至少6个突变基因型冬虫夏草菌在冬虫夏草子座中表达的动态变化

本研究检测了在冬虫夏草成熟过程中突变基因型冬虫夏草菌在子座中表达的动态变化。根据冬虫夏草菌基因内转录间隔区(ITS)序列中大量散在的点突变,设计了8个单核苷酸多态性(SNP)延伸引物,采用SNP质谱基因分型法测定各个SNP位点的单核苷酸延伸反应,观察冬虫夏草子座中转换突变和颠换突变基因型菌在冬虫夏草成熟过程中表达的动态变化。其中5个SNP延伸引物(067721-211,067721-240,067721-477,067721-531和067721-581)位于rDNA ITS1和ITS2段,用于区分GC和AT偏倚基因型,但不区分2个AT偏倚基因型。另外3个延伸引物(067740-324,067740-328和067740-360)位于rDNA 5.8S段,用于区分2个AT基因型。采用PCR+EcoRⅠ限制性酶切法检验2个GC偏倚基因型冬虫夏草菌在冬虫夏草成熟过程中表达的动态变化。结果表明:冬虫夏草子座rDNA ITS1-5.8S-ITS2片段的8个SNP位点的质谱图谱显示冬虫夏草子座中存在至少5个冬虫夏草菌转换突变和颠换突变等位基因。它们的表达在冬虫夏草成熟过程中呈现动态变化。067721-211和067721-477位点的AT偏倚型等位基因的峰高明显高于GC偏倚型;随着冬虫夏草的成熟,这2个位点的AT型等位基因峰高大幅度下降。SNP质谱图不仅显示转换突变的等位基因,颠换突变等位基因也有很高的检出率,它们的峰高在一些位点甚至超过GC和AT基因型等位基因;随着冬虫夏草的成熟,颠换突变等位基因的检出率和峰高趋于下降。在区分2个AT偏倚基因型的研究中,AB067744和AB067740 2个基因型的等位基因同时存在于未成熟冬虫夏草子座中。随着冬虫夏草的成熟,AB067744基因型等位基因的峰高明显降低,而AB067740基因型等位基因的峰高明显升高。PCR产物EcoRⅠ酶切试验发现子座中存在2组GC偏倚型菌,具有完全不同的成熟模式。综上,冬虫夏草子座中存在至少6个突变基因型冬虫夏草菌,其表达随着冬虫夏草的成熟呈现动态变化。这些突变基因型菌表达的动态变化对于冬虫夏草子座的萌发和成熟具有重要菌物学意义。     

冬虫夏草成熟过程中中国被毛孢和蝙蝠蛾拟青霉DNA共存及竞争增殖力、化学成分变化

文献报道从冬虫夏草中分离得到蝙蝠蛾拟青霉和中国被毛孢,本研究检测了蝙蝠蛾拟青霉和中国被毛孢真菌及其DNA在冬虫夏草中的共存,同时检测了2个真菌在冬虫夏草成熟过程中的竞争增殖力。应用色谱法和质谱法检测了冬虫夏草及中国被毛孢和蝙蝠蛾拟青霉菌丝体的化学成分指纹谱。在冬虫夏草中检测到蝙蝠蛾拟青霉和中国被毛孢活菌,在虫体和子座中检测到这两个真菌的DNA。从子座露出地表后,冬虫夏草的成熟伴有蛋白质和小分子有机化合物指纹谱的动态变化,中国被毛孢竞争增殖力下降(P<0.001)。中国被毛孢和蝙蝠蛾拟青霉菌丝体的化学成分指纹谱均不能与冬虫夏草的成分完全重叠,与中国被毛孢相比,蝙蝠蛾拟青霉菌丝体与冬虫夏草的指纹谱更相似。结论:中药冬虫夏草的成熟伴有蝙蝠蛾拟青霉和中国被毛孢真菌共存于它的虫体和子座,也伴有冬虫夏草化学成分的变化和中国被毛孢竞争增殖力的下降。     

冬虫夏草活性成分研究进展

本文系统综述了冬虫夏草Cordyceps sinensis的主要化学组成和药理学作用研究进展,重点对冬虫夏草多糖、核苷、肽类和甾醇的研究成果进行了总结,同时也针对有关冬虫夏草的一些问题进行了客观评述,为深入研究其有效成分与虫草相关新药物研发提供了依据和思路.     

