The Construction of Fusion Expression Vector Carrying GFP and TP of GGPPS from Ginkgo biloba L.

[Objective]The aim was to construct the fusion gene expression vector which consisted of GFP and TP gene of GGPPS from the Ginkgo biloba L.[Method]The transit-peptide（TP） sequence of GGPPS from cDNA of Ginkgo biloba L.was successfully cloned by using DNA recombination technology,which was then linked to the efficient plant expression vector p1304 ＋ to construct the fusion gene expression vector p1304 ＋-TP.Then engineering strain EHA105-p1304 ＋-TP was constructed by transformed p1304 ＋-TP to Agrobacterium rhizogenes EHA105 using freeze-thaw method.[Result]The fusion gene expression vector which consisted of GFP and TP gene of GGPPS from the Ginkgo biloba L.and engineering strain EHA105-p1304 ＋-TP were successfully constructed.[Conclusion]It lays a foundation for further study of subcellular localization of TP transit peptide,which can help to clarify the molecular mechanism of a key step in biosynthesis of ginkgolides precursors,and also provides an important basis for the research on metabolic engineering of ginkgolide.

Cloning and Functional Analysis of HDR Gene from Ginkgo biloba L

[Objective] The aims were to obtain cloning of HDR gene from Ginkgo biloba.and study its function.[Method] The coding sequence of HDR gene was cloned from G.biloba by reversed transcription polymerase chain reaction,which was designated as GbHDR （GenBank accession No.：DQ364231）.The cDNA full-length of GbHDR is 1 827 bp containing a 1 425-bp open reading frame （ORF） encoding a 474-amino-acid polypeptide and constructed into the prokaryotic expression vector pTrcGbHDR.The β-carotene biosynthetic pathway in E.coli strain XL1-Blue was reconstructed by transforming with pAC-BETA.This engineered XL1-Blue was transformed with pTrcGbHDR.[Result] A 1 441 bp GbHDR was obtained containing a 1 425-bp ORF encoding a 474-amino-acid residues of protein,the predicted molecular weight was 53.2 kD,and predicted isoelectric point was 5.76.Functional complementation assay indicated that GbHDR could promote theβ-carotene accumulation in engineered XL1-Blue harboring pTrcGbHDR and pAC-BETA,and as a result,the engineered bacteria showed the brightly orange given by β-carotene.This suggested that GbHDR had the typical function of known HDR genes.[Conclusion] A engineered bacteria of E.coli which could highly accumulate β-carotene was obtained,which will provide candidate genes and targets for realizing β-carotene metabolic engineering.

Preliminary Study on Water Physiological Characters of Male and Female Ginkgo biloba L.

[Objective] The experiment aimed to study the difference of water physiology of male and female Ginkgo biloba L. for discussing the strategy of water utilization as well as the important role of this difference during evolution process. [Method] The stem sap flow, stomatal conductance(Gs), transpiration rate(Tr) and water use efficiency (WUE) of male and female Ginkgo biloba L. were comparatively studied. [Result] The day-night processes of flow on male and female Ginkgo biloba L. were similar. The flow on male and female Ginkgo biloba L. in day were almost same while the flow at night on male Ginkgo biloba L. was bigger than that on female Ginkgo biloba L. The Tr and Gs of male and female Ginkgo biloba L. were high in morning and at night but low at noon ,while Tr and Gs of female Ginkgo biloba L. in morning and at night were higher than these of male Ginkgo biloba L. at the same time point. However, these indexes of female plant were lower than these of male plant from 11:00 to 14:00. WUE changing trends of male and female Ginkgo biloba L. were similar, while average water utilization rate of female Ginkgo biloba L. was slightly lower than that of male Ginkgo biloba L. [Conclusion] Compared with other companion plants, water physiology of male and female Ginkgo biloba L. had strong homoplasy. The phenomenon might be a survival strategy of dioecious plants under long term evolutionary pressure.

