HPLC测定苏黄止咳胶囊中紫苏醛含量

目的: 建立苏黄止咳胶囊中紫苏醛含量的HPLC测定方法. 方法: 采用高效液相色谱法,SHIMADZU Shim-pack VP-ODS C₁₈色谱柱 (4.6 mm × 250 mm, 5 μm),流动相甲醇-水(68:32),检测波长230 nm,流速0.8 mL·min^-1,柱温30 ℃. 结果: 紫苏醛在5.672~453.76 ng与其峰面积呈良好的线性关系,线性回归方程为Y=5.695X+1.28(r=0.999 5),平均加样回收率为99.57% (RSD 1.7%). 结论: 该方法准确、分离度好、专属性强,可用于苏黄止咳胶囊中紫苏醛的含量测定.     

HPLC测定3种葛根素注射液中葛根素含量

目的: 应用高效液相色谱法同时测定葛根素注射液、葛根素葡萄糖注射液和葛根素氯化钠注射液中葛根素的含量. 方法: 采用Merk,Purospher STAR RP-C₁₈色谱柱(4.6 mm×250 mm,5 μm),流动相甲醇-1%冰乙酸水溶液(25:75),流速1.0 mL·min^-1,柱温29 ℃,检测波长250 nm,进样量10 μL. 结果: 对照品葛根素在0.192~1.152 μg呈良好曲线关系,线性回归方程Y=468 374X+3 105.25(r=0.999 8),平均加样回收率为99.81%,RSD 0.42%. 结论: 3种注射液中的葛根素均可在选定的条件下得到较好的分离,线性关系良好.该方法简单快速、合理可行,可为葛根素注射液的质量控制提供科学依据.     

黄素馨化学成分及其抗氧化活性

对黄素馨Jasminum giraldii根皮的化学成分进行研究。采用正相硅胶、Sephadex LH-20以及Rp-HPLC制备柱色谱法,从黄素馨乙醇提取物的乙酸乙酯萃取部位中分离得到10个化合物,通过MS和NMR等谱学方法,鉴定结构为（+）-梣皮树脂醇（1）,（+）-丁香树脂醇（2）,（+）-丁香脂素-4’-O-β-D-葡萄糖苷（3）,齐墩果酸（4）,3-甲氧基-4-羟基桂皮醛（5）,芥子醛（6）,3,5-二甲氧基对羟基苯甲醛（7）,1-（4-甲氧基苯基）乙醇（8）,反式桂皮酸（9）,4-（1-甲氧基乙基）苯酚（10）。化合物1~3,5~8以及10为首次从素馨属植物中分离得到;化合物4和9为首次从该植物中分离得到。采用 DPPH自由基清除法对分离得到的化合物进行抗氧化活性的筛选。结果显示,化合物1显示出强的自由清除能力（IC₅₀为55.1 μmol·L^-1）,活性强于维生素C（IC₅₀为59.9 μmol·L^-1）,化合物2显示中等强度自由清除能力（IC₅₀为79.0 μmol·L^-1）,活性弱于维生素C,但强于2,6-二羟丁基对甲酚（IC₅₀为236 μmol·L^-1）。     

新型乌贝速释片的生物等效性研究

目的: 制备一种新型乌贝速释片,并研究该剂型与乌贝散在Beagle犬体内的药代动力学和生物等效性。方法: 采取粉末直接压片法制备乌贝速释片,将6只Beagle犬随机分为2组,分别口服给予乌贝速释片和乌贝散,采用LC-MS/MS测定给药后不同时刻血浆中贝母素甲的浓度,采用DAS 2.0软件按非房室模型处理得药动学参数,评价速释片剂与散剂的生物等效性。结果: 乌贝速释片中贝母素甲的主要药动学参数为C_max(7.4±2.3) μg·L^-1,AUC_0-t (59.13±15.25) μg·L^-1·h^-1,T_max(1.5±0.0) h,乌贝散中贝母素甲的主要药动学参数为C_max(8.0±1.7) μg·L^-1,AUC_0-t (68.78±16.27) μg·L^-1·h^-1,T_max(1.5±0.0) h,乌贝速释片中贝母素甲的lnAUC_0-t和lnC_max的90%置信区间分别为乌贝散相应参数的95.4%~104.6%,90.9%~109.1%。结论: 自制的乌贝速释片与乌贝散具有生物等效性,且生产工艺简单,服用方便。     

