HPLC-MS/MS法测定黄山贡菊花、叶和茎中酚类物质

采用HPLC-MS/MS检测技术分析了黄山贡菊花、叶、茎中酚类物质。结果表明,黄山贡菊花、叶、茎中酚类物质主要为黄酮及酚酸类化合物,其中贡菊花中含有13种黄酮类化合物,9种酚酸类化合物;叶中含有11种黄酮类化合物,6种酚酸类化合物;茎中含有8种黄酮类化合物,5种酚酸类化合物。贡菊花、叶、茎中共有的酚性成分包括:绿原酸、1,5-二咖啡酰奎尼酸、3,5-二咖啡酰奎尼酸、4,5-二咖啡酰奎尼酸、芹菜素-6-C-木糖-8-C-葡萄糖、芹菜素-6-C-葡萄糖-8-C-阿拉伯糖、芹菜素-6-C-阿拉伯糖-8-C-葡萄糖、木犀草苷、木犀草素-7-O-葡萄糖醛酸苷、香叶木素-7-O-6"-丙二酰-葡萄糖苷、金合欢素-7-O-6"-丙二酰-葡萄糖苷。金合欢素-7-O-半乳糖苷存在于贡菊茎、叶中,而在贡菊花中未发现;芹菜素-7-O-葡萄糖醛酸苷只存在于贡菊叶中,在贡菊花和茎中均未发现。     

培养基及培养条件对杜仲愈伤组织生长及次生代谢产物含量的影响

以MS、LS、B5、N6、H、Nitsch、White、1／2MS为基本培养基,分别添加0．5mg／L NAA和0．5mg／L BA,分析不同类型培养基对杜仲愈伤组织生长及次生代谢产物含量的影响,并以B5培养基进行光照条件、碳源、蔗糖浓度试验。结果表明：B5培养基不仅有利于愈伤组织生长,也有利于总黄酮的形成,而1／2MS培养基有利于绿原酸的积累;12h／d光照对愈伤组织的生长及绿原酸和总黄酮的合成有明显的促进作用,黑暗不影响愈伤组织的生长,但却抑制绿原酸和总黄酮的形成;3种碳源中,愈伤组织的增长量、绿原酸和总黄酮的含量均以蔗糖为碳源时最高,葡萄糖最低;蔗糖浓度在10～50g／L范围内绿原酸的含量随着糖浓度的升高而升高,40g／L时愈伤组织的增长量和总黄酮的含量最高。     

杜仲愈伤组织中次生代谢产物积累动态研究

以杜仲愈伤组织为材料,研究了不同接种量及培养时间、不同来源的愈伤组织及其继代次数对愈伤组织生长和次生代谢产物含量的影响。结果表明,愈伤组织继代培养时接种量在0．35g左右比较合适。愈伤组织的生长曲线大致呈S形,在20d时达到最大值,而总黄酮和绿原酸的含量均在16d时达到最大值。在继代培养中,茎和叶愈伤组织的增长量、绿原酸和总黄酮含量均在第三代达到最大值;下胚轴诱导的愈伤组织的增长量、绿原酸和总黄酮含量均在第四代达到最大值;子叶愈伤组织的增长量和总黄酮含量在第五代达到最大值,绿原酸含量于第四代达到最大值。不同来源的愈伤组织中,叶愈伤组织中绿原酸含最最高,下胚轴愈伤组织中总黄酮含量最高。     

培养基成分对杜仲愈伤组织生长及次生代谢产物含量的影响

以Bs＋0．5mg／L NAA＋0．5mg／L BA为基本培养基,研究了B5培养基中8种主要无机盐浓度对杜仲愈伤组织生长及绿原酸和总黄酮两种次生代谢产物含量的影响。结果表明：在1000～5000mg／L范围内增加培养基中KNO3的含量有利于愈伤组织生长,B5培养基中当KNO3的浓度达到2／3时,绿原酸和总黄酮含量及产量最高;(NH4)2SO4以4／3原浓度时对愈伤组织生长量、总黄酮含量及产量最高,对绿原酸的含量则是其为原浓度的1／3时最高;MgSO4以2／3浓度对生长量及1／3浓度对绿原酸、总黄酮积累最高;NaH2PO4、CaCl2和MnSO4以原浓度的愈伤组织生长和次生代谢产物合成最好;ZnSO4和FeSO4的原浓度愈伤组织的生长量最大,而1／3浓度的绿原酸和总黄酮含量最高。     