Determination of the Flavonoids from Ginkgo Biloba Extract by High Performance Liquid Chromatography

HPLC method for analysis of the flavonoids from ginkgo biloba extract (GBE) was studied. By suitable selection of columns. symmetrical chromatographic peaks were obtained without using acidic modifier in the mobile phase, which can eliminate the time for cleaning the chromatographic system and simplify the analystic method for GBE Experimental conditions: column: Hypersil BDS C-18, 5mumx4x250 mm: column temperature: 35degreesC; mobile phase: 46% methanol-54% water; flow rate: 0.7 mL/min; detection wavelength: 360nm.

Experimental Study on Inhibition of Neuronal Toxical Effect of Levodopa by Ginkgo Biloba Extract on Parkinson Disease in Rats

In order to observe neuronal toxical effect of Levodopa and investigate if using Levodopa together with Ginkgo Bilobar Extract (EGb) would be an workable method to treat Parkinson disease, rat models of Parkinson disease (PD) were made by injecting 6-OHDA stereotaxically to right side of the mesencephic ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Rotational behavioral observation, TUNEL, immunocytochemistry, Nissl's body staining were performed to measure the difference between group treated by Levodopa (50 mg/kg every day for 3 days, 5 days, 7 days, L-dopa group) and group treated by Levodopa combined with EGb (100 mg/ kg every day, E-D group). The results showed that in the L-dopa group, the numbers of apoptosis of substantial nigra, rings of rotational behavior were more than those in the E-D group (P<0. 05). The numbers of Nissl's cells in L-dopa group were fewer than in E-D group (P<0. 05). The results suggested that Levodopa had neur toxic effect and EGb may decrease the toxicity of levodopa. The combined use of EGb with Levodopa may be a workable method to treat PD and may be better than using Levodopa alone.

Extraction Process and Content Determination of Total Flavonoids in Ginkgo( Ginkgo biloba L. ) Leaves

[Objectives]This study was conducted to study the extraction process and the content determination of flavonoids in ginkgo( Ginkgo biloba L.) leaves.[Methods]Ethanol extraction and methanol extraction of total flavonoids in ginkgo leaves were studied,and the optimal extraction conditions for flavonoids were determined by orthogonal test; and with quercetin as reference substance,total flavonoid content in ginkgo leaves was determined by UV spectrophotometry.[Results]The optimal extraction process was 4 h of Soxhlet extraction with methanol; and the total flavonoid contents had a good linear relation in the range of 0. 006 5-0. 039 mg/ml( R^2= 0. 999 9),the average content was stabilized at 1. 135%,and the average recovery of the method was 102. 0%. [Conclusions]This study selected the optimal extraction process for total flavonoids in ginkgo leaves. The test method is simple with high accuracy and precision,and is suitable for the extraction and determination of total flavonoids in ginkgo leaves.

Dynamic changes of telomeric restriction fragment (TRF) lengths in cells during the developmental process from embryos to seedlings and a comparison with the embryonal calli in Ginkgo biloba L.

Telomeres are the structures that locate at the terminals of linear eukaryotic chromosomes. They can play essential roles in many cellular processes. The terminal location ofArabidopsis-type TTTAGGG tandem repeats were thought to be highly conserved. The terminal location of Ginkgo biloba L. consisting of TTTAGGG tandem repeats, were confirmed by Bal31 exonuclease degradation and Southern blotting. By comparing telomeric restriction fragment （TRF） lengths at different developmental stages from embryos to seedlings, a fluctuant tendency towards variation was found in these samples. The TRF length of embryos was also compared with that of embryonal calli and an upward trend was discovered in callus culture. The results suggest that there should be a telomerase mechanism or/and ALT mechanism for the maintenance of telomere length.

Content Determination of Quercetin in Ferment of Ginkgo biloba L. Leaves by HPLC

[Objectives] This study aimed to determine the content of quercetin in ferment of Ginkgo biloba L.leaves.[Methods]Bacillus licheniformis was selected for solid-state fermentation of G.biloba leaf powder,and the content of quercetin in ferment of G.biloba leaves was determined by reversed-phase high-performance liquid chromatography.First,the flavonoid glycosides were extracted with methanol.Then,the flavonoid glycosides were hydrolyzed with hydrochloric acid to prepare the test solution.The chromatographic conditions were as follows:Platisil ODS C_(18) column(150 mm × 4.6 mm,5 μm);V_(methonal)∶V_(water)(0.4% phosphoric acid solution) =55∶45;flow rate of 1 m L/min;Shimadzu UV detector;detection wavelength of 360 nm.[Results] Quercetin was used as a reference substance.In the range of 0.002 6-0.036 0 g/L,there was a good linear relationship,with correlation coefficient of 0.999 8 and RSD of 1.26%.[Conclusions] This method is simple,easy to operate,accurate,and reproducible.It is suitable for the determination of quercetin content in G.biloba leaves.