冬虫夏草化学成分分析

本研究建立了冬虫夏草中7类主要化学成分的分析方法,包括采用高效液相色谱法分析冬虫夏草中的多糖、糖醇、氨基酸和核苷,采用气相色谱法分析冬虫夏草中的甾醇、脂肪酸和挥发性成分,所建立的分析方法均成功运用于冬虫夏草样本分析。从冬虫夏草样本中鉴定了61个小分子化合物（2个糖醇、16个氨基酸、9个核苷、7个甾醇、18个脂肪酸、9个挥发性成分）;在对其多糖分析中发现其分子量主要分布于3.3-1 300kDa范围内,由3个主要色谱峰组成。同时,还比较分析了培植冬虫夏草和野生冬虫夏草的化学成分,结果显示两者小分子化合物种类一致,多糖分子量分布一致。     

青海省冬虫夏草资源特征分析

冬虫夏草作为青藏高原特有的生物资源,对生长环境有着极其特殊的要求.通过调查青海省冬虫夏草资源分布状况,选取玉树、果洛、黄南、海南等地区的冬虫夏草进行氨基酸、微量元素含量检测,以反映冬虫夏草资源特征.研究结果表明,青海省冬虫夏草含有丰富的氨基酸和微量元素,且冬虫夏草不同部位的氨基酸、微量元素含量有明显差别.将青海省冬虫夏草与其他产区的冬虫夏草氨基酸含量进行对比分析,青海省冬虫夏草氨基酸含量低于四川、云南产区的冬虫夏草,和西藏产区的冬虫夏草氨基酸含量基本一致.     

冬虫夏草生境选择主导因子

采用样方法对冬虫夏草生境的海拔、坡度、植被和土壤等环境因子进行了探索研究。结果表明:(1)冬虫夏草主要分布在海拔4000～4500m、坡度为15°～30°的上坡位高山草甸中。(2)PCA分析表明:植被和地形是影响冬虫夏草分布的主导因子。(3)不同深度土壤养分含量统计分析表明:在不同深度层次的土壤中,水分、pH值、全氮和全磷的差异不显著,有机质、有机碳、水解氮和有效磷的差异极显著。(4)对土壤养分含量判别分析表明:5cm土壤中,吸湿水、有机碳、有机质和水解氮显著影响冬虫夏草种群分布;15cm土壤中,全氮显著影响冬虫夏草种群分布;25cm土壤中,吸湿水、有机碳和有机质显著影响冬虫夏草种群分布;而自由水、pH值、全磷和有效磷对冬虫夏草种群分布的影响不显著。(5)植物盖度、植物数量和坡度直接影响冬虫夏草种群分布,而土壤则通过影响植物和土壤温湿度来间接影响冬虫夏草种群分布。     

冬虫夏草相关术语探讨

冬虫夏草是我国传统的名贵中药材,具有悠久的历史,不仅是真菌学、中药学及生态学研究的热点,也是藏区牧民收入的重要来源和新兴产业。然而,真菌分类学、生态学、中药学等不同学科以及产业领域和民众等对冬虫夏草的认识角度和层次不同,对冬虫夏草相关的名称、概念及其内涵的理解存在分歧,导致在交流过程中出现一些不一致,并对学术交流和产业发展带来不必要的负面影响。文中从不同学科、产业和民众的角度对冬虫夏草相关术语进行了论述和探讨,并建议规范化使用冬虫夏草的有关术语。     

人工培殖冬虫夏草D-甘露醇的含量测定

采用水提回流离心的方法,提取分离人工培殖冬虫夏草中D-甘露醇,用高碘酸钠与甘露醇反应产生黄色的3,5-二乙酰-1,4-脱氢二甲基吡啶,在波长412 nm处测定其最大吸收.甘露醇浓度在10～50 mg/L之间时,与吸光值(OD412)有良好线性关系,线性方程为C=79.172x-0.0713,相关系数r=0.9999.人工培殖冬虫夏草中D-甘露醇的含量分别为89.64 mg/g(2005年)、91.86 mg/g(2006年)、94.71 mg/g(2007年)、98.98 mg/g(2008年)、97.50 mg/g(2009年)、94.71 mg/g(野生)和96.84 mg/g(野生).人工培殖冬虫夏草与野生冬虫夏草D-甘露醇的含量基本相同.     