不同生长季节及生长年限银杏叶总黄酮甙含量的相关性研究

了银杏叶总黄酮工厂民不同生长季节及生长年限的相关性,结果表明,叶中总黄酮甙含量在８月份最高,幼苗叶的含量明显高于老树。     

高海拔地区不同海拔对菊花植株生长与品质的影响

通过田间测定引种栽培于四川省宝兴县不同海拔高度菊花植株的株高、地径、分枝数等营养生长指标及花数量、花重量、花直径、花厚度等生殖生长指标,采用高效液相色谱法测定菊花样品的绿原酸含量、木犀草苷含量、异绿原酸A含量等主要品质指标,并对测得数据进行方差分析和相关性分析,研究比较了高海拔地区不同海拔高度对菊花植株生长与品质的影响。结果表明:海拔梯度对菊花株高、花数、花重量、花直径、花厚度、木犀草苷含量和异绿原酸A含量均有显著影响(P0.05),对地径、分枝数和绿原酸含量无显著影响(P0.05),其中株高、花数和花直径与海拔梯度相关性显著(P0.05)。除分枝数和地径外,高海拔地区不同海拔高度对菊花植株的生长影响较大,在一定海拔范围内(2051~2 405m),海拔高度对菊花品质形成的影响较小,据2010版《药典净对菊花有效成分最低限量标准,海拔2051~2598 m所产的菊花均符合药用条件,以海拔2 329 m左右所产的菊花品质较佳。研究结果可为高海拔地区引种栽培菊花提供理论依据。     

银杏雌株高黄酮种质定向筛选

以46个银杏雌株为试验材料,研究了生育前期和生育后期单株间叶中总黄酮及其组分含量差异及变异,基于聚类分析筛选出高黄酮含量的春茶用、提取黄酮用及两者兼用的优株。主要研究结果如下:(1)生育初期的黄酮及其组分含量显著高于生育后期,两个生育时期的各银杏雌株间的叶中总黄酮及各组分含量均存在极显著性差异。(2)银杏雌株间的叶中总黄酮及各组分含量均存在较大的变异,其中生育前期叶中总黄酮及各组分含量变异系数为2106%～3335%,生育后期为2911%～5013%。(3)生育前期和生育后期的叶中总黄酮含量分别与对应生育期的槲皮素、山奈酚、异鼠李素含量均呈极显著正相关;生育前期和生育后期间叶中总黄酮及其组分含量均呈极显著高度正相关。(4)基于生育前期叶中的总黄酮及3个主要组分含量,筛选出了15、29、30、42、43、44、45、46、47、49、50、48等12个春茶用优株;基于生育后期叶中的总黄酮及3个主要组分含量,筛选出了42、46、43、44、45、47、48、50等8个提取黄酮用优株;基于生育前期和生育后期叶中总黄酮含量,筛选出了29、42、43、44、45、46、47、50、48等9个可作为银杏春茶及黄酮提取的兼用型优株。综上三类分别可作为春茶用、黄酮提取用及兼用型银杏种质,进一步无性系化扩繁、区试或推广。     

不同品种金银花中绿原酸和木犀草苷的含量测定

目的:测定不同季节‘济草堂一号’和‘九丰一号’金银花的花蕾、茎、叶中绿原酸和木犀草苷的含量。方法:按2010年版《中国药典》中金银花含量测定项规定的方法测定样品中绿原酸和木犀草苷的含量。结果:湘南产‘济草堂一号’和‘九丰一号’金银花都是绿原酸含量在花蕾中最高,木犀草苷含量在叶中最高。两个品种金银花的花蕾中绿原酸含量从5月到9月都迅速减少,而茎、叶中绿原酸含量变化趋势因品种而异;两个品种的花蕾、茎和叶中木犀草苷含量从5月到9月都逐渐减少。结论:湘南产‘济草堂一号’和‘九丰一号’金银花较佳的采收季节都是5月份。     

海螺沟引种金银花的药用成分含量对比研究

金银花是我国传统常用的中药材之一。近年来,在药用、香料、化妆品和保健食品等领域有极大的需求量,具有极高的药用价值和经济价值。为探索海螺沟适生金银花品种,从山东、河南、河北和四川引进21个主流栽培品种进行引种实验。采用高效液相色谱法(HPLC)测定了金银花花蕾、茎和叶中绿原酸和木犀草苷含量。结果如下:1)花蕾中绿原酸含量为1.617~3.394%,高于药典规定值(1.5%);茎和叶中绿原酸含量分别为0.613~1.712%和1.257~2.475%。花蕾中木犀草苷含量为0.009%~0.184%,山东的中金1号和蒙金1号的木犀草苷含量分别为0.009%和0.029%,低于药典规定含量(0.050%);茎和叶中木犀草甘含量分别为0.017~0.076%和0.406~0.562%。2)利用隶属函数综合评价花蕾、茎和叶中绿原酸和木犀草苷含量,山东鲁峰王3号得分最高,具有较高的品质,且生长茂盛,产量高,为可以引进的优良品种。     

