基于高通量测序454 GS FLX的丹参转录组学研究

本研究应用新一代高通量测序技术454 GS FLX Titanium对2年生丹参根的转录组进行测序, 研究其基因表达谱, 挖掘其功能基因。获得46 722表达序列标签 (express sequence tags, EST), 序列平均长度414 bp, 与Sanger测序的长度相当。所得序列与GenBank丹参EST合并拼接, 获得18 235条unigene, 其中, 454高通量测序发现了13 980条新的unigene。数据库中的序列同源性比较表明, 其中73.0% (13 308条) 与其他生物的已知基因具有不同程度的同源性。通过BLAST与Gene Ontology分析获得了可能参与丹参酮合成的序列27条 (编码15个关键酶), 参与丹酚酸合成的序列29条 (编码11个关键酶), 细胞色素P450序列70条, 转录因子序列577条。454高通量测序技术作为药用植物功能基因组研究的重要手段可在丹参功能基因的发现中发挥重要作用, 这些基因的发现为丹参酮和丹酚酸类化合物生物合成研究奠定了基础, 同时也为丹参的转录组研究提供了基础数据。     

西洋参PqERF1基因的克隆和生物信息学分析

乙烯响应因子(ethylene response factor,ERF)是具有PETALA2(AP2)结构域的转录因子。本研究通过西洋参(Panax quinquefolius L)的转录组高通量测序结果分析获得75条具有AP2结构域的转录因子序列,并克隆到一条开放阅读框为933对碱基的基因序列,定名为PqERF1。基因表达分析结果表明PqERF1基因受茉莉酸甲酯(MeJA)的诱导,这与人参皂苷含量的受诱导模式相同。蛋白结构预测结果表明PqERF1是定位于细胞核的具有完整保守的AP2结构域的转录因子,InterproScan蛋白功能结构域分析结果表明PqERF1很可能为发病机制相关蛋白;序列比对和进化关系分析结果也显示PqERF1与发病机制相关蛋白(烟草NtERF4)、次生代谢产物相关合成蛋白(野胡萝卜DcERF1)和花衰老相关蛋白(矮牵牛PhERF12)的相似性高,且系统进化关系较近。因此,推测PqERF1基因可能是参与西洋参次生代谢产物合成调节、抗病性和衰老相关的重要基因。     

一种新的小鼠Gabarap12 cDNA的克隆和鉴定(英文)

目的:克隆一个新的小鼠GABA_A受体相关蛋白相似蛋白2基因(Gabarapl2),并对其功能进行初步分析。方法:将已知的人GABARAPL2 cDNA序列为信息探针筛选GenBank小鼠EST数据库,将获得的一系列互相重叠的同源度较高的EST序列进行拼接。经过实验验证,在小鼠中分离和鉴定了新基因。自行制备小鼠RNA印迹膜,用杂交方法分析该基因在不同组织中的表达情况。结果:新基因在GenBank注册,登录号AF190644。同源比较显示,该推导蛋白与人GABARAPL2基因编码的蛋白完全相同,被基因命名委员会命名为小鼠Gabarapl2。该基因的推导蛋白含多个蛋白激酶磷酸化位点。杂交显示,该基因在脑、胸腺等组织表达较高,转录本大小约1.35kb。结论:克隆到一个新的小鼠Gabarapl2基因。     

一种新的小鼠Gabarapl2 cDNA的克隆和鉴定

目的：克隆一个新的小鼠GABAA受体相关蛋白相似蛋白2基因（Gabarapl2）,并对其功能进行初步分析。方法：将己知的人GABARAPL2 cDNA序列为信息探针筛选GenBank小鼠EST数据库,将获得的一系列互相重叠的同源度较高的EST序列进行拼接,经过实验验证,在小鼠中分离和鉴定了新基因,自行制备小鼠RNA印迹膜,用杂交方法分析该基因在不同组织中的表达情况。结果：新基因在GenBank注册,登录号AF190644。同源比较显示,该推导蛋白与人GABARAPL2基因编码的蛋白完全相同,被基因命名委员会命名为小鼠Gabarapl2,该基因的推导蛋白含多个蛋白激酶磷酸化位点,杂交显示,该基因在脑、胸腺等组织表达较高,转录本大小约1.35kb。结论：克隆到一个新的小鼠Gabarapl2基因。     

