三叶半夏(Pinellia ternata)雄配子败育的遗传分析

从减数分裂过程，小池子发育和染色体数目与组成三方面探讨了三叶半夏雄配子败育的原 因。三叶半夏（Ｐｉｎｅｌｌｉａ ｔｅｒｎａｔａ）体细胞染色体数目２ｎ＝９０，其减数分裂异常，多数同源染色体能配对形 成二价体，部分同源染色体呈单价体形式或配对成多价体。减数分裂的各时期都能观察到导致遗传物 质不均衡分离的异常结构，如落后染色体，染色体桥和微核等。根据结果推测：三叶半夏是具有染色体 结构变异的多倍体复合种。染色体结构变异是三叶半夏雄配子败育的主要原因。小孢子母细胞都能完 成减数分裂过程，且单核小孢子能通过两次有丝分裂形成三核花粉粒。成熟花粉粒中可育花粉不足 １％。三核期是雄配子败育的时期。

Oxalic acid in ginger specifically denatures the acrid raphides in the unprocessed dried tuber of Pinellia ternata

Objective:Pinellia Tuber,the dried tuber of Pinellia ternata,is widely used in Japanese Kampo medicines and traditional Chinese medicines.The unprocessed Pinellia Tuber is known to cause very strong acrid irritation at oral and laryngopharynx mucosa.Recent studies have shown that the sharp needle-like crystals called raphides,that are composed of calcium oxalate and proteins,are the main causative substances of the irritation.Ginger,the rhizome of Zingiber officinale,has been used in the processing to reduce the acridity of Pinellia Tuber since before the sixth century,however,the mechanisms of reducing acridity have not been scientifically proved yet.Methods:We developed the raphides denaturation assay(RDA)to quantify the degree of denaturation in the raphides to cause irritation.By their lipophilic characters,the raphides could be extracted in petroleum ether(PE)layer from powdered Pinellia Tuber suspended in water,and the contents of the raphides in PE layer were measured by the absorbance.By this assay,we conducted the activity-guided fractionation from the boiling water extract of ginger to find the ingredients to denature the raphides.We also conducted the gustatory tests to detect the change of the irritation of the denatured raphides.Results:The treatment of powdered Pinellia Tuber suspension with ginger extract reduced the distribution of raphides in PE layer in RDA in a concentration-dependent manner.The activity-guided fractionation using RDA revealed that oxalic acid was the main active ingredient in ginger extract to denature the raphides of Pinellia Tuber.Oxalic acid reduced the lipophilicity of the raphides in the thermo-,time-,and concentration-dependent manners,and its activity was affected by pH.The treatment of powdered Pinellia Tuber suspension with oxalic acid significantly reduced its acrid irritation in gustatory test in human.Conclusions:We found that oxalic acid is the main active ingredient in ginger to reduce the acrid irritation of Pinellia Tuber.

Research Progress of the Wild Medicinal Plant,Pinellia ternata

Based on literature reviews and analysis of research reports on Pinellia ternata found locally and abroad in recent years,this article summarizes and arranges them.The research on Pinellia ternata mainly focuses on its cultivation,tissue culture,and so on.There are only a few research on its active components and its regulation mechanism.The wild resources of Pinellia ternata are gradually decreasing,hence it is urgent to take effective measures to protect these wild resources as well as to establish germplasm resources bank and nursery.In order to meet the needs of the domestic market,it is necessary to investigate the distribution of wild Pinellia ternata resources,explore the best growing environment and conditions,artificially cultivate Pinellia ternata,as well as implement resource industrialization,sustainable development,and utilization.

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

Characterization and biological activities of lectin isolated from <i>Pinellia ternata</i>

In this paper, Pinellia ternata lectin (PTL) purified from 95% saturated ammonium sulfate precipitation by applying mannose-Sepharose 4B affinity column chromatography was first described. The mannose-Sepharose 4B affinity adsorption gel has already been designed and prepared before the experiments according the combinative characterization between lectin and mannose. Mass spectrometry analysis result shows that PTL is a glycoprotein with the molecular weight of 12.165 kD. It was conclude that PTL had strong agglutination effects on mouse red blood cells, and the minimum reaction concentration was 25 μg/ml according the Hemagglutination. Results of automatic amino acid analysis indicated that PTL mainly contained 15 varieties of amino acids, of which the minimum content was cysteine and aspartate was the maximum. Cell experiment results suggested that PTL of low concentrations (0.004 mg/ml, 0.02 mg/ml and 0.1 mg/ml) promoted HeLa cell proliferation, but the effect weakened with the concentrations and treated-time increasing. However, the HeLa cells pro- liferation was intensively inhibited by higher PTL concentrations (0.5 mg/ml and 1 mg/ml), and the effect increased in a dose and time dependent manner.

