Identification and Analysis of the WRKY Transcription Factor Gene Family in Verbena bonariensis

The WRKY transcription factor gene family is one of the unique gene families in plants.It plays an important role in response to abiotic stresses such as cold and drought,hormone signal transduction,regulation of biosynthesis,leaf senescence seed germination,etc.However,little information is available about WRKY transcription factors in Verbena bonariensis.In this study,70 VbWRKY genes were identified from the whole genome.The phylogenetic analysis of the WRKY gene family in V.bonariensis and Arabidopsis shows that the WRKY genes in V.bonariensis can be divided into three groups:I,II,and III,which contain 13,47,and 10 members,respectively.Group II can be further divided into five subclasses:IIa(5),IIb(10),IIc(18),IId(6),and IIe(8).Conservative motif analysis showed that 64 proteins encoded by the VbWRKY gene had conserved motifs 1,2 and 3,and the same subclass motif elements were approximately the same.The collinearity analysis showed that there were 44 homologous gene pairs among the VbWRKYs,and these homologous gene pairs may have the same function.Promoter sequence analysis showed that the VbWRKY gene has multiple cis-acting elements,including not only cis-acting elements related to low-temperature and light responses,but also cis-acting elements related to hormone regulation,Among them,most VbWRKY genes contain response elements about low-temperature,and 30 VbWRKY genes contain low-temperature response elements(LTR),and 61 VbWRKY genes contain abscisic acid response elements(ABRE),indicating that VbWRKY plays a crucial role in plant growth and abiotic stress.According to the expression of VbWRKY in the cold stress and different tissues transcriptome,70 VbWRKY genes played their respective roles in various tissues and stages to regulate plant growth,Also,some of them participated in the process of cold stress tolerance,52 VbWRKYs showed significant differences in expression under cold stress,and 37 VbWRKY genes were up-regulated under cold stress.9 VbWRKY genes were selected for quantitative real-time PCR(qRT-PCR)analysis under low-temperature stress,and the results showed that all 9 genes were upregulated under low-temperature stress.Ultimately,the present study provides a comprehensive analysis of the predicted V.bonariensis WRKY genes family,which provided a theoretical basis for the study of low-temperature resistance and growth and development of V.bonariensis.

细叶美女樱(Verbena Tenera Spreng)对镉的耐性和富集特征研究

为探讨观赏花卉应用于植物修复技术的可行性,以细叶美女樱(Verbena Tenera Spreng)为材料,采用盆栽试验,研究了细叶美女樱对镉的耐性、吸收和富集能力,以期为今后观赏植物应用于重金属污染土壤修复提供理论依据。结果表明,随着镉处理浓度的增加,细叶美女樱干重呈先升高后降低的趋势,20~80 mg·kg^(-1)镉处理能促进植株的生长,100~120 mg·kg^(-1)镉处理对生长无影响,表明细叶美女樱对镉有较强的耐性。镉处理下,根系中丙二醛(MDA)含量和电解质渗透率分别于20和60 mg·kg^(-1)镉处理时开始显著高于对照,叶片中MDA含量和电解质渗透率分别于120和80 mg·kg^(-1)镉处理时开始显著高于对照,说明细叶美女樱受到了氧化伤害,且根系受伤害程度较大;氧化伤害更大程度地刺激了抗氧化防御系统,根系中POD、CAT活性的升高幅度大于叶片中CAT、APX活性的升高幅度,但抗氧化酶不能完全消除膜质过氧化引起的伤害。细叶美女樱各器官中镉含量表现为根>茎>叶的分布格局,植株对镉的转运系数小于1;但是地上部镉富集量高于根系,地上部最大富集量为778.31μg·pot^(-1)。这些结果表明细叶美女樱对镉有很强的耐性和富集能力,是一种良好的修复镉污染土壤的观赏绿化植物。