青海沙棘不同部位总黄酮含量比较研究

研究了青海沙棘不同部位总黄酮含量的差异。用超声提取作为沙棘总黄酮的制备方法,以芦丁为对照品,采用分光光度法作为沙棘总黄酮的测定方法。结果表明:沙棘根、茎和叶中总黄酮含量分别为5.12、11.37和95.87 mg/g,叶中总黄酮含量远高于根和茎。与其他植物和其它地区沙棘叶片中总黄酮的含量相比,青海沙棘叶片中总黄酮含量都较高,因此青海沙棘具有较大的开发价值和应用前景。     

菊花R病毒杭白菊分离株全基因组序列测定与分析

杭白菊作为著名的中药“浙八味”之一,种植规模和产区不断扩大,但其病毒病的发生也日益严重,对其产量和品质造成严重影响。本研究利用双链RNA（double stranded RNA,dsRNA）和非序列依赖PCR扩增（sequence independent amplification,SIA）等方法,对感病杭白菊病原物进行鉴定,为杭白菊病毒病原的检测构建一套快速和简便的方法。结果表明,感病杭白菊被菊花R病毒（Chrysanthemum virus R,CVR）侵染,将其命名为CVR-TX。通过对其全基因组进行序列扩增与分析,获得其全长基因组为8 872 bp,编码6个ORF,具有Carlavirus属病毒的典型特征。基于全基因组核酸序列以及复制酶、外壳蛋白氨基酸的序列比对发现,CVR-TX与CVR-BJ同源性最高,分别为85.5%、96.0%和96.3%;与Carlavirus属其他病毒同源性分别在48.2%～54.4%、46.9%～55.3%和36.8%～59.5%,因此CVR被确定为一种新的Carlavirus属病毒。系统进化分析表明,基于全长基因组、复制酶（replicase）基因和外壳蛋白（coat protein,CP）基因与CVR-BJ聚为一簇,亲缘性最近。本研究获得了CVR-TX的全长基因组,丰富了CVR的基因组信息,通过生物信息学分析明确其种属关系和区域变化情况,从而为建立CVR可靠灵敏的分子检测手段和有效的防控措施提供理论基础。     

不同品种菊花和贵州产野菊花中木犀草素的含量比较

目的测定并比较不同品种菊花和贵州产野菊花中木犀草素的含量。方法用高效液相色谱法测定木犀草素的含量。色谱柱为ODS柱（4．6mm×250mm,5μm）,流动相为甲醇-水-冰醋酸（体积比45：55：0．4）,检测波长为254nm,流速1．0mL／min。结果7个菊花样品的木犀草素含量,以贵州野菊花的木犀草素含量最高（3．876mg／g）,杭菊的木犀草素含量最低（0．302mg／g）。结论贵州产野菊花中木犀草素的含量均高于其他品种菊花,具有较高的药用价值。     

菊花R病毒杭白菊分离株全基因组序列测定与分析

杭白菊作为著名的中药“浙八味”之一,种植规模和产区不断扩大,但其病毒病的发生也日益严重,对其产量和品质造成严重影响。本研究利用双链RNA（double stranded RNA,dsRNA）和非序列依赖PCR扩增（sequence independent amplification,SIA）等方法,对感病杭白菊病原物进行鉴定,为杭白菊病毒病原的检测构建一套快速和简便的方法。结果表明,感病杭白菊被菊花R病毒（Chrysanthemum virus R,CVR）侵染,将其命名为CVR-TX。通过对其全基因组进行序列扩增与分析,获得其全长基因组为8 872 bp,编码6个ORF,具有Carlavirus属病毒的典型特征。基于全基因组核酸序列以及复制酶、外壳蛋白氨基酸的序列比对发现,CVR-TX与CVR-BJ同源性最高,分别为85.5%、96.0%和96.3%;与Carlavirus属其他病毒同源性分别在48.2%～54.4%、46.9%～55.3%和36.8%～59.5%,因此CVR被确定为一种新的Carlavirus属病毒。系统进化分析表明,基于全长基因组、复制酶（replicase）基因和外壳蛋白（coat protein,CP）基因与CVR-BJ聚为一簇,亲缘性最近。本研究获得了CVR-TX的全长基因组,丰富了CVR的基因组信息,通过生物信息学分析明确其种属关系和区域变化情况,从而为建立CVR可靠灵敏的分子检测手段和有效的防控措施提供理论基础。     

Polyamine metabolism,floral initiation and floral development in Chrysanthemum (Chrysanthemum morifolium Ramat.)