Physiological and transcriptional responses to heat stress in a typical phenotype of Pinellia ternata

Pinellia ternata is an important medicinal plant,and its growth and development are easily threatened by high temperature.In this study,comprehensive research on physiological,cytological and transcriptional responses to different levels of heat stress were conducted on a typical phenotype of P.ternata.First,P.ternata exhibited tolerance to the increased temperature,which was supported by normal growing leaves,as well as decreased and sustained photosynthetic parameters.Severe stress aggravated the damages,and P.ternata displayed an obvious leaf senescence phenotype,with significantly increased SOD and POD activities(46%and 213%).In addition,mesophyll cells were seriously damaged,chloroplast thylakoid was fuzzy,grana lamellae and stroma lamellae were obviously broken,and grana thylakoids were stacked,resulting in a dramatically declined photosynthetic rate(74.6%).Moreover,a total of 16808 genes were significantly differential expressed during this process,most of which were involved in photosynthesis,transmembrane transporter activity and plastid metabolism.The number of differentially expressed transcription factors in MYB and bHLH families was the largest,indicating that these genes might participate in heat stress response in P.ternata.These findings provide insight into the response to high temperature and facilitate the standardized cultivation of P.ternata.

三叶半夏(Pinellia ternata(Thunb.)Breit)的间歇浸没培养

目的:利用间歇浸没培养反应器对药用植物三叶半夏(Pinellia ternata(Thunb.)Breit)进行组培快繁研究。方法:以增殖倍数、鲜重、平均鲜重、折干率和组培苗状态为指标,一方面对三叶半夏叶片、叶柄及丛生芽体进行培养,考察适合间歇浸没培养的外植体类型;另一方面以叶柄为外植体对三叶半夏进行间歇浸没培养、固体培养和液体摇瓶培养,考察三种培养方式对其生长的影响。结果:不同的外植体在反应器内培养时,从增殖情况来看,丛生芽作为外植体得到的增殖倍数高达39.15,叶柄为27.65,叶片为18.05。鲜重方面与增殖倍数具有相同的规律。从平均鲜重和折干率来看,无显著差异。由组培苗形态来看,以叶柄诱导的组培苗最为健壮,而以丛生芽诱导的组培苗则较为细弱。不同培养方式下的增殖倍数分别为:间歇浸没培养方式最高为24.73,其次固体培养为14.75,液体摇瓶培养为12.26,差异显著。平均鲜重和折干率均为间歇浸没培养最优。由组培苗形态来看固体培养优于间歇浸没培养,反应器得到的组培苗较为细弱。在保卫细胞长宽和叶绿体个数上两者没有显著性差异。而液体摇瓶培养玻璃化导致叶绿体个数减少,不适合培养三叶半夏的。结论:较适合间歇浸没培养的外植体类型为叶柄和丛生芽,以叶柄为优。间歇浸没培养方式相对于传统的固体和液体培养在各项生理指标上表现较好,具有大规模培养三叶半夏的潜力。

Cation Selectivity of Channels Formed at Planar Lipid Bilayer by Pinellia ternata Lectin

By means of the two-compartment system. PTL-channels, the first lectin channels formed on planar lipid bilayers by Pinellia ternata lectin (PTL) have been studied. The results show (i) PTL-channels are voltage-independent and have apparent subunits; (ii) in 50 mmol/L KC1 and 25 mmol/L BaCl2 solutions, a single channel has unit conductance of 21 pS and 42 pS, respectively; (iii) the channel exhibits a slightly higher permeability to divalent than monovalent cation (PBa/PK=4.1), and (iv) the selectivity among divalent or monovalent cations is poor. The cation selectivity sequence for the channel will follow PBa(7. 0)-Psr (6. 4)~PMg(6. 4)】PK(1. 7)】PN.(1. 0)~PLi(l. 0).Moreover, these data also give explanation to the facilitatory action of PTL on the release of acetyl-choline from motor nerve terminals.