美女樱(Verbena hybrida)茎尖组织培养

以2个美女樱品种的茎尖为外植体,筛选适宜的消毒方法和初代培养、继代培养及生根培养的最佳培养基,建立了现代美女樱优良品种的离体快繁体系。结果表明,外植体消毒以70%酒精浸泡15 s、升汞浸泡4 min为宜,污染率较低（10%）,萌发率很高（87.5%）。供试的2个美女樱品种初代萌发和继代培养对不同激素浓度配比有一定差异。大花品种Quartz的适宜继代培养基为MS＋BA1.0 mg/L＋NAA0.2 mg/L,小花品种Aztec的适宜继代培养基为MS＋BA0.5 mg/L＋NAA0.1 mg/L。对于同一品种而言,较高浓度6-BA可以直接从叶上诱导出愈伤组织,并由愈伤组织形成少量不定芽。2个品种在1/2MS＋NAA0.05 mg/L培养基上均能生根,生根率达96%~98%。

海滩马鞭草(Verbena littoralis H.B.K.)的组织构造显微鉴别

目的首次对海滩马鞭草的组织构造和显微特征进行鉴别。方法取干燥海滩马鞭草的茎、根茎、叶制成横切面永久切片 ,将全草粉末用水合氯醛透化、将根茎用铬酸 硝酸解离后 ,置光学显微镜下观察。结果绘制出海滩马鞭草的根茎、茎和叶的组织构造详图和全草粉末的显微特征详图。结论为鉴别海滩马鞭草药材提供科学的依据。

Effective component from verbena officinalis L.inhibits proliferation and induces apoptosis of human choriocarcinoma JAR cells

Objective:To examine the action of the effective component,4'-methylether-scutellarein,from Verbena officinalis L.(VOL)on the proliferation and apoptosis of human choriocarcinomaJAR cells.Methods:Cell proliferation was measured by MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl tetrasodium bromide,MTT]assay and the incorporation of tritiated thymidine(~3H-TdR).Apoptosis of cell was evaluated by flow cytometry(FCM)and the characteristic apoptoticDNA ladder by agarose gel electrophoresis,and the morphological changes of apoptotic JAR cellswere observed under fluorescence microscopy and electron microscopy(EM).Expressions of ap-optosis proteins,poly(ADP-ribose)polymerase(PARP)and caspase-3,-8,and -9 were deter-mined with Western blot.Results:The effective component from VOL inhibited the proliferation of JAR cells in a dose-and time-dependent manner.The treated cell cycle was arrested in S phase and an apoptotic peakwas found in S phase using FCM analysis.A typical DNA ladder appeared in the treatment groupwhen analyzed by agarose gel electrophoresis.Using fluorescence microscopy,the percentage ofapoptotic cell was 0.9%,6%,and 14% after treatments of 10,20,and 40 mg·L^(-1) of the effec-tive component,respectively,for 48 h.Typical apoptotic changes,such as condensed chromatinand presence of apoptotic bodies,were observed under EM.Treatment with effective componentfor 48 h and 72 h also induced protein expression of PARP and caspase-3,-8,and -9 as seen byWestern blot.Conclusions:The effective component from VOL inhibits cell proliferation and induces apop-tosis in human choriocarcinoma JAR cells.

Investigation of the volatile oils,lipid constituents and biological activity of Ballota andreuzziana,Teucrium zanonii and Verbena tenuisecta in Libya

Objective:To determine the chemical composition of essential oils and lipid constituents of Ballola andreuzziana(B.andreuzziana),Teucrium zanonii(T.zanonii) and Verbena tenuisecta(V. tenuisecta) growing in Libya,and to test the antibacterial activity of different extracts of Teucrium zanonii.Methods:The volatile oils of all plants were extracted by hydrodistillation method and analyzed by GC/MS method.The lipid constituents of plants were obtained by extraction with petroleum ether and fractionated into fatty alcohols,fatty acids and unsapoinfiable matters. Antibacterial activity of T.zanonii extracts and antioxidant activity of different extracts of T. zanonii were also studied.Results:The volate oil of B.andreuzziana was found to consists of 20 compounds in which caryophyllin is the main one(63.1%),the volatile oil of T.zanonii consists of 74 compounds in which germacrene-D was the main compound,while the volatile oil of V. tenuisecta consists of 13 compounds with l-octen-3- ol as a major constituent(52.87%).The study of antimicrobial activity of different extracts of V.tenuisecta showed that,both methanol and butanol extracts exhibited the highest activity against Mycobacterium phlei(M.pblei) and Candida albicans(C.albicans) respectively,while petroleum ether,fatty alcohols and unsaponifiable fractions had no antimicrobial activity against all the tested microorganisms. Investigation of the antioxidant activity of different extracts of T.zanonii using DPPH method proved that,the ethyl acetate and butanol fractions showed the highest activity where the inhibition percentage(1%) are 93.6 and 92.1 respectively.Conclusions:This is the first report about the volatile oils of these plants where T.zanonii have the highest content and the highest number of the identified compounds.The study of antioxidant T.zanonii extracts proved that,the ethyl acetate,butanol and aqueous extracts have the highest antioxidant activity.Methanol and butanol extracts of V.tenuisecta exhibited the highest activity against M.phlei and C.albicans respectively.