HPLC同时测定半枝莲中对香豆酸和肉桂酸的含量

目的:建立半枝莲药材中同时测定对香豆酸和肉桂酸含量的测定方法。方法:采用Eclipse SB-C₁₈色谱柱(4.6 mm×250 mm,5 μm),流动相乙腈-0.2%磷酸水梯度洗脱,流速1 mL·min^-1,检测波长310,280 nm,柱温26℃。结果:对香豆酸和肉桂酸分别在0.148~1.776 μg(r=0.9998),0.062~0.992 μg(r=0.9996)呈良好的线性关系,平均加样回收率分别为99.4%(RSD 1.5%),99.3%(RSD 1.8%),供试品溶液在12 h内稳定。通过对8批半枝莲药材的测定,结果发现湖北(批号130501)样品中2种成分含量总体优于其他产地样品。结论:该含量测定方法精密度良好,准确度高,适用于半枝莲药材中对香豆酸和肉桂酸的含量测定。     

HPLC同时测定止喘灵口服液中氢溴酸东莨菪碱、硫酸阿托品、盐酸麻黄碱和盐酸伪麻黄碱的含量

目的: 建立同时测定止喘灵口服液中氢溴酸东莨菪碱、硫酸阿托品、盐酸麻黄碱和盐酸伪麻黄碱含量的高效液相色谱法. 方法: 采用Agela Durashell RP-C₁₈色谱柱(4.6 mm×250 mm,5 μm),流动相为乙腈-磷酸钠缓冲液(含17.5 mmol·L^-1十二烷基硫酸钠的0.07 mol·L^-1磷酸钠溶液,用磷酸调节pH 6.0)(30:70),流速0.9 mL·min^-1,检测波长207 nm,柱温25℃. 结果: 氢溴酸东莨菪碱、硫酸阿托品、盐酸麻黄碱和盐酸伪麻黄碱分别在0.021 21~1.060 5,0.011 14~0.557, 0.200 56 ~10.028,0.070 33~3.516 5 μg与峰面积呈良好的线性关系,r分别为0.999 3,0.999 6,0.999 7,0.9996;平均回收率(n=6)分别为101.9%,99.80%,100.3%,100.2%. 结论: 该方法快速简便,重复性好,专属性强,可作为止喘灵口服液的质量控制方法之一.     

藿香与广藿香抗氧化活性研究

目的: 研究藿香和广藿香的茎、叶提取物抗氧化活性,总多酚与总黄酮含量与抗氧化活性的关系,并分析二者药理药效是否具有可替代性. 方法: 采用DPPH法、ABTS法和FRAP方法进行研究. 结果: 发现总黄酮含量与抗氧化总能力有一定相关性(r=0.609 9).通过比较藿香与广藿香的抗氧化能力发现,广藿香乙酸乙酯部位清除DPPH自由基能力最强[IC₅₀(79.6±3.14) mg·L^-1],藿香乙酸乙酯部位清除ABTS自由基能力最强[IC₅₀ (4.41±0.23) mg·L^-1],广藿香石油醚部位Fe³⁺的还原能力最强[TEAC (1 822.99±1 141.13) μmol·g^-1]. 结论: 广藿香与藿香均具有一定抗氧化能力,且差别不大.     