Discrimination of toxic ingredient between raw and processed Pinellia ternata by UPLC/Q-TOF-MS/MS with principal component analysis and T-test

Objective: To investigate the toxicity difference between raw and processed Pinelliae Rhizoma(Banxia in Chinese, BX), the rhizoma of Pinellia ternata, from the view of chemical composition.Methods: Sixteen samples of raw and processed BX were prepared and analyzed by UPLC/Q-TOF-MS/MS.The discrimination(chemical marker) between the two group was investigated by principal component analysis(PCA) and T-test analysis. According to the accurate charge-to-mass ratio, MS/MS fragments, and comparison of corresponding data with the reference or database, the chemical markers were identified preliminarily.Results: Liquiritin, liquiritigenin, and lysophosphatidylcholine(LPC) were identified as the characteristic markers. The reducing of LPC in processed BX was one of the main reasons for detoxification because LPC could induce the inflammatory response;Liquiritin and liquiritigenin showed the anti-inflammatory effect and reduced liver injury, therefore the appearance of them in processed BX was an another reason for detoxification.Conclusion: An approach to explain the mechanisms of reducing the toxicity in medicinal plants by processing was proposed. Moreover, the chemical markers of toxicity could be used to differentiate the raw material from processed herbs for the quality control and safety application in clinical practice.

Effect of Pinellia ternata Lectin on Membrane Currents of Mouse Motor Nerve Terminals

Pinellia ternata lectin (PTL) extracted from the fresh juice of rhizome of pinellia ternata used as a traditional Chinese medicine facilitated the quantal release of acetylcholine (ACh) in the mouse motor nerve terminals and formed cation channels in artificial lipid bilayer. Here we report the action of PTL on presynaptic membrane currents of motor nerve terminals.The experiments were performed on the intercostal nerve triangularis sterni muscle preparations. By means of the perineurial recording, the effects of PTL on the sodium current in the preterminal part , three potassium currents and two calcium currents generated from the nerve terminals were investigated. The results show that PTL increases voltage-dependent fast Ca2+ current (ICa,f), Na+ current (INa) and Ca2+-acti-vated K+ current (IK,Ca) without action on either the voltage-dependent fast K+ current (IK,f) or the slow K+ current (IK,S). These effects are irreversible, but can be reversed by mannan, the specific binding sugar for PTL.The

油樟精油对半夏开放式组培愈伤组织诱导及分化的作用

以半夏(Pinellia ternata)叶柄、叶片作为外植体,采用组培方法研究不同浓度油樟精油对半夏愈伤组织诱导及分化的作用。结果表明,在基本培养基MS配方基础上,TDZ 0.3 mg/L和油樟精油体积浓度为0.20 mL/L的配方组合,半夏叶柄及叶片愈伤组织诱导率分别为92.3%、95.7%;TDZ 0.3 mg/L和油樟精油体积浓度为0.10 mL/L的配方组合时,分化率为100.0%,平均丛生苗数达5.25个,分化效果最佳。发现油樟精油体积浓度为0.20 mL/L时的促进效果相对较好,可以有效促进愈伤组织的诱导,提高愈伤诱导率,愈伤组织质地较紧密,呈浅绿色,增殖效果较好,分化率达100%,愈伤组织出现的芽点相对明显,且数量相对较多。综合培养成本来看,油樟精油在半夏开放式组培体系的最适浓度为0.20 mL/L。

5%苯醚·咯菌腈悬浮种衣剂的制备及其在半夏种子处理中的应用

为探明成膜剂对悬浮种衣剂成膜效果的影响,采用湿法研磨法,对成膜剂进行筛选,研究5%苯醚·咯菌腈悬浮种衣剂配方的稳定性和对半夏种子安全性。确定5%苯醚·咯菌腈悬浮种衣剂的配方为:咯菌腈3%、苯醚甲环唑2%、脂肪醇聚氧乙烯醚1%、十二烷基苯磺酸钠1%、环氧大豆油1%、乙二醇1%、黄原胶0.5%、壳聚糖0.5%、碱性玫瑰精0.5%,水补足100%。所制悬浮种衣剂各项指标均符合产品质量标准的要求,成膜效果良好,对半夏种子发芽率无影响。