冬虫夏草与韩芝液体发酵混合培养特性研究

平板培养基上比较了冬虫夏草、韩芝及树舌的菌丝生长速度和对淀粉的利用情况,结果显示三者的生长速度分别达到1.28、0.64和0.55 cm/d,且韩芝与树舌对淀粉的利用均优于冬虫夏草;混合培养结果得出菌种的最佳菌龄比为虫草:韩芝=3 d:5 d,最佳的接种比例为2:3;正交试验混合发酵最佳的培养基配方(%):葡萄糖1.0、淀粉2.0、黄豆粉2.0、酵母粉0.5,在此条件下,菌丝生物量达到最大值1.97 g/100 mL。     

冬虫夏草提取物的美白作用

以冬虫夏草提取物(Chinese cordyceps extract)为研究对象,通过蘑菇酪氨酸酶活性抑制试验和小鼠皮肤黑色素瘤细胞(B16-F10)黑素合成抑制试验考察冬虫夏草提取物的美白活性。结果显示冬虫夏草提取物(质量浓度40～200 mg/m L)呈剂量依赖性抑制蘑菇酪氨酸酶的活性,且在安全剂量(质量浓度0.1～0.5 mg/m L)下显著抑制小鼠皮肤黑色素瘤细胞(B16-F10)内的黑色素合成(P0.05)。说明冬虫夏草提取物能通过抑制酪氨酸酶活性有效阻滞黑色素的合成,从而实现美白作用。     

Altered Proteomic Polymorphisms in the Caterpillar Body and Stroma of Natural Cordyceps sinensis during Maturation

Objective

To examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem.

Methods

The surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). Results: SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%–4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster.

Conclusion

Proteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs.     

Prokaryotic Expression and Bioinformatics Analysis of Cytosolic Malate Dehydrogenase from Camellia sinensis(Theaceae)

The complete gene of cytosolic malate dehydrogenase (cMDH) from Camellia sinensis, called Cs cMDH, was obtained by RT PCR and rapid amplification of cDNA ends (GenBank accession number GQ845406). This gene was 1 235 bp in length, encoding a protein of 332 amino acids with the putative molecular weight of 355 kD. The Ecoli Rosetta (DE3) harboring pGEX MDH was induced by 05 mmol·L 1 IPTG at 32℃ for 3 hours, and a 615 kD glutathione Stransferase (GST) fused MDH was obtained in soluble form. The results of NCBI BLAST revealed that Cs cMDH shared 88%-93% of amino acid sequence identity with other cMDH from different higher plants. According to the multiple sequence alignment based on the three dimensional structure of protein, Cs cMDH was predicted to be a dimer with thirteen β sheet and thirteen α helix of each subunit. Cs cMDH contains typical fingerprint sequence (G12AAGQIG18) as all MDHs. The amino acid D43 in Cs cMDH is conserved in all NAD MDHs. Cs cMDH also has some conserved sequence units homologous to other NAD MDHs, such as NAD+ binding sites, catalytic motif and substrate binding sites. Moreover, Cs cMDH contains six Cys which are highly conserved in all plant NAD cMDHs. Therefore, Cs cMDH was inferred to be NAD dependent cMDH. The present study may provide the fundament for the further functional characterization of Cs cMDH.     