离子色谱法测定维生素B1注射液中的EDTA及硝酸根离子

目的： 建立测定维生素B₁注射液中EDTA及硝酸根离子含量的离子色谱法。 方法： 采用IonPac AS 11-HC色谱柱（4.0 mm ×250 mm）,IonPac AS 11-HC guard保护柱（4.0 mm×50 mm）和ASRS 300抑制器（4 mm）,淋洗液为12 mmol·L^-1氢氧化钠,流速1.0 mL·min^-1,抑制电流60 mA,电导检测器温度35℃,柱温30℃,进样体积25 μL。 结果： EDTA和硝酸根离子检出限分别为0.29,0.01 mg·L^-1,EDTA在1.19～23.8 mg·L^-1,线性关系良好（r=0.999 7）,回收率为98.2%～100.6%;硝酸根离子在1.0～10.0 mg·L^-1线性关系良好（r=0.999 6）,回收率为97.9%～99.5%。 结论： 该实验方法选择性高,灵敏度好,测定结果准确,并且简便,快速,可用于注射液中ETDA及硝酸根离子的测定研究和质量控制。     

LC-MS/MS测定大鼠血浆中莫诺苷血药浓度

莫诺苷是从中药山茱萸Cornus officinalis Sieb. & Zucc.中提取分离获得的环烯醚萜苷类化合物,具有神经保护等多种药理活性.本研究首次采用LC-MS/MS技术建立了大鼠血浆中莫诺苷浓度的测定方法.血浆样品采用蛋白沉淀法,以金丝桃苷作为内标,色谱柱为Inertsil C8-3色谱柱(2.1 mm×50 mm,5 μm),流动相为水(含1 mmol·L^-1甲酸钠)-乙腈梯度洗脱,流速0.4 mL·min^-1.采用电喷雾离子源(ESI),正离子模式,多反应监测(MRM).莫诺苷在2~5 000 μg·L^-1 (r= 0.995 7)线性关系良好,最低定量限为2 μg·L^-1.方法精密度、准确度、回收率和基质效应均符合生物样品测定的要求,适合大鼠血浆中莫诺苷浓度的测定.应用该方法进行莫诺苷在大鼠体内的药代动力学研究,给药剂量为20 mg·kg^-1,绘制血药浓度-时间曲线,并采用DAS 2.0 计算得主要药代动力学参数:AUC_0-∞为(587.6±290.7) μg·min·L^-1,C_max为(334.2±148.0) μg·L^-1,T_max为(0.6±0.3) h,t_1/2为(0.7±0.3) h.     

杜仲叶化学成分的研究

杜仲叶来源于杜仲科植物杜仲Eucommia ulmoides的干燥叶。为了深入了解杜仲叶中的活性成分,该研究采用D-101大孔树脂,MCI树脂,反相ODS,Sephadex LH-20,Rp-HPLC制备柱色谱法和重结晶等方法,从杜仲叶乙醇提取物的正丁醇萃取部位中分离得到10个化合物,通过MS和NMR等谱学方法,鉴定结构为山柰素-3-O-β-D-葡萄糖苷（1）,槲皮素-3-O-β-D-葡萄糖苷（2）,槲皮素（3）,槲皮素-3-O-β-D-木糖基-（1→2）-β-D-葡萄糖苷（4）,山柰酚-3-O-α-L-鼠李糖基-（1→6）-β-D-葡萄糖苷（5）,（2S,3S）-（-）-花旗松素-3-O-β-D-葡萄糖苷（6）,4-羟基肉桂酸（7）,（+）-环橄榄脂素（8）,松脂素-β-D-葡萄糖苷（9）,角鲨烯（10）,其中化合物1,5~7,10为首次从该属植物中分离得到。采用DPPH自由基清除法对分离得到的化合物进行抗氧化活性研究。结果显示,其中化合物 2 表现出显著的自由基清除能力（IC₅₀为13.7 μmol·L^-1）,活性强于Vit C（IC₅₀为59.9 μmol·L^-1）;化合物1,3,9显示中等强度自由基清除能力（IC₅₀分别为161,137,214 μmol·L^-1）,活性弱于Vit C,但强于2,6-二羟丁基对甲酚（IC₅₀为236 μmol·L^-1）;化合物4,6自由基清除能力较弱（IC₅₀分别为264,299 μmol·L^-1）;提示杜仲的药理作用,可能与其黄酮和木脂素类化学成分具有能够体内清除体内活性氧及抗氧化成分有关。     

