Determination of Neurotoxin β-ODAP and Non-protein Amino Acids in Lathyrus Sativus by High-Performance Liquid Chromatography with Precolumn Derivatization with 6-Amino quinolyl-N-hydroxysuccinimidyl Carbamate (AQC)

A new method was developed for the quantitative determination of the neurotoxic nonprotein amino acid, 3-N-oxalyl-L-2,3-diaminopropioni acid (β-ODAP), its nontoxic α-isomer and other non-protein amino acids in the plant samples of Lathyrus sativus after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) by reversed-phase high-performance liquid chromatography (HPLC). 2-Amino butyric acid (ABA) was used as an internal standard.The RP HPLC detection limit for both isomers is 1.8 ng with good response linearity. The results are compared with a colorimetric method.

HPLC Determination of Neurotoxin β-N-oxalyl-L-α, β-diaminopropionic acid and Its α -Isomer in Lathyrus sativus by Precolumn Derivatisation with 1-Fluoro-2,4-dinitrobenzcne

A rapid and simple method is presented for determining β-N-oxalyl-α. β- diaminopropionic acid (β -ODAP) and its much less toxic α -isomer (α -ODAP) in Lathyrus sativus. Seed and foliage extracts of Lathyrus sativus were treated with 1-fluoro-2,4-dinitrobenzene (FDNB) and a reversed-phase high-performance liquid chromatographic method for the separation of the derivatives in the pmol range is reported.

Effect of Dietary Administration of Lathyrus sativus Pulse on Intestinal Biochemical Parameters in Normal and Scorbutic Guinea Pigs

Objective In order to investigate that ascorbic acid deficiency is responcible for lathyrus toxicity, the effect of dietary feeding of lathyrus pulse in normal and scorbutic guinea pigs for 3 months, on intestinal biochemical parameters was undertaken. Methods The intestinal brush border membrane (BBM) marker and xenobiotic metabolising enzymes (XME) were assayed. Results Exposure to 80% lathyrus alone and in scorbutic conditions showed significant inhibition of alkaline phosphatase (28%-30%), sucrase (19%) and γ-glutamyl transpeptidase (GGT) (15%-27%) enzymes, while Ca2+-Mg2+-ATPase was significantly inhibited (38%) in scorbutic plus lathyrus treated group. The phase I XME (AHH) remained unchanged while the phase II enzyme glutathione-S-tranferase (GST) was significantly decreased (20%-22%) in lathyrus and scorbutic plus lathyrus treated groups. Quinone reductase (QR) activity was found to be significantly decreased in lathyrus exposed group (20%). The intestinal biomarker contents including hexose (25%-34%) and phospholipids (20%-40%) were significantly reduced in lathyrus and scorbutic plus lathyrus exposed animals, while sialic acid showed a significant decrease (28%) in scorbutic plus lathyrus treated group. However, cholesterol levels were signifcantly enhanced (15%-28%) in lathyrus and scorbutic plus lathyrus treated animals. Conclusion The results indicate that oral feeding of lathyrus pulse to guinea pigs can alter BBM parameters as well as XME, which may result in the intestinal toxicity. Further, ascorbic acid deficiency could be one of the pre-disposing factors of lathyrus toxicity.

L-Homoarginine Accumulation in Grass Pea (<i>Lathyrus sativus</i>L.) Dry Seeds. A Preliminary Survey

Grass pea (Lathyrus sativus L.) has great agronomic potential as grain and forage legume, and presently is con-sidered as a model crop for sustainable agriculture. However, the development into an important food legume has been hin- dered by the presence of the neurotoxic amino acid β-N-Oxalyl-α, β-diaminopropionic acid (β-ODAP). Recent studies reported that homoarginine (Har) can counteract this toxic action. This research was undertaken to shed light on the variation of Har amount within grass pea. The influence of the environment and of the year-to-year variation of cli- matic conditions was also investigated. Seven Italian grass pea ecotypes were evaluated for two subsequent growing seasons in two locations of southern Italy. In contrast with previous studies collected data evidenced a significant variation of Har amount among the tested ecotypes. Moreover, a significant positive correlation between Har and ODAP level was observed. The effect of year-to-year variation of temperature and rainfall quantity is also discussed.