马鞭草苯乙醇苷类成分研究

目的研究马鞭草Verbenae Herba的苯乙醇苷类成分。方法马鞭草80%乙醇提取物采用硅胶、Sephadex LH-20、TLC、半制备HPLC进行分离纯化,根据理化性质及波谱数据鉴定所得化合物的结构。结果从中分离得到9个化合物,分别鉴定为verbofficoside A(1)、肉苁蓉苷D(2)、广防风苷A(3)、毛蕊花糖苷(4)、异毛蕊花糖苷(5)、肉苁蓉苷C(6)、肉苁蓉苷F(7)、去咖啡酰基毛蕊花苷(8)、jionoside C(9)。结论化合物1为新化合物,化合物3、6~9均首次从该植物中分离得到。

柳叶马鞭草离体快繁体系建立

本研究以柳叶马鞭草盆栽苗为实验材料,探索其快繁体系。结果表明,将5~6 cm的带腋芽茎段以75%酒精与20%NaClO组合设计试验,发现用75%酒精消毒30 s, 20%NaClO消毒8 min的效果最好,7 d后污染率为12.78%,死亡率为4.44%,成活率达82.78%。将3~4 cm腋芽放在添加不同浓度6-BA、NAA的MS培养基中进行增殖诱导培养,发现在培养基为MS+1.5 mg/L 6-BA+0.10 mg/L NAA培养时,30 d后诱导率为100%,增殖系数达4.77,产生的丛生芽数目多。将3~4 cm的丛生芽剪成单芽,在培养基为MS+0.10 mg/L NAA进行培养时,15 d后株高差为4.64 cm,芽苗健壮且无枯萎现象。将长势好、高度5 cm左右的柳叶马鞭草无根苗置于培养基中探索IBA、NAA激素浓度,在MS+0.10 mg/L IBA+0.05 mg/L NAA条件下,15 d后生根率为100%,平均生根数为40.43,平均根长为3.27 cm;45 d后,平均根长为14.33 cm。进一步以蛭石代替琼脂作为基质,15 d后生根率为100%,平均根长可达4.50 cm, 45 d后平均根长可达42.33 cm,显著高于琼脂基质中的平均根长。自然光条件下炼苗4 d后,移栽至配比为V_(蛭石)∶V_(营养土)=1∶2的基质上,30 d后成活率为93.33%。

马鞭草的生物学功能及其在动物生产中的应用研究进展

马鞭草作为一种药用价值较高的植物,常用于活血、解毒和治疗疟疾,具有抗菌、抗氧化、抗病毒和镇痛效果。马鞭草能够提升动物生产性能,改善畜产品品质,预防疾病,促进饲料消化吸收,增强动物机体健康。马鞭草在动物生产中具有广阔的应用前景,值得进一步研究。文章综述了马鞭草的生物学功能及其在动物生产中的研究进展,以期为马鞭草在动物生产中的进一步应用提供参考。

基于三种入侵植物风险评价体系评估柳叶马鞭草的入侵风险

柳叶马鞭草(Verbena bonariensis L.)植株高大、种子量大、繁殖方式多样、抗旱能力较强,城市公园绿地引种后已出现多处逸生的植株,其具有演变为类似加拿大一枝黄花这样失控的外来入侵植物的潜力。基于野外调查和植物性状解剖,采用3种外来物种风险评估指标体系[《外来草本植物安全性评估技术规范》(NY/T 3669—2020)、《外来入侵植物杂草化风险“五阶评估法”体系》《外来草本植物引入风险评估技术规范》(NY/T 1851—2010)]分别对柳叶马鞭草进行风险评估,并同时评估了外来入侵植物加拿大一枝黄花(Solidago canadensis L.)和外来植物番茄(Solanum lycopersicum L.)的风险,以此作为参照。在3种风险评估标准中,《NY/T 3669—2020》评估柳叶马鞭草的风险值(R)为63.6,为严禁引入。《外来入侵植物杂草化风险“五阶评估法”体系》评估柳叶马鞭草的入侵风险值(R)为47.5,必须严格限制引入的目的、区域、数量和次数,而且引入后必须有足够措施限制其逃逸和扩散,并加强监测工作。《NY/T 1851—2010》评估柳叶马鞭草的风险值(R)为1.07,风险等级为中,须增加评估内容或暂缓引入。3种评价体系均表明,柳叶马鞭草具有较高的外来入侵风险,应加强监控和管理,防止演变为恶性入侵杂草。