Investigation of plant extracts in traditional medicine of the Brazilian Cerrado against protozoans and yeasts

Aim of the study

To investigate the activities of the 217 plant extracts in traditional medicine of the Brazilian Cerrado against protozoans and yeasts.

Materials and methods

Plant extracts were prepared by the method of maceration using solvents of different polarities. The growth inhibition of chloroquine-resistant Plasmodium falciparum strain (FcB1) was determined by measuring the radioactivity of the tritiated hypoxanthine incorporated. Activity against Leishmania (Leishmania) chagasi and Trypanosoma cruzi was measured by the MTT colorimetric assay. The antifungal tests were carried out by using the CLSI method. The active extracts were tested also by cytotoxicity assay using NIH-3T3 cells of mammalian fibroblasts.

Results

Two hundred and seventeen extracts of plants were tested against Plasmodium falciparum. The eleven active extracts, belonging to eight plant species were evaluated against L. (L.) chagasi, Trypanosoma cruzi, yeasts and in NIH-3T3 cells. The results found in these biological models are consistent with the ethnopharmacological data of these plants. The ethyl acetate extract of Diospyros hispida root showed IC₅₀ values of 1 μg/mL against Plasmodium falciparum. This extract demonstrated no toxicity against mammalian cells, resulting in a significant selectivity index (SI) of 435.8. The dichloromethane extract of Calophyllum brasiliense root wood was active against Cryptococcus gattii LMGO 01 with MIC of 1.95 μg/mL; and Candida albicans ATCC 10231 and Candida krusei LMGO 174, both with MIC of 7.81 μg/mL. The same extract was also active against Plasmodium falciparum and L. (L.) chagasi with IC₅₀ of 6.7 and 27.6 μg/mL respectively. The ethyl acetate extract of Spiranthera odoratissima leaves was active against Cryptococcus gattii LMGO 01 with MIC of 31.25 μg/mL, and against Plasmodium falciparum with IC₅₀ of 9.2 μg/mL and Trypanosoma cruzi with IC₅₀ of 56.3 μg/mL.

Conclusion

The active extracts for protozoans and human pathogenic yeasts are considered promising to continue the search for the identification and development of leading compounds.     

Effect of feeding Mucuna pruriens on helminth parasite infestation in lambs

Ethnopharmacological relevance

Mucuna pruriens is a tropical legume anecdotally reputed to have anthelmintic properties. This study was conducted to examine the validity of such claims.

Aim of the study

The aim of this study was to determine if ingestion of Mucuna seeds reduces helminth parasite infestation in lambs.

Materials and methods

Thirty-six Dorper × Katahdin ram lambs were assigned to three treatments, a cottonseed meal based control diet, a diet in which Mucuna replaced cottonseed meal and the control diet with levamisole (7.5 mg/kg body weight) administration. All diets were isonitrogenous and isocaloric. The 12 lambs in each treatment were assigned randomly to 4 pens, each containing 3 lambs. Lambs were trickle infected three times per week by gavage with infectious Haemonchus contortus larvae (2000 larvae/lamb) for 3 weeks.

Results

Levamisole treatment decreased fecal egg counts by 87% and abomasal worm counts by 83%. Mucuna intake did not statistically affect fecal egg counts or abomasal worm counts, though numerical (P > 0.10) reductions of 7.4% and 18.1%, respectively were evident. Anemia indicators, feed intake, and lamb growth were unaffected by treatment.