Effects of Supplementing Finger Millet (<i>Eleucine coracana</i>) Straw with Grass Pea (<i>Lathyrus sativus</i>L.) Hulls and Concentrate Mixture on Feed Intake, Digestibility and Body Weight Change in Washera Sheep

The productivity of sheep in Ethiopia is very low mainly due to a serious shortage of feedstuff. As a result, it is necessary to look for cheap locally available feedstuffs to sustain sheep production in the country. Non-conventional feeds like grass pea (Lathyrus sativus L.) hulls (GPH) could partly fill the gap in the feed supply, decrease competition for food between humans and animals and reduce feed cost. Therefore, this experiment was designed to evaluate effects of different proportions of grass pea hulls and concentrate mixture (CM) supplementation on feed intake, digestibility, and growth of Washera sheep fed finger millet straw (FMS) as a basal diet. Diets consisted FMS alone fed ad libitum (T1), 100% CM (T2), 30% GPH mixed with 70% CM (T3), 30% CM mixed with 70% GPH (T4), and 100% GPH (T5). For all treatments, FMS was fed ad libitum. Experimental sheep were arranged in a randomized complete block design and treatment feeds were assigned to the animals within a block. The results showed that FMS contained 5.5% CP and 68.9% NDF. The supplements contained 21%, 20%, 16% and 14% CP;and 36%, 43%, 52% and 59% NDF for T2, T3, T4 and T5, respectively. Supplementation improved (p < 0.0001) DM and nutrient digestibility. Average daily gain (ADG) was 60, 84, 51, and 28 g/day for T2, T3, T4 and T5, respectively, which showed that T3 recorded the highest ADG and T5 resulted in the lowest ADG (p < 0.0001). While, animals in T1 lost a bodyweight of 5.6 g/day. As the proportion of GPH supplementation was greater than 30%, there was reduction (p < 0.05) in DM intake, DM and CP digestibility and consequently depressed ADG in sheep. Partial budget analysis results showed that net return and change in net return were in the order of T3 > T2 > T4 > T5. Therefore, from biological point of view as well as based on partial budget analysis, T3 was found to be recommendable for smallholder sheep producers.

山黧豆AAE超家族基因鉴定及LsAAE3的酶活检测

【目的】三七素(β-N-草酰-L-α,β-二氨基丙酸,β-ODAP)是山黧豆、三七等物种中的神经活性物质,其生物合成与草酰辅酶A密切相关。研究基于全基因组鉴定山黧豆草酰辅酶A合成酶的编码基因,为β-ODAP生物合成的调控研究奠定基础。【方法】用AMP结合结构域隐马尔科夫模型在山黧豆基因组中鉴定酰基激活酶超家族(acyl-activating enzyme superfamily,AAE)基因,在此基础上,克隆山黧豆草酰辅酶A合成酶基因LsAAE3,用多序列比对、原核表达及酶活检测等方法分析LsAAE3基因功能。【结果】山黧豆中至少存在22条AAE家族基因;系统发育分析表明:LsAAE3与拟南芥AtAAE3等聚为一支,隶属于酰基辅酶A合成酶家族。基因克隆及序列分析表明:LsAAE3基因cDNA全长为1566 bp;LsAAE3和AtAAE3的保守结构域类似,均含有AMP结合位点、CoA结合位点及AAE保守结构域。SDS-PAGE检测表明所获融合蛋白条带单一,大小在56 kD左右;用His标签抗体进行Western blot分析发现,诱导后和纯化的融合蛋白均能检测到特征条带,说明所获融合蛋白为LsAAE3。体外酶活检测表明:LsAAE3具有草酰辅酶A合成酶活性,其活性发挥依赖于ATP、Mg^(2+)。【结论】研究在全基因组水平鉴定山黧豆AAE超家族基因,并验证了LsAAE3具有草酰辅酶A合成酶活性,为进一步分析山黧豆LsAAE3在β-ODAP生物合成过程中的功能奠定基础。