人参皂苷合成生物学关键元件HMGR基因克隆与表达分析

3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme-A reductase,HMGR)是甲羟戊酸途径中的第一个限速酶,直接催化人参皂苷的生物合成,是人参皂苷合成生物学研究中的重要元件之一。本研究克隆了人参(Panax ginseng)中一个新HMGR(命名为PgHMGR2)的编码区序列,并对其编码的蛋白进行生物信息学预测及基因表达分析。PgHMGR2与GenBank中的另一条人参HMGR的序列同源性仅为71.6%,因此,PgHMGR2是人参中HMGR基因家族的一个新成员。PgHMGR2基因全长1 770 bp,编码589个氨基酸残基。生物信息学预测PgHMGR2编码蛋白不含信号肽,包含两个跨膜区,具有HMGR催化作用的活性中心结构域。PgHMGR2编码蛋白与西洋参(Panax quinquefolius)的HMGR(GenBank登录号为ACV65036)具有99%的序列相似性。实时荧光定量PCR检测到PgHMGR2在人参花中表达量最高,其次为叶和根,茎中表达量最低。本研究是国内外首次获得的人参PgHMGR2基因的全长序列,为进一步鉴定PgHMGR2的功能及开展人参皂苷的合成生物学研究奠定基础。     

生物信息学在新基因HCA520研究中的作用

利用生物信息学对新基因HCA520的基因和蛋白序列进行分析，探讨生物信息学在新基因研究中的作用。以人类基因组数据库为基础，利用电子PCR和SAGE对HCA520进行染色体定位和组织表达分布分析；BLAST程序进行HCA520的基因结构分析和相似序列搜索；ORF finder和Gene Runner3.05程序对HCA520编码蛋白进行序列预测和功能分析。RT-PCR检测HCA520的组织表达谱。通过以上分析得出HCA520的全长cDNA为2396bp，定位于染色体16p12.2，其最长的开放读码框架为591bp，编码196个氨基酸。编码氨基酸含有典型的EF-hand保守序列，与CHP具有很高的同源性，为一胞内蛋白。在多种组织中有表达。提示HCA520可能是新的钙离子结合蛋白家族成员，可能通过调节Na^ /H^ 交换子的活性影响细胞的生物学功能。     

西洋参皂甙的生物合成研究进展

西洋参为名贵中药材,中国是西洋参的第一大消费国和第二大生产国。皂甙是西洋参的主要活性成分,也是影响西洋参品质的主要因素。目前分离得到的西洋参皂苷已超过30种,不同皂苷具有不同的药理作用,主要包括抗肿瘤,抗氧化,抗衰老,抗疲劳,增强免疫力,改善心血管系统功能等作用。由于一些种类的西洋参皂苷天然含量较低,无法通过化学提取获得足够的产物来完成临床试验,更无法满足正常药物生产的需要。通过化学方法合成皂苷受到成本和产量的制约,利用植物细胞培养或毛状根培养生产皂苷的研究已经进行了数十年,但始终未获得突破性进展,与天然西洋参相比,培养物中的皂苷含量没有显著提高。因此,皂苷来源的困难成为制约皂苷药物研究和生产的瓶颈。在西洋参中,皂苷合成途径是异戊二烯代谢途径的一个分枝,分枝点为氧化鲨烯经氧化鲨烯环化酶催化形成达玛烷或β-香树脂,然后经过多个细胞色素P450和糖基转移酶的催化作用形成终产物皂苷。本课题组在国内外率先开展了对西洋参皂甙合成相关基因的克隆和研究。已经完成了达玛烷合成酶和β-香树脂合成酶的克隆和鉴定工作,并通过cDNA文库构建,EST序列分析和RACE扩增等方法获得了多个细胞色素P450和糖基转移酶的候选基因,有关基因的进一步筛选和鉴定正在进行中。     