Conclusions

Levamisole reduced the Haemonchus parasite burden in lambs significantly but feeding Mucuna reduced the burden by levels unlikely to eliminate the clinical effects of parasitism.     

Screening of the topical anti-inflammatory activity of the bark of Acacia cornigera Willdenow, Byrsonima crassifolia Kunth, Sweetia panamensis Yakovlev and the leaves of Sphagneticola trilobata Hitchcock

Ethnopharmacological relevance

An investigation of topical anti-inflammatory activity was undertaken on plants used in Central America traditional medicine.

Aim of study

Four herbal drugs used in the folk medicine of Central America to treat inflammatory skin affections (Acacia cornigera bark, Byrsonima crassifolia bark, Sphagneticola trilobata leaves and Sweetia panamensis bark) were evaluated for their topical anti-inflammatory activity.

Materials and methods

Petroleum ether, chloroform and methanol extracts were obtained for herbal medicines and then extracts were tested on Croton oil-induced ear dermatitis model in mice.

Results

Almost all the extracts reduced the Croton oil-induced ear dermatitis in mice and the chloroform ones showed the highest activity, with ID₅₀ (dose giving 50% oedema inhibition) values ranging from 112 μg/cm² (Byrsonima crassifolia) to 183 μg/cm² (Sphagneticola trilobata). As reference, ID₅₀ of the non-steroidal anti-inflammatory drug indomethacin was 93 μg/cm².

Conclusions

Lipophilic extracts from these species can be regarded as potential sources of anti-inflammatory principles.     

中国石斛属植物文献计量研究

石斛是珍稀濒危中药材,目前正处于快速发展阶段。为全面了解我国石斛属植物研究的历史和发展现状,作者以1954～2010年"中国知网中国学术期刊网络出版总库"收录的石斛研究文献为依据,采用文献计量学的原理和方法,对我国石斛属植物研究文献从文献年代分布、期刊分布与被引频率、主题分布、研究对象分布、作者与研究机构分布等方面进行了统计与分析。结果表明,我国石斛研究明显分为起步(2个)、停滞、平稳发展、快速上升5个阶段;期刊分布存在离散性与集中性并存的现象,已形成核心期刊研究群,并以《中国中药杂志》、《中草药》和《陕西中医》为代表;研究主题广泛涉及临床与药理、组织培养与种苗繁育、成分分析等多个领域,已经形成比较稳定的研究机构和团队,但研究对象差异显著,以铁皮石斛、金钗石斛和霍山石斛最为集中。我国石斛属植物的研究已取得显著成果,但种植产业发展缓慢,供需矛盾突出,预计种苗繁育与人工种植、产品开发、化学与药理等方面是未来的研究热点,其文献报道仍将进一步上升。     

Antifungal activity of Brazilian medicinal plants involved in popular treatment of mycoses

A survey of medicinal plants used to treat common mycoses was done in the Curituba district, Sergipe State, Brazil. One hundred inhabitants were interviewed by health agents and traditional healers. Four different plants were the most cited (more than 50% of the citations): Ziziphus joazeiro, Caesalpinia pyramidalis, Bumelia sartorum and Hymenea courbaril. The aqueous extracts obtained following traditional methods and using different parts of these plants, were submitted to drop agar diffusion tests for primary antimicrobial screening. Only the water infusion extract of Ziziphus joazeiro and Caesalpinea pyramidalis presented a significant antifungal activity against Trichophyton rubrum, Candida guilliermondii, Candida albicans, Cryptococcus neoformans and Fonsecaea pedrosoi, when compared to the antifungal agent amphotericin B. The minimal inhibitory concentration (MIC) of the bioactive extracts was evaluated by the microdilution method. Best activity with a MIC of 6.5 microg/ml for both extracts was observed against Trichophyton rubrum and Candida guilliermondii. Ziziphus joazeiro and Caesalpinea pyramidalis extracts presented also low acute toxicity in murine models. The present study validates the folk use of these plant extracts and indicates that they can be effective potential candidates for the development of new strategies to treat fungal infections.     