不同产地半夏农艺性状及光合生理特性研究

为分析不同产地半夏在同一试验地的农艺性状及光合生理特性差异,以2年生20个不同产地的半夏为试验材料,测定其农艺性状、光合生理指标及叶绿素荧光参数。结果表明,不同产地半夏在农艺性状上存在显著差异,其中,陕西商洛产半夏的植株最高、种球最大;贵州贵阳产半夏的叶面积最大;河北保定产半夏的农艺性状整体较低。不同产地半夏的光合参数也存在明显差异,推测其光合特性和光能利用率具有较大差别,四川和贵州产半夏具有较高光合潜力。相关性分析发现,净光合速率(net photosynthetic rate,Pn)与气孔导度(stomatal conductance,Gs)、水分利用率(water use rate,WUE)、最大荧光(maximal fluorescence,Fm)、电子传递速率(electron transport rate,ETR)和叶绿素a(chlorophyll a)含量呈极显著(P<0.01)正相关,与叶绿素b(chlorophyll b)含量呈显著(P<0.05)正相关。主成分分析发现,种球直径、中裂叶长、株高、蒸腾速率(transpiration rate,Tr)、光化学淬灭系数(photochemical quenching,qP)和胞间CO_(2)浓度(intercellular CO_(2) concentration,Ci)是影响半夏生长的主要指标。聚类分析发现,四川、贵州、甘肃、河南、湖南和陕西等产地的半夏农艺性状表现较好,且具有较强的光合潜力,被聚为第Ⅰ类;保定、邢台、张家口和文山等产半夏的各项指标较低,被聚为第Ⅱ类。综上所述,四川、贵州、甘肃、河南、湖南和陕西的半夏种源具有明显优势,有良好的培育前景。以上研究结果为半夏良种选育提供了理论依据。

利用组培技术构建半夏杂交体系

本研究通过构建半夏品系的杂交体系并研究杂交种苗的扩繁周期,以解决半夏自然杂交成功率低、杂交种苗繁育效率低的问题。对消毒过的半夏花粉和雌蕊以TTC法测定其活力,并通过对比实验确定雌蕊的最佳培养基,对3种新的半夏品系持续培养出F_(1)代杂交苗,通过杂交苗继代探究转接周期和成苗周期。结果表明,半夏雌蕊和花粉的最佳消毒时间为6 min;雌蕊的最佳培养基配方是半糖无琼脂MS培养基20.00 g/L+蔗糖15.00 g/L+琼脂5.00 g/L+6-BA 1.00 mg/L+NAA 0.01 mg/L,pH=5.8。以此为基础,培育出F_(1)代苗;4种半夏品系的F_(1)代杂交苗与亲本组培苗叶型有明显区别,经过扩繁发现了两个杂交种苗的扩繁周期。本研究建立了基于植物组织培养技术的半夏杂交体系并获得杂交苗,并探明杂交种苗的繁育周期。

半夏炮制过程中毒性凝集素蛋白的基质辅助激光解吸电离飞行时间质谱分析

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)受灵敏度和重复性的影响,对完整蛋白的定量分析存在困难。本研究采用纤维素纳米晶体(CNC)辅助MALDI-TOF MS,结合内标法定量分析半夏炮制过程中半夏凝集素(PTL),通过优化CNC辅助样本制备的时长和比例,以麦胚凝集素(TVL)为内标,直接分析不同炮制程度半夏各炮制品及其浸泡上清液中PTL含量。结果表明,CNC辅助MALDI样本制备有效提升了MALDI-TOF MS定量分析的线性和重复性。随着炮制时间的延长,姜半夏、清半夏和法半夏炮制品的PTL含量逐渐下降,浸泡上清液中PTL含量逐渐上升。其中,由于pH值的改变,法半夏炮制品浸泡上清液中PTL含量呈先上升后下降的趋势。此外,将该方法应用于不同饮片生产企业的多批次半夏炮制品饮片中,均未检出PTL。该方法操作简便,可有效分析半夏炮制过程中PTL含量的变化,为研究半夏炮制过程对PTL的影响提供了参考。

不同产地半夏主要药理作用分析

[目的]比较不同产地半夏主要药理作用,为半夏的质量评价和临床用药提供试验依据。[方法]分别对国内6个主产区(湖北天门、江苏南通、河南唐河、四川蓬溪、河北安国、贵州赫章)半夏进行主要药效研究,采用气管酚红排泌法、浓氨水引咳法、冰醋酸致小鼠扭体、二甲苯致小鼠耳肿胀试验方法建立痰液分泌模型、咳嗽模型、镇痛模型及炎症模型,探讨不同产地半夏的祛痰、镇咳、镇痛及抗炎作用的差异。[结果]在止咳祛痰作用方面,贵州赫章、江苏南通、四川蓬溪3个产区半夏能够明显增加小鼠气管酚红排泌量,显著减少小鼠咳嗽次数及延长咳嗽潜伏期(P<0.01);在镇痛抗炎作用方面,四川蓬溪、湖北天门、河南唐河3个产区半夏均能够明显延长疼痛潜伏期及减少扭体次数,显著降低小鼠耳肿胀度(P<0.01);贵州赫章、河北安国产半夏镇痛抗炎作用次之,江苏南通产半夏镇痛抗炎作用弱于其他产地。[结论]6个产区半夏均有不同程度的祛痰、镇咳、镇痛、抗炎作用,其中贵州赫章、江苏南通、四川蓬溪3个产区半夏在止咳祛痰作用明显优于湖北天门、河南唐河、河北安国;在镇痛抗炎作用方面,四川蓬溪、湖北天门所产半夏强于其他产地。