山黧豆还田与氮肥减施对稻田土壤活性有机碳组分及酶活性的影响

为探明绿肥山黧豆(Lathyrus sativus)还田配施氮肥在改良土壤方面的效应,明确合适的绿肥配施氮肥比例,设置2×4双因素试验,研究绿肥的不同翻压量[15000(M_(1))、22500(M_(2))、30000(M_(3))、37500(M_(4))kg·hm^(−2)]和氮肥的不同施氮量[常规施氮量的60%(N_(1))和80%(N_(2))配比]对土壤活性有机碳库各组分、碳库管理指数和酶活性等指标的影响。结果表明:与常规施肥(CF)处理相比,翻压一定量的绿肥并配施减量氮肥能有效提升稻田总有机碳、活性有机碳、可溶性有机碳、微生物生物量碳含量及碳库管理指数,提升效果随配施比例的不同存在差异,其中M_(4)N_(1)、M_(4)N_(2)处理提升效果最佳。在相同施氮水平下,有机碳各组分含量、碳库管理指数及总体酶活性均呈现出随翻压量增加而增加的趋势。与CF处理相比,翻压绿肥并配施氮肥对稻田土壤过氧化氢酶无显著影响(P>0.05),对纤维素酶、蔗糖酶和β-葡萄糖甘酶活性均具有显著影响(P<0.05)。总体酶活性均表现为M_(4)N_(2)>M_(4)N_(1)>M_(3)N_(1)>M_(3)N_(2)>M_(2)N_(1)>M_(2)N_(2)>M_(1)N_(1)>M_(1)N_(2)>CF>CK处理。各有机碳组分之间具有显著(P<0.05)或极显著(P<0.01)的相关性,β-葡萄糖甘酶、纤维素酶与土壤活性有机碳各组分均呈正相关关系(P<0.05)。对土壤活性有机碳组分含量及土壤酶活性影响因素的灰色关联度综合分析结果表明,60%氮肥+37500 kg·hm^(−2)绿肥模式的综合评价效果最好。

家山黧豆及其毒素ODAP的研究

家山黧豆作为人类的食物及动物的饲料,最早可追溯到新石器时代.长期以来,因其营养丰富,具有耐旱、耐寒等优良生产性状,广受世界各地的青睐.特别是在大旱年份,在其它粮食作物绝收的情况下,仍有较好的收成,因此,被视为干旱及半干旱地区首选的优良作物.但由于其含有毒素β-ODAP,使山黧豆的种植受到限制.近年来,由于人类对肉质品的量与质的多元化的需求,家山黧豆这一潜在的、丰产的、高营养的豆科作物,已引起各国学者的广泛关注.从家山黧豆在植物学、遗传学、分子生物学、生态学、营养学、山黧豆中毒、毒理学、毒素ODAP、ODAP的分析方法、ODAP的生物合成途径、养殖业等几个方面的研究进展作了简要综述.

水分胁迫下山黧豆中ABA及ODAP的积累研究

用PEG、PEG +ABA、ABA分别处理 15d龄的山黧豆幼苗 ,取其叶片为实验材料 ,测定内源ABA、O DAP、MDA和H2 O2 含量以及几种抗氧化酶活性 ,结果表明 ,与对照相比处理材料叶片中ABA和ODAP含量显著增加 ;外源ABA的加入降低了PEG胁迫引起的MDA和H2 O2 含量的增加 ,延缓了PEG胁迫引起的CAT活性的衰减 ,提高了GR活性 .用外源ABA长时间处理山黧豆 ,发现叶片中ABA含量显著增加 ,随后出现ODAP的积累 ;ABA处理初期 (0～ 3d)对叶片中活性氧代谢影响不大 ,随着ABA处理时间的延长 (7～ 15d) ,可引起叶片中SOD、POD、CAT、GR活性的降低 ,MDA、H2 O2 含量的增加 ,表明ABA确实可促进ODAP的积累 .