应用抑制消减杂交技术筛选动脉粥样硬化相关基因

目的　筛选动脉粥样硬化 (AS)相关基因。方法通过菌落原位杂交技术筛选用抑制消减杂交法构建的传代培养前后牛主动脉内皮细胞差异表达基因消减cDNA文库 ,用聚合酶链反应方法进一步筛选出有插入片段的阳性克隆 ,将阳性克隆进行DNA测序和同源性比较分析。建立兔AS模型 ,采用RNA点杂交技术鉴定部分筛选到的cDNA序列的AS相关性。结果　从消减文库中随机挑取的 75 0个白色克隆中筛选出 88个阳性克隆 ,DNA测序获得 6 1个cDNA序列 ,其中 2 6个为已知牛基因序列 ,19个与已知人基因具有高度同源性 ,其他 16个是未知基因片段。通过基因同源性分析 ,选择部分cDNA序列进行组织表达差异及其与AS病变的关系分析 ,结果表明 ,转录调节因子DEAD box ,Ⅻa因子抑制蛋白 ,淋巴调适蛋白和转位复合蛋白 β基因为AS相关基因。结论　这 4种基因为本文首次报道的AS相关基因。     

Ezrin分子生物学特点及其与肝细胞肝癌关系的研究进展

Ezrin又称细胞绒毛蛋白(cytovillin),是细胞骨架相关蛋白,其编码基因Vil2,定位于染色体6q25、2q26.Ezrin是Ezrin亚家族的原型,主要存在于小肠上皮细胞的刷状缘和胎盘的微绒毛中.后续研究发现,Ezrin和根蛋白(RadiLxin)和膜突蛋白(Moesin)在结构和功能关系非常紧密,故称之为ERM家族.     

基于转录组测序的白鼠尾草2-ODD基因家族鉴定与表达分析

酮戊二酸(2OG)/Fe(Ⅱ)依赖双加氧酶(2-ODD)在植物初生和次生代谢过程中发挥着重要作用。本研究基于高通量测序技术平台Illumina NovaSeq 6000进行了白鼠尾草(Salvia apiana Jepson)转录组测序,对测序结果进行de novo拼接后,共获得38 534个独立基因(unigenes)。将获得的独立基因进行基因功能注释,有29 982个独立基因获得功能注释。通过生物信息学方法在白鼠尾草转录组数据中进行2-ODD基因家族鉴定,并对鉴定得到的基因序列进行序列特征分析,包括序列的同源性、理化特征、信号肽、跨膜结构域、亚细胞定位、二级结构和三级结构预测等,另对它们的进化关系及表达模式进行分析。结果显示,从白鼠尾草转录组中共鉴定得到39个具有完整序列结构的2-ODD家族基因,这些基因编码蛋白质的平均长度为320个氨基酸,分子质量约为36.00 kDa,多为亲水性蛋白。系统进化分析将白鼠尾草2-ODD基因分为多个亚家族,这些基因在白鼠尾草不同部位均有表达,多数基因在根中的表达量明显高于叶中的表达量。本研究可为白鼠尾草2-ODD基因的深入研究奠定基础。     

高效液相色谱法用于西洋参与白参的分析和鉴别

采用高效液相色谱法测定了１０个不同地区栽培的西洋参与白参中人参皂苷 Ｒｂ１的含量,并利用相对保留时间和相对含量对两者进行定性鉴别     

Comparison of qualitative and quantitative in vitro ginsenoside production in callus cultures of three Panax species

The qualitative and quantitative difference in the various ginsenoside constituents of the crude butanol-soluble saponin fractions of callus cultures of two Indian species of Panax namely P. sikkimensis and P. pseudoginseng have been compared with that of P. quinquefolium (American ginseng). The 45-50 days old calli of the two Indian species, though found to accumulate crude ginsenoside at levels (0.9% and 1.1%, respectively) comparable to that in P. quinquefolium (1.2%), P. pseudoginseng callus showed high productivity of ginsenosides Rf (40.57%) and Ro (19.60%). P. quinquefolium calli on the other hand accumulated more of Rb and Rg group of ginsenosides but while the former appeared to be a Rg (2) accumulator the callus of the later was rich in the Rg (1) fraction.     

Comparison of the pharmacological effects of Panax ginseng and Panax quinquefolium