半夏开花物候与结实特征分析

为深入理解半夏独特的开花物候特征和生殖特性,对半夏花的形态学特征、个体开花散粉习性、居群开花物候及结实特征进行了统计分析。结果表明,半夏群体开花规律呈单峰型,花期集中在5月下旬到6月下旬,花期长约58 d;半夏开花与散粉的日变化规律均呈倒单峰型,开花高峰期为7:00—11:00,而花药散粉高峰期为7:00—9:00;散粉持续时间为47.22 h,个体花期持续时间、花序开放进程、花序开花持续时间和种群花期历时分别为18、13、5.3、51.5 d;花序开放进程与花序开花持续时间分别以14 d和6 d出现的频率最高。半夏花不具虫媒、风媒花特征,传粉完全依靠花粉的自然散落,这可能是其结实率低的主要原因;进一步相关性分析发现,始花时间分别与开花率和结实率存在显著负相关关系,花期长度和开花率分别与结实率呈极显著正相关;开花率、结实率与半夏个体的块茎大小密切相关。综上,人工辅助延长半夏的花期和提高开花率有助于提高其结实率,进而提高有性繁殖效率。

桔梗-姜半夏配伍促进小鼠NK细胞在肺脏募集并抑制肿瘤肺脏转移

肺脏肿瘤是一种高发病率、高致死率疾病[1],在发展早期主要依赖NK细胞等淋巴细胞发挥抗肿瘤作用[2],其中CXCR3^(+)NK细胞可依赖CXCL9/10等趋化因子的招募作用[3]而快速迁移,并大量分泌IFN-γ和穿孔素等效应因子,发挥免疫监视等作用[4]。桔梗-姜半夏配伍(the combination of Platycodon Grandiflorum and Pinella Ternata,PG+PT)常用于肿瘤的临床治疗中[5]。本研究旨在探讨PG+PT对肺脏肿瘤微环境中NK细胞、特别是CXCR3+NK的免疫药理作用,以明确该配伍发挥抗肿瘤作用的部分药理机制。

中国野生半夏的遗传多样性和遗传结构研究

第四纪气候波动以及地理和环境隔离深刻地影响了现代植物的遗传多样性、遗传结构和地理分布格局。该研究采用分子谱系地理学的研究方法对药用植物半夏19个居群共212个个体的3个叶绿体片段psb K-psb I、atp F-atp H和trn L-F进行分析,探究半夏的遗传多样性、遗传结构、地理分布格局模式及成因,并探讨其居群历史动态。结果表明:(1)半夏总单倍型多样性H d为0.882,总核苷酸多样性π为1.23×10-3,在物种水平上表现出较高的遗传多样性。(2)分子方差分析(AMOVA)结果显示,半夏遗传变异主要发生在居群间,显著的遗传分化(F ST=0.909,P<0.001)和较低的种群内遗传多样性(H S=0.134);种群间遗传分化系数N ST=0.913>G ST=0.855(0.01<P<0.05),表明叶绿体单倍型具有明显的谱系地理结构。(3)中性检验结果显示,Tajima s D值、Fu and Li s D值以及Fu and Li s F值均为不显著正值,Fu s Fs值为不显著负值且失配分析曲线呈双峰,表明半夏居群整体没有经历过扩张事件。(4)单倍型地理分布显示,西南地区和中-东部地区具有单倍型多样性较高,并存在特有单倍型,故推测第四纪冰期时在这两个区域存在冰期避难所。总之,通过3个叶绿体基因对不同区域半夏的分析,阐明了其遗传多样性、遗传结构和地理分布格局,为半夏优良种源的分子筛选和保护提出了科学的建议和保护策略。

基于转录组分析鉴定半夏叶片机械损伤应答基因

【目的】探明半夏叶片在机械损伤胁迫应答方面的基因表达差异,为进一步研究半夏胁迫应答机制提供参考。【方法】对半夏在机械损伤胁迫下的叶片进行转录组测序,并比较分析基因表达与调控通路。【结果】从3个组织共获得246711个单基因(Unigene),其中117798个基因在Nr、COG等4个数据库中获得注释。剪切型机械损伤和针刺型机械损伤分别识别7895个和22972个应答基因。在应答基因中,发现多个机械损伤胁迫的基因成员。【结论】半夏机械损伤应答过程可能与防御应答、损伤应答和叶绿体类囊体膜等多个过程有关。