Experimental study on anti-inflammatory activity of a TCM recipe consisting of the supercritical fluid CO2 extract of Chrysanthemum indicum, Patchouli Oil and Zedoary Turmeric Oil in vivo

Ethnopharmacological relevance

Chrysanthemum indicum (Compositae) Linné, Pogostemon cablin (Blanco) Benth and Curcuma wenyujin (Zingiberaceae) Y. H. Chen et C. Ling are three of the extensively used herbal remedies among traditional Chinese medicines for the purpose of anti-inflammation. A traditional Chinese medicine (TCM) recipe named CPZ consisting extracts of the above three herbs, has shown noteworthy anti-influenza activity, which is closely related to its anti-inflammatory feature.

Aim of this study

To investigated the anti-inflammtory activity of CPZ in vivo for a further exploration of the recipe's anti-inflammatory properties.

Materials and methods

The anti-inflammatory property of CPZ on acute inflammation was evaluated by inflammatory models of dimethylbenzene (DMB)-induced ear vasodilatation and acetic acid-induced capillary permeability enhancement in mice, as well as the carrageenan-induced paw edema rat model, in which inflammation-related cytokine including prostaglandin E₂ (PGE₂), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in the edematous paw tissue were determined by enzyme linked immunosorbent assay (ELISA). Moreover, effect of CPZ on chronic inflammation was observed through granuloma formation in rats subjected to cotton pellet implantation.

Results

CPZ (340, 170, and 85 mg/kg for mice, p.o.) not only decreased the DMB-induced ear vasodilatation but also attenuated capillary permeability under acetic acid challenge in mice. And the significant inhibition on carrageenan-induced paw edema was observed. Further more, the ELISA results showed that CPZ (170, 85, and 42.5 mg/kg for rats, p.o.) could up-regulate the level of IL-1β in the edema paw tissue of rats significantly while down-regulate that of PGE₂, but no apparent effect on TNF-α or NO was observed in the test. Besides, CPZ had a certain degree of restraining effect on the cotton pellet-induced granuloma formation in rats and the highest dose of 170 mg/kg even showed a significant suppression on it.

Conclusion

The above results indicated that CPZ possessed a potent anti-inflammatory activity, which is indicated to be closely associated with its regulation on IL-1β and PGE₂ thereby mediating the inflammatory response acting at an appropriate level.     

Regulation effects on abnormal glucose and lipid metabolism of TZQ-F,a new kind of Traditional Chinese Medicine

Aim of the study

Based on the recipe of the traditional anti-diabetic formula TZQ, we developed TZQ-F, a new formula including 8 fractions isolated from Red Paeony root, Mulberry leaf, Lotus leaf, Danshen root and Hawthorn leaf with a good quality assurance. The study was aimed at fraction preparation and effects of the fractions on abnormal glucose and lipid metabolism.

Materials and methods

The active fractions were obtained by macroporous resin, ion-exchange resin and polyamide resin column chromatographies. HPLC analyses were used for quality control. In vitro mechanism study included DPPH radical scavenging, AGEs formation inhibition, α-glucosidase inhibition and lipase inhibition, and rats on high-fat diet were used for in vivo study.

Results

In vitro mechanism study showed that among the 8 fractions, three of them had inhibition effects on intestinal disaccharase, three with inhibition effects on lipase, and five with effects of free radical scavenging. In vivo study showed that after 4 weeks of treatment, TZQ-F significantly decreased the levels of serum total cholesterol, TG, glucose, LDL-C and HDL-C in rats on high-fat diet. Consistent with the in vitro and in vivo results, histology study demonstrated that TZQ-F alleviated hepatic steatosis induced by high-fat diet.