Effects of Coupling Treatment of Acid and Aluminum under Soilless Culture on Water Content of Ginkgo biloba L.

In this experiment,two-factor 7-level uniform design scheme was applied.Separate treatment of each factor was performed for single effect test.Then,it analyzed the effects of acid and aluminum stress under soilless culture on the water content of plant during the growth of Ginkgo biloba L.seedlings,so as to provide a theoretical basis for studying the connection between plant water and physiological stress.The results showed that the water content of G.biloba plants declined with the decrease of p H,but the water content of G.biloba plants declined with the increase in the concentration of aluminum treatment,indicating that the stronger the acid and aluminum stress,the lower the water content of the plants,affecting the normal absorption of water content of G.biloba plants,accordingly leading to lack of water.According to the analysis of the degree of influence on the water content,p H is the first factor influencing the growth of G.biloba plants.If p H is lower than 3.5,G.biloba plants will not growth,while p H is higher than 5.0,aluminum has no significant effect on the water content of G.biloba plants.In conclusion,the water content of G.biloba plants can be used as an essential indicator for acid and aluminum stress.

Determination of the Content of Kaempferol from Fermented Ginkgo( Ginkgo biloba L.) Leaves

[Objectives] This study was conducted to determine kaempferol content in ginkgo( Ginkgo biloba L.) leaves subjected to microbial fermentation.[Methods]Bacillus licheniformis was selected for solid-state fermentation of ginkgo leaves,and the content of kaempferol in ginkgo leaves was determined by RPHPLC method. At first,methanol was used to extract flavonoid glycosides,which were then hydrolyzed by hydrochloric acid solution. HPLC was performed with Platisil ODS column C18( 150 mm ×4. 6 mm,5 μm) using mobile phase Vmethanol∶ Vwater( 0. 4% phosphoric acid solution) = 55∶45 at a flow rate of 1 ml/min,and the eluate was detected with a shimadzu HPLC ultraviolet detector at 360 nm. [Results]With kaempferol as the reference substance,the correlation coefficient was0. 999 2 in the range of 0. 001 06-0. 016 96 g/L. The content in the fermented product was less than that in the non-fermented product by 28%. [Conclusions]The method is simple,accurate,and is suitable for determination of kaempferol. This study will provide an experimental basis for the development and utilization of ginkgo.

Cloning and Analysis of Telomere-associated Sequences of Ginkgo biloba L..

Total genomic DNA was extracted from leaves of Ginkgo biloba L. by the method of hot CTAB. After determining quantification of DNA sample by microclorimetric spectrophotography, Arabidopsis-type telomere primer and Sau3AⅠ cassette primer were used to isolate telomere-associated sequences from G. biloba L. by the method of cassette-ligation-mediated polymerase chain reaction (PCR). Using this method, two telomere-associated sequences, TAS1 and TAS2, were isolated. The authors preformed Southern hybridization of EcoRⅠ-treated G. biloba genomic DNA with each clone. The hybridization pattern showed that the clones obtained were derived from G. biloba genomic DNA. There are the Arabidopsis-type TTTAGGG tandem repeats in telomeres of G. biloba.

Historical Changes of Ginkgo Biloba L. Culture

Ginkgo Biloba L.is a rare species endemic to China,strengthening the study of Ginkgo culture is of great significance to eco-economic development.This paper uses the historical research methods to study the Ginkgo and its cultural development process in China.According to the characteristics of the development of Ginkgo culture,the process can be divided into three stages:Shang and Zhou Dynasties to the Northern and Southern Dynasties(which is named theological era),Sui and Tang Dynasties to early Qing Dynasty(which is named the literature era),and the modern China which is named the scientific era.The history of Ginkgo culture is a history of Ginkgo being gradually recognized,and the Ginkgo culture's connotation and extension are gradually deepened and developed.The construction of Ginkgo today's culture should be in the inheritance of historical culture,and combined with the needs of the times comprehensive innovation,take the science and human harmonious development road.