A comparative study on the production of exopolysaccharides between two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures

AIMS: The present study comparatively investigates the optimal culture conditions for the production of exopolysaccharides (EPS) and cordycepin during submerged mycelial culture of two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis. METHODS AND RESULTS: Fermentations were performed in flasks and in 5-l stirred-tank fermenters. In the case of C. militaris, the highest mycelial biomass (22.9 g l(-1)) and EPS production (5 g l(-1)) were achieved in a medium of 40 g l(-1) sucrose, 5 g l(-1) corn steep powder at 30 degrees C, and an initial pH 8.0. The optimum culture conditions for C. sinensis was shown to be (in g l(-1)) 20 sucrose, 25 corn steep powder, 0.78 CaCl2, 1.73 MgSO4.7H2O at 20 degrees C, and an initial pH 4.0, where the maximum mycelial biomass and EPS were 20.9 and 4.1 g l(-1) respectively. Cordycepin, another bioactive metabolite, was excreted at low levels during the early fermentation period (maximum 38.8 mg l(-1) in C. militaris; 18.2 mg l(-1) in C. sinensis). CONCLUSIONS: The two fungi showed different nutritional and environmental requirements in their submerged cultures. Overall, the concentrations of mycelial biomass, EPS and cordycepin achieved in submerged culture of C. militaris were higher than those of C. sinensis. SIGNIFICANCE AND IMPACT OF THE STUDY: C. militaris and C. sinensis are representative insect-born fungi which have been longstanding and widely used as traditional medicines in eastern Asia. Comparative studies between two fungi are currently not available and this is the first report on the optimum medium composition for submerged culture of C. sinensis.     

冬虫夏草无性型—中国被毛孢固态发酵条件的初步研究

采用以麦角甾醇含量为指标间接测定固态发酵产物生物量的方法,对冬虫夏草无性型—中国被毛孢的固态发酵条件进行了研究。正交试验结果表明固态发酵最佳培养基组成为：大米5g,玉米粉2g,蚕蛹粉1g,麸皮2g;单因素试验结果表明：当培养温度20℃、料水比1：1.5、料层厚度2cm、无光照时对菌体生长较为有利。发酵条件优化后,菌丝体中麦角甾醇的含量可达0.5911mg/g,比优化前提高了38.6%。     

基于RNA-seq的能源植物芒转录组分析

芒(Miscanthus sinensis Anderss)是多年生C4草本植物,可为能量和纤维素产品生产提供高品质的木质纤维素材料,是一种理想的能源植物。采用Illumina Hi Seq?2000高通量测序技术,对芒花芽和叶芽进行转录组分析。经拼接组装共获得98 326个Unigene,序列平均长度822 bp,N50为1 337 bp。将Unigene序列与NR、NT、Swiss-Prot、KEGG、GO和COG数据库进行比对(Evalue1e-5),共有74 134条Unigene获得了基因注释,占总Unigene的75.40%。其中,通过GO功能分类,45 507个Unigene映射到GO不同的功能节点上;通过KEGG pathways分析,共有36 710个Unigene参与了128个代谢通路;比对到同源序列比例最高的物种分别为高粱(37 731,60.86%)、玉米(16 258,26.22%)、水稻(3 065,4.94%),共占所有同源序列的92.02%。此外,获得了芒C4关键酶相关基因24个。这些注释信息的完成为芒功能基因及相关候选基因的发掘提供了重要依据。     

Clonorchis sinensis: immunolocalization of 26 kDa glutathione S-transferase in adult worms

A mu-class glutathione S-transferase (Cs26GST) of molecular mass 26 kDa was characterized from Clonorchis sinensis. In adult C. sinensis, the distribution of the Cs26GST was investigated by immuno-histochemistry and electron microscopy. Cs26GST was localized to the tegument and parenchyma. Immunogold labeling was strong in the tegumental cell bodies and moderate in the tegument and ova in the oviduct. It is suggested that Cs26GST plays a role in the metabolism and fecundity of C. sinensis.     

确证冬虫夏草无性型的分子生物学证据I.中国被毛孢与冬虫夏草之间的关系

从青海的冬虫夏草子实体上分离出中国被毛孢［Ｃｏｒｄｙｃｅｐｓ　ｓｉｎｅｎｓｉｓ（Ｂｅｒｋ．）Ｓａｃｃ．］,并利用ＲＡＰＤ－ＰＣＲ技术,筛选出８种引物,获得了冬虫夏草和中国被毛抱相应的基因组ＤＮＡ指纹图谱,两者相似率高达９６％,从而表明冬虫夏草的无性型为中国被毛孢。