Conclusions

TZQ-F possesses the potential regulation effects on abnormal glucose and lipid metabolism.     

常用藏药“蒂达”(藏茵陈)基原物种药用合理性及资源利用价值评价

目的:对“蒂达”各品种不同基原植物药用合理性及其资源利用价值作出评价.方法:采用HPLC指纹图谱共有特征峰、结合SPSS聚类分析方法,评价“蒂达”不同品种基原植物的成分组成相似性.结果:“蒂达”不同品种涉及的来源有龙胆科獐牙菜属、花锚属、扁蕾属及肋柱花属共15种1变种33个样品,指纹图谱全谱相似度较差;以10个共有峰成分的相对面积聚类,其基原植物可分别聚为4个组;上述4个属的属间成分组成差异较大;其中獐牙菜属不同种间成分组成变化较大,而其他各属种间成分组成相似度较高.结论:上述龙胆科4属植物均含有具有保肝利胆生物活性的环烯醚萜类、(山)酮类及三萜类成分,藏医学将其归为“蒂达”一类药材使用具有一定的物质基础;鉴于“蒂达”类药材不同基原各品种间在成分组成上差异较大,分别制定各品种的HPLC指纹图谱,是鉴别和控制各品种质量的有效手段,可有效控制各基原品种质量.     

乌拉尔甘草HMGR基因cDNA的克隆与序列分析

目的:对乌拉尔甘草3.羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3methylglutary CoA reductase,HMGR)的cDNA克隆并进行序列分析.方法:根据NCBI数据库中的豆科其他物种HMGR的cDNA保守区设计引物,利用同源扩增和cDNA末端快速扩增技术从甘草根中获得目的基因;利用BLAST进行序列比对,ORF Finder寻找开发阅读框,Prosite分析蛋白质的基本结构域,Clustal x比对已有HMGR的氨基酸序列,并构建进化树.结果:得到1个全长为1 842 bp的HMGR的cDNA序列,含有1 722 bp的开放阅读框(open reading frame,ORF),编码573个氨基酸,具有HMGR家族的特异序列,推测的氨基酸序列与豌豆、蒺藜苜蓿的氨基酸序列一致性分别为84%,76%.结论:对甘草HMGR基因的cDNA进行了克隆,为进一步研究3-羟基-3-甲基戊二酰辅酶A在甘草酸生物合成途径中的作用提供了理论依据.     

虫草属EST-SSR标记系统的建立研究

目的:通过球孢虫草、蛹虫草EST设计EST-SSR引物,建立虫草属EST-SSR标记系统.方法:从NCBI公共数据库下载获得虫草EST,利用Sequece Seiners 1.2软件去除冗余序列并设计引物,进行PAGE电泳.结果:通过去除EST总序列中低质量的和冗余的序列后,得到全长为2 953 173 bp的4 556条无冗余球孢虫草EST.从中发掘出718个EST-SSR,分布于616条EST中,出现频率是15.8％.平均分布频率是每4 096 bp出现1个,三核苷酸重复序列有419个,是出现最多的重复类型.蛹虫草EST去冗余后得到1 363条无冗余EST,共含有1 117个EST-SSR,出现频率为81.95％,出现最多的重复类型是A核苷酸重复.根据球孢虫草EST-SSR序列,设计合成50对引物,有扩增产物的引物为34对,占总设计引物数的68％.根据蛹虫草EST-SSR,设计合成40对引物,有扩增产物的引物为39对,占总设计引物数的97.5％.基于SSR标记进行聚类分析,7种虫草无性型均能分开,且分为4支.结论:虫草属EST-SSR出现频率较高、类型较丰富、多态性潜能较高,具有较高的利用价值.球孢虫草和蛹虫草EST开发的SSR标记在虫草属有较好的转移性与通用性,可以很好的应用于虫草种间遗传关系的研究.应用虫草物种EST建立分子标记是一条简便而又有效的途径.