Medical application of Panax ginseng was first found in "Shen-Nong Herbal Classic"around 200 AD Panax quinquefolium was first introduced in "Essential of Materia Medica" in 1694 in China. The most important bioactive components contained in P ginseng and P quinquefolium are ginseng saponins (GS). The contents of ginsenoside Rb1, Re, and Rd in P quinquefolium are higher than they are in P ginseng. In P ginseng, the contents of Rg1,Rb2, and Rc are higher than they are in P quinquefolium. P ginseng had a higher ratio of Rg1: Rb1, and which was lower in P quinquefolium. After steaming for several hours, the total GS will decrease. However, some ginsenosides (Rg2, 20R-Rg2, Rg3, Rh1 and Rh2) increase, while others (Rb1, Rb2, Rb3, Rc, Rd, Re, and Rg1) decrease. However, variation, especially in P quinquefolium, is high. P ginseng and P quinquefolium are general tonics and adaptogens. Rg1 and Rb1 enhance central nervous system (CNS) activities, but the effect of the latter is weaker. Thus, for the higher contents of Rg1, P ginseng is a stimulant, whereas the Rb1 contents of P quinquefolium are mainly calming to the CNS. Re, Rg1, panaxan A and B from P ginseng are good for diabetes. Re and Rg1 enhance angiogenesis, whereas Rb1, Rg3 and Rh2 inhibit it. Rh2, an antitumor agent, can be obtained from Rb1 by steaming. The content of Re in P quinquefolium are higher than in P ginseng by 3-4 times. The vasorelax, antioxidant, antihyperlipidemic, and angiogenic effects of Re are reported. Thus, for the CNS "hot," wound healing and hypoglycemic effects, P ginseng is better than P quinquefolium. For anticancer effects, P quinquefolium is better.     

Polyacetylenes in hairy roots of a panax hybrid

From the hairy roots of an interspecific hybrid ginseng ( Panax ginseng x P. quinquefolium) known as Pgq, three polyacetylenes were isolated: panaxynol, panaxydol and 1,8-heptadecadiene-3,10-diol. These compounds isolated from the hairy roots were used for quantitative analysis to investigate the polyacetylene production of the hairy roots cultured in Gamborg B5 (B5) and 1/8 Murashige-Skoog (MS) liquid media. Maximum growth of the hairy roots was observed (ca. 5.4 g fresh weight/100 mL flask) at 8 weeks of culture in B5 medium. The highest total content of total polyacetylenes was 0.18 % of dry weight at week 8 when cultured in 1/8 MS medium. In addition, we compared the yields of polyacetylenes and ginsenosides in hairy roots cultured in B5 with those in 1/8 MS media and found the highest yields were obtained in the hairy roots cultured in B5 medium (1.24 mmol/flask polyacetylenes and 4.45 mmol/flask ginsenosides at week 8).     

The inhibitory effects of ginsenosides on protein tyrosine kinase activated by hypoxia/reoxygenation in cultured human umbilical vein endothelial cells

27 individual ginsenosides and aglycones, together with five extracts from ginseng roots, ginseng leaves, American ginseng roots, American ginseng leaves and non-saponin fraction from roots of Panax ginseng, were tested for their effects on protein tyrosine kinase (PTK) activation induced by an in vitro hypoxia/reoxygenation (H/R) model in cultured human umbilical vein endothelial cells (HUVEC). The results indicated that ginsenoside-Rb1 (3), -Rd (7), -Ra1 (1) and -Ro (27) showed significant inhibitory effects on PTK activation induced by H/R. Dose-response experiments revealed that ginsenoside-Rb1 was the most active compound and it completely blocked PTK activation at a wide range of concentrations. Most protopanaxadiol-type ginsenosides and some protopanaxatriol-type saponins also showed significant effects on PTK activation. However, the crude extracts did not protect against H/R-induced PTK activation.     

Auxins affected ginsenoside production and growth of hairy roots in Panax hybrid

Hairy roots of interspecific hybrid ginseng (Panax ginseng x P. quinquefolium), induced by Agrobacterium rhizogenes ATCC 15834, grew well in B5 liquid media supplemented with 2.5 microM auxins (3-indole butyric acid (IBA), 1-naphtaleneacetic acid (NAA) and 3-indoleacetic acid (IAA)). The hairy roots cultured in B5 liquid medium supplemented with 2.5 microM IBA showed best growth (6.39 g fresh weight per a flask, at week 8). The highest content of the total ginsenosides was 1.63% as dry weight at week 8 when cultured with 2.5 microM NAA. The different auxins affected the numbers and lateral branching roots. Especially, 2.5 microM IBA promoted the lateral root formation (43.7+/-4.0 roots, at week 8), and 2.5 microM NAA promoted the lateral root growth (45.3+/-5.6 mm, at week 8). The growth and ginsenosides production of 8-week old hairy roots cultured in B5 liquid media supplemented with IBA and NAA combinations were also investigated. Hairy roots produced higher amounts of ginsenosides in B5 liquid media supplemented with 0.5-1.0 microM IBA and NAA combinations than that cultured in B5 liquid media supplemented with only IBA and NAA. The highest yield of ginsenoside was obtained when cultured with 0.5 microM IBA and 1.0 microM IBA combination (6.38 mg per a flask, at week 8).     