Mechanism of Ginkgo biloba L.leaf in the treatment of ischemic stroke based on network pharmacology,bioinformatics and molecular docking

Ginkgo biloba L.leaf(GBL)has been reported to protect against ischemic stroke(IS),one of the leading causes of death and longterm disability worldwide,while there is a lack of systematic study on the exact mechanism.Here,network pharmacology and bioinformatics were used to predict the active components,important targets,and potential mechanisms of GBL in the treatment of IS.Active compounds of GBL were screened based on drug-like index and oral bioavailability,key target genes were screened based on network pharmacology and gene chip,downstream pathways for the regulation of key target genes were predicted based on gene set enrichment analysis,and the interaction between key targets and active compounds was verified based on molecular docking.The results showed that GBL played a protective role in cerebral ischemia with mainly 14 active compounds,such as isoquercitrin,luteolin-4’-glucoside,beta-sitosterol,campesterol,diosmetin,ginkgolide B,ginkgolide C,ginkgolide J,ginkgolide M,isogoycyrol,laricitrin,luteolin,sesamin,and stigmasterol.Further studies revealed that GBL played important role in immunomodulation and inflammation inhibition after cerebral ischemia by acting on its peripheral targets ARG1 and MMP9 to regulate Toll-like receptor,Chemokine and Notch signaling pathway.Meanwhile,GBL played important role in reducing neuroinflammation and blood-brain barrier damage after cerebral ischemia by acting on its central targets,CCL2,PTGS2,IL6,IL1B and MMP9 to regulate the Cytokine-cytokine receptor interaction,Jak-STAT,and Toll-like receptor signaling pathway.Additionally,molecular docking verified that the active compounds mentioned above could bind to ARG1,MMP9,CCL2,PTGS2,IL6,and IL1B.The present study shows the multicomponent,multitarget and multichannel pharmacological effects of GBL on cerebral ischemia and provides a new strategy for the treatment of IS.

孑遗植物银杏(Ginkgo biloba L.)伴性光合生理特征与进化生态

银杏类是一类古老的雌雄异株植物,目前仅存单科单属。由于银杏在系统发育等方面的独特地位,吸引了科学家从不同方面进行了广泛的研究。在适宜的条件下,测定了生长在野外环境下的成年银杏的雌雄个体的光合特性。研究结果表明,银杏的雌雄植株对光具有相同的表现趋势,光饱和点,光补偿点,光下呼吸速率等均没有明显差异。但是雌性银杏的净光合速率明显大于雄性银杏的净光合速率。这种差异可能与雌性个体在繁衍后代时需要投入更多的能量有关。研究也表明,在与其它的裸子植物和被子植物比较时,银杏的光合能力并没有明显的弱势,因此光合能力可能并不是银杏在第三纪分布受限的直接原因.

孑遗植物银杏(Ginkgo biloba L.)雌雄株水分生理特征初步研究

[目的]研究银杏雌雄株在水分生理上的差异，探讨其水分利用策略，以及在进化过程中的重要作用。[方法]对银杏雌雄株的树干液流（sap flow）、气孔导度（Gs）、蒸腾速率（Tr）以及水分利用效率（WUE）进行对比研究。[结果]雌雄银杏的液流昼夜进程具有相似的规律，且白天雌雄株液流基本相同，夜间雄株液流大于雌株；不同性别银杏的丹和岱同样呈现为早晚高、午间低的曲线趋势，早晚雌株有比雄株更高的Tr和Gs，但中午11：00-14：00之间雌株的值低于雄株；雌雄银杏的WUE曲线变化趋势相似，但雌株的平均水分利用效率略小于雄株。[结论]与其他伴性植物比较分析得知，不同性别银杏的水分生理有较强的趋同特征。这可能是在长期进化的压力下，雌雄异株植物的一种生存策略。

Biotransformation of 14-Deacetoxy-13-oxo sinenxan A by Ginkgo Cell Cultures

Deacetoxy-13-oxo sinenxan A (1) was converted to 9a-hydroxy-13-oxo-2a, 5a, 10b-triacetoxy-4(20),11-taxadiene (2) and 10b-hydroxy-13-oxo-2a,5a,9a-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures in 45% and 15% yields, respectively.

