed germination.     

Enhancing Biological Control of Leafrollers (Lepidoptera: Tortricidae) by Sowing Buckwheat (Fagopyrum esculentum) in an Orchard

Two successive trials were carried out in stone-fruit and apple orchards in the summers of 1994-95 and 1995-96 respectively to determine the effect of floral resources on leafroller parasitoid numbers. Orchards were divided into eight blocks, each containing one replicate of a control and a treatment. The treatment had buckwheat (Fagopyrum esculentum Moench) drilled (as 3 m 2 m plots) in the centre of each replicate, while the control areas were kept weed free with herbicides. Catches of parasitoids in yellow pan traps were compared between the control and treatment areas. In the first trial, on average 10 times more parasitoids were captured in the buckwheat plots than in the controls. However, few of the parasitoids were natural enemies of leafrollers; the catches were dominated by Anacharis sp., a parasitoid of the brown lacewing ( Micromus tasmaniae Walker), itself a beneficial insect. Leafrollers were very rare in this orchard, which explains the absence of their parasitoids in the traps. In the second season, with the experiment conducted in an orchard with a history of leafroller populations, higher numbers of Anacharis sp. were again caught in buckwheat than in control plots. However, significantly higher (34% compared with 20%) levels (P< 0.05) of parasitism (by Dolichogenidea tasmanica (Cameron)) of released leafroller larvae were observed in buckwheat plots than in controls, despite the small plot size. Also, higher numbers of D. tasmanica were captured in yellow sticky traps 2 m above the buckwheat than in the equivalent position in the control plots.     

外源水杨酸和茉莉酸对荞麦幼苗耐盐生理特性的效应

以盐敏感荞麦品种‘TQ-0808’幼苗为材料,采用100mmol·L^（-1）NaCl并添加不同浓度水杨酸（sA）和茉莉酸（JA）的处理方法,测定荞麦耐盐生理指标,确定外源SA和JA对荞麦耐盐生理特性的效应。结果表明,外源SA和JA均能明显降低盐胁迫下荞麦叶片质膜透性和丙二醛（MDA）含量,外源SA降低的幅度略高于JA,外源SA最适浓度为0．6mmol·L^（-1）外源SA和JA均能明显增加盐胁迫下荞麦叶片超氧化物歧化酶（SOD）和抗坏血酸过氧化物酶（APX）活性及叶片净光合速率,外源JA增加的幅度明显大于SA,外源JA最适浓度为40gmol·L^（-1）。外源sA和JA明显改善盐胁迫下荞麦的生理特性,对盐胁迫具有较好的缓解作用,说明通过适当浓度的外源SA和JA处理来提高荞麦耐盐性是可行的。     

Somatic Embryogenesis and Bud Formation on Cultured Fagopyrum esculentum Hypocotyls

An indirect somatic embryogenesis via the development of proembryogenic cell complexes (PECC) was observed in the in vitro cultured hypocotyl explants of 4–5-day-old buckwheat (Fagopyrum esculentum Moench.) seedlings. PECC development was shown to depend on culturing conditions, including 2,4-D concentration and the period of explant exposure to 2,4-D, sucrose concentration, and explant density. The culturing protocol was designed to ensure the development of buckwheat somatic embryos in two-month period. The cytogenetic analysis demonstrated that this protocol did not affect the chromosome numbers in the regenerated plants.