山黧豆叶片蛋白质双向电泳技术的建立

以山黧豆叶片为材料,比较分析了蛋白质的不同提取方法,在此基础上着重于样品制备.对IPG胶条的选择,第一向等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳的电泳程序及参数、染色方法等相关技术进行了比较和条件优化.结果显示:采用TCA-丙酮沉淀法提取蛋白质,裂解液中加入Tris-base作为蛋白酶抑制剂,等电聚焦电泳时延长低电压的电泳时间(30 V1、2 h,500 V1、h,1 000 V2、h)以促进盐离子泳出的方法对山黧豆叶片蛋白质进行双向电泳,并用考马斯亮蓝和银染复合染色法进行凝胶染色,能够获得蛋白点清晰的双向电泳图谱,说明用优化后的方法建立起的山黧豆叶片蛋白质双向电泳技术,蛋白质样品制备质量好,电泳分辨率高,完全适合于进一步的蛋白质组学研究.

山黧豆毒素ODAP的生物合成及与抗逆性关系研究进展

有关山黧豆毒素ODAP的生物合成途径的前体物和合成程序已经证实,但其关键合成酶尚未分离与鉴定,因而无法克隆相应基因和利用反义RNA技术以控制其生物合成。研究表明,利用相应的抑制剂控制ODAP生物合成前体物可降低ODAP的积累量。山黧豆中ODAP含量与其抗逆性之间密切相关,这与其能有效地清除山黧豆中.OH自由基有关,外源ODAP处理获同样效果。此外,因ODAP既是含氮化合物,又是游离氨基酸,极易溶于水,可在逆境胁迫下与脯氨酸和多胺一样大量而迅速地积累,推测它也可能作为细胞渗透调节物质和防脱水剂,并在氮代谢和能量代谢方面起重要作用。对今后该领域的重点研究方向也进行了探讨。

山黧豆根系对H_2O_2诱导氧化胁迫的生理应答

以水培7d苗龄的山黧豆幼苗为材料,向水培溶液中施加不同浓度H2O2处理山黧豆幼苗24h,分析山黧豆根系受氧化胁迫的程度与抗氧化系统的应答特征,以揭示山黧豆对氧化胁迫的耐受机制。结果显示:(1)随外源H2O2处理浓度的不断增加,山黧豆幼苗侧根的数目无显著变化,而其根的鲜重则显著降低。(2)同时,根系组织的内源H2O2染色范围和程度显著增高,但根尖区域始终保持较低水平的H2O2;相反,O-·2染色范围和程度明显减少,根尖区域却始终保持较高水平的O-·2。(3)同期根系抗坏血酸(ASC)含量及过氧化氢酶(CAT)、过氧化物酶(POD)与抗坏血酸过氧化物酶(APX)的活性均表现出了先升高后降低的趋势,而超氧化物歧化酶(SOD)一直表现为持续上升的趋势。研究表明,在外源H2O2胁迫条件下,山黧豆根系O-·2的积累可能与其生长和活力呈正相关,而根系H2O2的积累则与其受氧化胁迫程度呈正相关;低浓度的H2O2处理可以提高山黧豆抗氧化系统对体内活性氧的清除能力。

干旱条件下两种山黧豆气孔特性及种子ODAP,粗蛋白和淀粉积累的研究

对干旱条件下永寿山黧豆(YS)和定西山黧豆(DX)的气孔特性、光合特性和种子物质的积累进行了研究.结果表明,干旱使两种山黧豆气孔密度(SD)显著升高,同时显著减小气孔开度(SA),SA在30%以上的气孔所占的比例显著减小,而气孔阻力(r)却显著增加;;干旱条件下,两种山黧豆的光合速率(Pn)和蒸腾速率(E)均较正常水分条件下的小,而叶片水平水分利用效率(WUE)却显著上升;;干旱使两种山黧豆种子千粒重降低,而种子ODAP,粗蛋白和淀粉含量均有一定程度增加.相比之下,干旱更严重地影响了YS的SD,SA,r,Pn,E以及种子ODAP含量,而更显著地提高DX的WUE和种子粗蛋白、淀粉含量.分析认为,干旱通过影响气孔密度和开度而影响光合速率、蒸腾速率和水分利用效率;;干旱也增加了气孔阻力,而气孔阻力的增加大幅度地减小了蒸腾速率;;光合速率、蒸腾速率和水分利用效率的变化最终影响种子千粒重以及种子ODAP,粗蛋白和淀粉等物质的积累;;DX可能较耐旱,水分亏缺对YS的影响较DX大.