Transformation of Ginkgo Hairy Root and Establishment of Its Suspension Culture Clone

Tender leaves,buds and stems of ginkgo biloba L .were transformed successfully by Agrobacterium rhizogenes with Ri plasmid,and its suspension cultured clone was eastablished.The result of detection of opines suggested that A.rhizogenes had integrated the T DNA of the Ri plasmid into genomic DNA of ginkgo cells.A new way is offered to exploit ginkgo resources with biological technology.

Studies on Biological Characteristics of Ginkgo Hairy Root Culture

The comparative studies on properties of growth and cultivated conditions of seven transformed ginkgo hairy root clones were reported. Different clones display various phenotypes characterized by growth rate.The results show that the suitable inoculum is benefical to the growth of ginkgo hairy root.NH + 4/NO - 3, pH ,sucrose, and inositol have important effects on the growth of ginkgo hairy root.

基于高通量测序与UPLC-Q-TOF/MS技术的不同产地银杏叶内生菌与其主要化学成分相关性探究

目的:利用高通量测序,通过比较不同产地银杏叶内生菌的差异,并结合化学成分相关性分析,从微生态的角度区分不同产地银杏叶的品质差异。方法:以都江堰、平邑、崇明3个产地4—7月的银杏叶片为研究对象,采用16S rDNA、内转录间隔区(ITS)扩增子高通量测序方法分析叶片内生细菌、真菌的多样性及群落组成,再基于超高效液相色谱-四级杆-飞行时间质谱法(UPLC-Q-TOF/MS)对银杏叶主要化学成分含量进行检测,并对其内生菌多样性及主要化学成分含量进行相关性生物信息学分析。结果:3个产地银杏叶内生真菌的多样性和丰富度均为都江堰>崇明>平邑;内生细菌的多样性随着月份整体呈降低趋势,丰富度在6月份最高;不同产地叶片间优势内生真菌与优势内生细菌均一致。相关性分析发现,银杏叶片内生细菌与黄酮醇苷大多呈显著正相关,如假单胞菌(Pseudomonas)、罗姆布茨菌(Romboutsia)、葡萄球菌(Staphylococcus)等;银杏叶片内生真菌中,黄酮醇苷与链格孢属(Alternaria)、枝孢属(Cladosporium)、球腔菌属(Mycosphaerella)呈显著正相关。结论:通过高通量测序免培养的方法获得了较全面的内生菌信息,优于传统培养法。药用银杏在不同地域的内生优势菌较为一致,这可能与其稳定生成的次生代谢化合物有关。可通过属水平的群落结构区分3个产地银杏叶;化学成分与内生菌的相关性可以预测出与药效成分有关的菌群。研究结果为进一步筛选银杏叶内生菌提供了实验基础,为探讨药用银杏道地性的形成机制提供了新思路。

γ-氨基丁酸/L-瓜氨酸增强白果分离蛋白凝胶性

以白果分离蛋白(GSPI)为成胶基质材料,将γ-氨基丁酸(GABA)和L-瓜氨酸(L-Cit)分别加入到GSPI水溶液中,均质后再将溶液pH分别调至5.0、6.0和7.0,制备了热诱导凝胶。采用物化分析法、机械力学、蛋白凝胶电泳、SEM及FTIR表征了溶胶的物性、凝胶性和微观结构,并探讨GABA、L-Cit对GSPI凝胶性能的影响及其机制。结果表明,GABA、L-Cit对GSPI溶解度无提升作用,但能显著降低蛋白疏水性;GABA促进溶胶聚集并降低其ζ-电位,而L-Cit在pH 7.0时则促进解聚和提高ζ-电位;两者均能促进蛋白分子疏水基团向内折叠。在pH5.0和pH7.0时,以上两种氨基酸特别是L-Cit显著提高蛋白凝胶性能,而在pH6.0时则弱化凝胶性能。其影响机制可能是氨基酸与GSPI之间的静电作用,而这种效应依赖于所在体系的pH。