Red American ginseng: ginsenoside constituents and antiproliferative activities of heat-processed Panax quinquefolius roots

Red Asian ginseng ( Panax ginseng C. A. Meyer, Araliaceae) is used in many Oriental countries. In this study, the saponin constituents and anticancer activities of steamed American ginseng ( Panax quinquefolius L.) roots were evaluated. The contents of 12 ginsenosides in the roots were determined using high performance liquid chromatography (HPLC). After the steaming treatment (100 - 120 degrees C for 1 h and 120 degrees C for 0.5 - 4 h), the quantity of 7 ginsenosides decreased and that of 5 others increased. The content of ginsenoside Rg3, a previously recognized anticancer compound, increased significantly when the root was steamed at 120 degrees C for 0.5 - 3 h. The antiproliferative effects of unsteamed and steamed (120 degrees C for 1 h and 2 h) American ginseng root extracts were assayed by the modified trichrome stain (MTS) method using three cancer cell lines (SW-480, HT-29, NSCLC). Heat-processing increased the antiproliferative effect of American ginseng significantly, and the activity of the extract from roots steamed for 2 h was greater than that of roots steamed for 1 h. Chemical constituents and antiproliferative activities of white and red Asian ginseng have also been evaluated. Five representative ginsenosides, Rb1, Rd, Re, Rg2 and Rg3, were studied. Ginsenoside Rg3 had the most potent effect. The antiproliferative activities of red American ginseng are augmented when ginsenoside Rg3 is increased.     

AFLP法构建人参、西洋参基因组DNA指纹图谱

目的　采用扩增片段长度多态性DNA(AFLP)分子遗传标志技术,分析人参、西洋参基因组DNA多态性。方法　人参、西洋参干燥根基因组DNA,经EcoRI/MseI酶切并与其相应的人工接头连接后,使用选择性引物进行PCR扩增。结果　经变性聚丙烯酰胺凝胶电泳检测,成功构建出多态性丰富和重复性好的人参、西洋参DNA指纹图谱。结论　AFLP法有望成为一种独立的切实可行的手段,将在人参、西洋参等药用植物的鉴定、生物进化、系统发育研究及指导道地性药材的科学栽培等方面发挥重要作用。     

高效液相色谱法对淫羊藿及人参复方产品的定性定量分析

目的　建立一种快速有效的分析方法 ,达到鉴定淫羊藿及人参复方产品中黄酮苷类及人参皂苷类成分 ,并测定各成分含量的目的 ,最终控制复方中淫羊藿及人参的比例。方法　通过反相液相色谱 ,对包括药典所收载的淫羊藿药材及其产品进行分析。淫羊藿中的主要黄酮苷类成分 ,以淫羊藿定A、淫羊藿定B、淫羊藿定C、淫羊藿苷为对照品 ;对主要的人参皂苷类成分 ,以人参皂苷Rg1 、Re、Rf、Rb1 、Rb2 、Rd为对照品。结果　在同一色谱条件下 ,对主要黄酮苷类及人参皂苷类成分均可同时测定且分离良好。结论　该方法适用于淫羊藿、人参及其复方产品中淫羊藿黄酮苷类、人参皂苷类成分的定性、定量分析     

Ginsenoside extraction from Panax quinquefolium L. (American ginseng) root by using ultrahigh pressure

A new method of ultrahigh pressure extraction (UPE) was used to extract the ginsenosides from Panax quinquefolium L. (American ginseng) root at room temperature. Several solvents, including water, ethanol, methanol, and n-butanol were used in the UPE. The ginsenosides were quantified by a HPLC equipped with UV-vis detector. The results showed that ethanol is the most efficient solvent among the used ones. Compared with other methods, i.e., Soxhlet extraction, heat reflux extraction, ultrasound-assisted extraction, microwave-assisted extraction, and supercritical CO2 extraction, the UPE has the highest extraction yield in the shortest time. The extraction yield of 0.861% ginsenoside-Rc in 2 min was achieved by the UPE, while the yields of 0.284% and 0.661% were obtained in several hours by supercritical CO2 extraction and the heat reflux extraction, respectively.