水分胁迫对山黧豆萌发中蛋白质和游离氨基酸的影响

对山黧豆 (L athyrus sativus)进行不同浓度 PEG胁迫处理 ,研究水分胁迫对山黧豆种子萌发过程中蛋白质和游离氨基酸的影响。结果表明 ,水分胁迫能导致山黧豆根芽和子叶可溶性蛋白质含量增加 ,非可溶性蛋白质分解受到抑制 ;子叶游离氨基酸含量降低 ,而根芽游离氨基酸含量增高 ,尤其是丙氨酸、异亮氨酸、脯氨酸和精氨酸。随着种子萌发时间的延长 ,根芽 β- ODAP含量逐渐增加 ,而子叶 β- ODAP却降低 ;在水分胁迫下子叶和根芽

山黧豆抗逆性研究进展

山黧豆是一种栽培历史悠久、种植广泛的豆科作物,具有很高的蛋白质含量和极强的耐旱能力,因而成为一些干旱、贫困和人口众多地区的主要饲料和粮食作物,且被考虑作为可持续发展农业的模式作物。随着人类对粮食、蛋白质和新型食物资源需求的日益增长,山黧豆的研究引起了世界各国学者的重视。该文对国内外近年来有关山黧豆在干旱、盐、重金属及生物胁迫下抗逆性方面的研究进展作了概述,同时简要介绍了山黧豆与干旱相适应的形态特征,并对亟需重点研究的几个方向作了展望。

黑籽南瓜砧木对黄瓜生长结实、抗病性及营养元素含量的影响

为了探索黑籽南瓜(CucurbitaficifoliaBouch啨)对黄瓜(CucumissativusLinn.)生长的影响,以前者为砧木,'津绿21 1'和'津研4号'2个黄瓜品种为接穗,以自根植株为对照,对嫁接植株的形态指标、抗病性、单株产量及叶片营养元素含量进行研究。结果表明,嫁接植株在不同时期的形态指标均高于自根植株;嫁接黄瓜的单株产量显著高于自根黄瓜;枯萎病、病毒病、霜霉病和灰霉病4种病害在嫁接植株上的病情指数和发病率均低于自根植株;2个品种嫁接植株叶片的N、P、K、Ca和Mg含量均显著或极显著高于自根植株。结果表明,黑籽南瓜作为优良砧木可显著促进黄瓜的生长结实,提高其抗病性及对营养元素的吸收能力。

山黧豆蛋白质的提取及营养价值研究

优化碱溶酸沉提取山黧豆蛋白的方法,得到山黧豆粗蛋白,并分析其氨基酸组成,同时对山黧豆蛋白质进行了综合的营养价值评价。结果显示,山黧豆蛋白质的较佳提取条件为:提取温度40℃,pH值10,提取时间1 h,料液比1∶20 g/mL。此条件下,山黧豆蛋白质的提取率最高,蛋白质质量分数高达26.1%。山黧豆蛋白的等电点为pH值5;山黧豆蛋白质中含有18种蛋白质氨基酸,其中谷氨酸质量分数最高为17.4%,其次是天冬氨酸10.0%。其必需氨基酸占总氨基酸的比值EAA/TAA为42%(>40%)、必需氨基酸与非必需氨基酸的比值EAA/NEAA为72.41%(>60%),即均大于FAO/WHO提出的参考蛋白量。山黧豆蛋白中的氨基酸评分、化学评分、氨基酸比值系数以及必需氨基酸比率,除蛋氨酸+胱氨酸、苏氨酸、色氨酸、缬氨酸外,其他均高于WHO/FAO标准;蛋氨酸+胱氨酸、色氨酸分别为第一和第二限制性氨基酸。山黧豆蛋白质的必需氨基酸指数EAAI值为1.16,根据蛋白质的评价标准:EAAI值大于0.9为优质蛋白源,说明山黧豆蛋白质属于优质蛋白。