超声波法提取马鞭草黄酮条件的优化及其抗菌活性研究

采用超声波法提取马鞭草(Verbena officinalis)中总黄酮,设计单因素试验和正交试验考察超声波功率、乙醇体积分数、超声波处理时间和料液比对总黄酮提取效果的影响,优化提取工艺条件。并采用纸片琼脂扩散法考察马鞭草提取物对几种微生物的抑制作用。结果表明,优化的马鞭草总黄酮提取条件为乙醇体积分数60%、超声波功率350W、超声波处理时间50min、料液比m马鞭草∶V乙醇=1∶30(g/mL)。抑菌试验表明,马鞭草黄酮类化合物对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、白假丝酵母(Candida spp.)、青霉(Penicillium citrimum)、黑曲霉(Aspergillusniger)均有一定的抑制作用。

马鞭草化学成分的研究

目的:分离鉴定马鞭草的化学成分。方法:乙醇提取,大孔树脂、硅胶、聚酰胺柱色谱分离,所得单体成分通过UV,IR,NMR及MS波谱分析确定结构。结果:分得4个化合物,分别鉴定为芹菜素(Ⅰ), 4′-羟基汉黄芩素(Ⅱ),马鞭草苷(Ⅲ),戟叶马鞭草苷(Ⅳ)。结论:化合物Ⅰ,Ⅱ为首次从该属植物中分离得到。

马鞭草化学成分及药理活性研究进展

马鞭草为中国传统中药,具有活血散瘀、解毒、利水、退黄等功效,临床上常用于水肿、疟疾、黄疸、肝炎、咳嗽、流行性感冒等症的治疗。马鞭草所含化学成分众多,其中黄酮类、环烯醚萜类、苯乙醇苷类、三萜类、挥发油类等成分为其主要活性成分。现代药理研究表明,马鞭草具有抗炎、抗病毒、抗菌、镇咳、抗肿瘤及调节免疫等作用。文章总结了近20年来国内外有关马鞭草化学成分、药理及毒性相关文献,对其研究进展进行综述,为马鞭草的药物开发及临床应用提供依据。

人早孕绒毛滋养层细胞的分离纯化及马鞭草对其分泌绒毛膜促性腺激素的影响

从正常妊娠６～８周早期胎盘Ｐｅｒｃｏｌ梯度离心分离出滋养层细胞进行体外培养，观察马鞭草对培养的滋养层细胞形态及绒毛膜促性腺激素（ＨＣＧ）分泌功能的影响，探讨了马鞭草抗早孕的细胞学作用机理。结果表明２５，５０ｍｇ／ｍｌ马鞭草乙醇提取液对滋养层细胞ＨＣＧ分泌有明显的抑制作用，给药后２４ｈ即见部分细胞变圆，４８ｈ大多固缩死亡。一定浓度的马鞭草可直接杀伤滋养层细胞，抑制ＨＣＧ的分泌而中止早孕。

马鞭草黄酮类化学成分的研究

从马鞭草Verbena officinalsL.中分离得到5个化合物,经理化鉴定和光谱分析确定其结构,结果均为黄酮类化合物,分别为:木犀草素(1)、山柰酚(2)、槲皮素(3)、芹菜素(4)和4′-羟基汉黄芩素。其中化合物1,2和3为首次从该植物中分离得到。