In the short-day plant Chrysanthemum (Chrysanthemum morifolium Ramat. variety Pavo) putrescine and spermidine conjugates appeared in the apical bud before the first observable transformation of the meristem into floral structures. These compounds accumulated on floral initiation and well before floral evocation. Spermidine conjugates were predominant during floral initiation whereas free amines did not accumulate to any significant extent. Different associations of amides were observed during floral initiation as compared with the reproductive phase. 3,4-Dimethoxyphenethylamine conjugates (water-insoluble compounds) were the predominant amine conjugates observed during flower development. These compounds decreased drastically after fertilization. In vegetative buds from plants grown in long days polyamine conjugates were very low and appeared as plants aged. We present evidence that ornithine decarboxylase (ODC) regulates putrescine biosynthesis during floral initiation and floral development. When ODC action was blocked by DFMO (-DL-difluoromethylornithine, a specific, irreversible inhibitor of ODC), flowering was inhibited, and free and conjugated polyamines were not detected. This treatment led to a slight enhancement of ADC activity. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with DFMA (-DL difluoromethylarginine, a specific, irreversible inhibitor of ADC) did not affect flowering and the polyamine titers. The results suggest that ODC and polyamine conjugates are involved in regulating floral initiation in Chrysanthemum.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine     

Evaluating self-incompatibility in Chrysanthemum

Summary The impact of ovule number on seed set calculations for self-incompatible (SI) species was investigated. Diploid Chrysanthemum was chosen for this study because accurate counts of the potential number of ovules could be made. Individuals in populations of C. carinatum, C. coronarium, C. c. subsp. spatiosum, and C. segetum were crossed in complete diallels. All species exhibited similar results. Therefore, only the diallel data from C. coronarium subsp. spatiosum were presented. The seed set data with and without ovule counts were processed by SIGMAS, a computer program designed to analyze SI data. Incorporation of the actual number of ovules into seed set diallels provided the most realistic representation of values for self-incompatibility studies. Data derived from equations excluding ovule counts might lead to inaccurate genetic interpretations. Ovule counts were significant between and within genotypes for self (disc and ray florets), but not cross (ray florets only) pollinations. The disc florets in self-pollinations were found to be responsible for increasing the variability in ovule number. The statistics indicate that the disc and ray florets composed two distinct populations. At the diploid level with a single daisy flower type, the disc floret numbers were variable, whereas ray florets were relatively static. This was not the case with polyploid chrysanthemums, where both ovule populations were dynamic and interactive. The conservative nature of percent pseudo-self-compatibility (%PSC) deems it necessary to obtain an accurate measure of female fertility. Values for this could be obtained using a bulk pollination or a tester with unmatched S alleles.Scientific Journal Series paper no. 15,880 of the Minnesota Agricultural Experiment Station.     

MicroRNA Expression Profile during Aphid Feeding in Chrysanthemum (Chrysanthemum morifolium)

MicroRNAs (miRNAs) are important regulators of gene expression, affecting many biological processes. As yet, their roles in the response of chrysanthemum to aphid feeding have not been explored. Here, the identity and abundance of miRNAs induced by aphid infestation have been obtained using high-throughput Illumina sequencing platform. Three leaf small RNA libraries were generated, one from plants infested with the aphid Macrosiphoniella sanbourni (library A), one from plants with mock puncture treatment (library M), and the third from untreated control plants (library CK). A total of 7,944,797, 7,605,251 and 9,244,002 clean unique reads, ranging from 18 to 30 nucleotides (nt) in length, were obtained from library CK, A and M, respectively. As a result, 303 conserved miRNAs belonging to 276 miRNAs families and 234 potential novel miRNAs were detected in chrysanthemum leaf, out of which 80, 100 and 79 significantly differentially expressed miRNAs were identified in the comparison of CK-VS-A, CK-VS-M and M-VS-A, respectively. Several of the differentially abundant miRNAs (in particular miR159a, miR160a, miR393a) may be associated with the plant''s response to aphid infestation.     

黄山贡菊的组织培养与快速繁殖

1　植物名称　菊 (Ｃｈｒｙｓａｎｔｈｅｍｕｍｍｏｒｉｆｏｌｉｕｍ)品种黄山贡菊。2　材料类别　茎尖。3　培养条件　 (1 )诱导愈伤组织培养基 :ＭＳ 6 ＢＡ 3ｍｇ·Ｌ- 1 (单位下同 ) ＮＡＡ 0 .1 ;(2 )丛生芽增殖培养基 :ＭＳ 6 ＢＡ 0 .5 ;(3 )生根培养基 :1 /2ＭＳ。上述培养基均加入 0 .8%琼脂和 3 %的蔗糖 ,ｐＨ值为 6 .0 ,在 1 2 1℃高温高压下灭菌 3 0ｍｉｎ。培养温度为 (2 4± 2 )℃ ,光照时间 1 2ｈ·ｄ- 1 ,光照度为 2 0 0 0ｌｘ。4　生长与分化情况4.1　愈伤组织的诱导　从无病虫害、生长健壮的植株上选取茎尖 ,用稀释 …