Pb胁迫对香豌豆幼苗部分生长和生理生化指标的影响

采用盆栽方法研究了土壤中0(CK)、150、300、450和600 mg.kg-1铅(Pb)对香豌豆(Lathyrus odoratus Linn.)幼苗部分生长和生理生化指标的影响。结果表明:随Pb质量浓度的提高,香豌豆株高、根长以及地上和地下部分的干质量均呈逐渐下降趋势,耐性指数呈低质量浓度条件下升高、高质量浓度条件下降低的趋势;其中,在450和600 mg.kg-1Pb胁迫条件下,香豌豆幼苗根长以及地上部分和地下部分的干质量均显著低于对照(P<0.05),说明植株的生长受到明显抑制。随Pb质量浓度的提高,幼苗地上和地下部分的相对电导率和SOD活性呈逐渐增加的趋势,其中,在600 mg.kg-1Pb胁迫条件下相对电导率和SOD活性均最高。各处理组地上和地下部分的脯氨酸(Pro)含量呈低质量浓度条件下降低、高质量浓度条件下增加的趋势,而POD活性、谷胱甘肽(GSH)和抗坏血酸(AsA)含量则呈现低质量浓度条件下增加、高质量浓度条件下降低的趋势。其中,在300 mg.kg-1Pb胁迫条件下Pro含量最低且显著低于对照;在450 mg.kg-1Pb胁迫条件下POD活性最高;在150 mg.kg-1Pb胁迫条下GSH含量最高,且各处理组间POD活性和GSH含量有显著差异;但各处理组AsA含量均与对照无显著差异。研究结果表明:香豌豆对Pb胁迫具有一定的耐性,可用于轻度Pb污染环境的修复。

水分胁迫下山黧豆多胺代谢与 β-N-草酰-L-α ,β-二氨基丙酸积累相关性的研究

为了研究水分胁迫下山黧豆 (LathyrussativusL .)叶片中多胺代谢与 β_N_草酰_L_α,β_二氨基丙酸 (ODAP)积累的相关关系 ,利用聚乙二醇 (PEG)对山黧豆幼苗进行水分胁迫处理 ,同时加入腐胺 (Put) ,α_二氟甲基精氨酸(DFMA)和Put+DFMA。实验结果表明 ,随PEG处理时间的延长 ,山黧豆幼苗叶片中Put、亚精胺 (Spd)和精胺 (Spm)含量逐渐增加 ,特别是Spm含量增加显著 ,同时ODAP逐渐积累 ;在PEG处理的同时 ,加入Put使得Put、Spd含量显著增加 ,但对Spm影响不大 ,同样对ODAP含量影响也较小 ;加入DFMA可显著抑制Put、Spd、Spm的积累 ,同时也抑制了ODAP的积累 ;加入Put+DFMA ,Put可以部分地减缓DFMA对两种内源多胺 (Put和Spd)合成的抑制作用 ,但对Spm所受DFMA的抑制作用影响不大 ,这时ODAP的积累也受到抑制。由此可见 ,水分胁迫对山黧豆幼苗叶片中多胺特别是Spm含量的增加与ODAP的积累密切相关。

山黧豆中还原型谷胱甘肽的荧光检测

对应用荧光分光光度法测定山黧豆中还原型谷胱甘肽含量的方法进行了研究。以邻苯二甲醛与还原型谷胱甘肽反应形成荧光体系,研究了室温下溶液酸碱度、邻苯二甲醛浓度、反应时间等对测定结果的影响,从而确定了荧光法测定山黧豆中还原型谷胱甘肽含量的最佳检测条件:激发波长为347nm,发射波长为425nm;溶液pH为8.0;反应体系中邻苯二甲醛浓度为0.30mg·mL-1;衍生化反应时间为20min。还原型谷胱甘肽含量在0.05～0.80μg·mL-1范围内与荧光强度有良好的线性关系,回归方程为y=1084x+63.148,相关性系数0.9990。该法快速、灵敏、准确,适合批量样品检测。