马鞭草提取液对孕早期人滋养层细胞功能影响的研究

正常妊娠６～８周人绒毛滋养层细胞以Ｐｅｒｃｏｌ分离、纯化及体外培养为实验手段，观察马鞭草提取液对滋养层细胞功能的影响，进一步探讨了马鞭草抗早孕的细胞学作用机理。结果表明：２５ｇ／Ｌ、５０ｇ／Ｌ的马鞭草提取液能阻碍细胞生长，培养液中人绒毛膜促性腺激素（ＨＣＧ）含量明显减少。琥珀酸脱氢酶（ＳＤＨ）组织化学及ＨＣＧ免疫组织化学染色，ＮＹＤ－１０００显微图像分析系统定量分析显示，细胞内ＳＤＨ的活性及ＨＣＧ分泌有明显抑制作用，其抑制率随加药剂量增加而增大。超微结构观察也显示细胞明显受损。一定浓度的马鞭草提取液能干扰细胞能量代谢，抑制ＨＣＧ的合成和分泌。

HPLC测定马鞭草中熊果酸的含量

目的由于2000年版中国药典中马鞭草项下对其有效成分熊果酸的含量测定采用薄层扫描法,在长期使用中,发现有一些不足,为此本文建立重现性好,灵敏度高并且比较稳定的高效液相色谱法测定马鞭草中熊果酸含量,并进行方法学考察。方法HypersilODS色谱柱(4.0mm×250mm,5μm);柱温35℃;流动相为乙腈-0.1%磷酸水溶液(75∶25);流速为1.0mL·min-1;检测波长为210nm。结果熊果酸进样量在0.624～3.120μg呈良好线性关系,平均回收率97.02%,RSD为0.94%(n=5);结论本法准确、灵敏度高、重现性好,更适用于马鞭草药材质量控制。

马鞭草抗早孕作用的动物实验研究

给早孕小鼠灌服马鞭草提取液,光镜观察细胞生长情况,细胞的变化及微血管分布情况。观察其对早孕小鼠子宫、胎盘、胎仔的影响,并作形态描述。实验表明:马鞭草能明显抑制胚胎生长使其固缩死亡,胚胎重量0 .129 ±0 .075 g ,与空白对照组比较有显著差异( P< 0 .05) ;胎仔长0 .32 ±0 .14 cm ,与空白对照组比较有极显著差异( P< 0 .01) ,与米非司酮组比较无显著差异( P> 0 .05) 。光镜显示:胎盘滋养层细胞退变凋亡,可见核固缩,染色质向细胞核膜下集聚。光镜下采用NYD1000 型图像分析Q 系统测定胎盘组织微血管分布面积,明显小于空白对照组( P< 0 .01) ,与米非司酮组比较无显著差异( P> 0 .05) 。结果表明马鞭草的抗早孕可能与其抗孕酮作用有关。

马鞭草药材中齐墩果酸、熊果酸的RP-HPLC测定

目的:采用重复性好、精密度高且较稳定的反相高效液相色谱法测定马鞭草中齐墩果酸、熊果酸含量,并进行方法学考察,建立有效控制马鞭草质量的方法。方法:液相条件:Agilent ZORBAX80AExtend-C18分析柱(4.6mm×250mm,5μm),流动相:甲醇-磷酸盐缓冲液(89∶11);柱温40℃;流速0.8ml/min;检测波长210nm。结果:齐墩果酸与熊果酸进样量在0.1192￣4.768μg范围内均呈良好线性关系,其中齐墩果酸平均回收率为97.1%,RSD为2.4%(n=5);熊果酸平均回收率为98.9%,RSD为3.0%(n=5)。结论:本方法使马鞭草中质控成分齐墩果酸和熊果酸达到基线分离,操作简便、结果可靠,可为马鞭草质量控制和评价提供有效手段。

GA_3和6-BA对美女樱种子萌发特性及幼苗生长的影响

试验采用不同浓度的GA3和6-BA处理美女樱种子,研究其对美女樱种子发芽势、发芽率、生理生化活性、种子浸提液的电导率、幼苗干鲜重等方面的影响。结果表明,美女樱种子经过GA3和6-BA浸种处理后,种子发芽率、发芽势提高,种子TTC还原力增强,POD酶活性提高,种子浸提液的电导率降低,根系长度增加,干物质积累增多,种子活力高于对照。各指标提高的效果均表现为6-BA好于GA3,6-BA的处理最佳使用浓度为10mg/L,种子的发芽率为89.6%,GA3的处理最佳使用浓度为300mg/L,种子的发芽率为87.5%。