沈阳地区北虫草野生与市售菌株人工培育子实体的蛋白质组学比较

【背景】北虫草作为冬虫夏草的代用品,具有与冬虫夏草类似的药理活性,其富含的蛋白质和氨基酸通常作为衡量真菌营养价值的重要指标,从中分离纯化具有潜在临床应用价值的蛋白质或多肽,已成为一个研究热点。【目的】检测沈阳北虫草野生与市售菌株人工培育子实体的蛋白质组成,分析相同培育条件下获得的蛋白种类、数量及其功能的差异,为深入研究鉴定沈阳地区北虫草药用蛋白和针对性驯化提供了蛋白质组学数据基础。【方法】采集沈阳棋盘山野生北虫草菌株,与市售人工栽培北虫草菌株同期分别经组织分离、液体发酵后培育获得子实体,通过蛋白提取、胰酶酶解后,采用非标定量技术液相色谱-质谱联用方法,对野生和市售来源培育的子实体样本进行定量蛋白组的研究。【结果】共鉴定到9 233条特异性肽段和1 923个蛋白,其中含有1 163个可定量蛋白,野生来源培育子实体有214个蛋白表达发生上调,181个蛋白表达发生下调,对这些差异蛋白进行功能富集分析发现,其主要参与能量生产/转换、氨基酸转运/代谢、抗氧化功能。在相同的营养摄取条件下,野生来源培育菌种在各个能量代谢、氨基酸代谢功能中的相关蛋白表达量高于市售来源培育的菌种。野生来源培育菌种的一种抗氧化重要蛋白(Gene Name:ISF_02112)表达量远远高于(Fold Change9)市售来源培育菌种。同时与抗氧化和代谢功能相关的差异蛋白有22个。【结论】沈阳地区北虫草野生菌株经适当人工培育会保留部分优良的生物学特性,2种来源菌株培育的子实体具有丰富及优异抗氧化功能的蛋白,子实体蛋白的抗氧化能力与其整体代谢能力相关。本研究结果为深入研究鉴定北虫草药用蛋白和针对性驯化提供蛋白质组学数据基础。     

Preservation affects the vegetative growth and fruiting body production of <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fungal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.     

野生蛹虫草菌株培养特性及子实体多糖含量比较分析

蛹虫草(Cordyceps militaris)是一种药食两用真菌,为获得高产优质的菌种资源,以采集分离的5株蛹虫草野生菌株(XY002、XY008、XY011、XY029、XY032)为研究对象,通过对5株菌株的分子鉴定、交配型基因分子检测、培养特性及子实体多糖含量测定,确定5株菌株皆为蛹虫草菌株,除XY008只含有MAT1-1-1交配型基因外,其他菌株都含有两种交配型基因MAT1-1-1和MAT1-2-1,5株菌株在菌丝生长速度、分生孢子数量、子实体形态、产量及子实体多糖含量均存在较大差异。综合培养特性及多糖含量分析的结果表明,菌株XY011子实体产量较高达29.60 g/瓶,多糖含量最高达100.79 mg/g,子实体长度最长达11.41 cm,而且子实体粗壮,发育周期短,确定为优势菌株,具有较好的开发价值。     

蛹虫草表型多态性对子实体产生及虫草菌素的影响

为选育高产子实体和虫草菌素的蛹虫草菌株,从野生蛹虫草中分离单孢子并进行分型,之后进行产子实体实验;同时对蛹虫草子实体进行了产孢结构的观察,并用高效液相色谱仪检测了子实体和培养基中的虫草菌素。结果表明,菌落背面颜色为橙铬色的菌株容易产生子实体;出发(原代)菌株所产子实体在形态上更接近野生蛹虫草,而角变株的子实体畸形;出发菌株产子实体能力要比该菌株未发生角变部分分离株和角变部分分离株都强。在虫草菌素的产量上也以出发菌株为高,表明表型多态性对蛹虫草产子实体和虫草菌素有很大的影响。这对于优势菌种的筛选和生产实践有借鉴意义。     

Hypoglycemic activity of the fungi Cordyceps militaris, Cordyceps sinensis, Tricholoma mongolicum, and Omphalia lapidescens in streptozotocin-induced diabetic rats

Crude extracts were prepared from fruiting bodies and mycelia of the medicinal fungus Cordyceps militaris, and a polysaccharide-enriched fraction was obtained after extraction with hot water and ethanol precipitation. Polysaccharide-enriched fractions were similarly prepared from Cordyceps sinensis, Omphalia lapidescens, and Tricholoma mongolicum. The various aforementioned preparations were orally administered into different groups of adult rats 24 h before an intraperitoneal injection of streptozotocin (40 mg/kg body weight), and subsequently daily for another 4 days. The dosage used was 10 mg/kg body weight for polysaccharide-enriched preparations and 100 mg/kg body weight for crude extracts. Control rats received distilled water instead of crude extract or polysaccharide-enriched preparation. It was found in the control rats that plasma glucose level rose from about 90 mg/dl before streptozotocin injection to levels that were maintained at about 300 mg/dl postinjection. All preparations produced hypoglycemic effects. C. militaris polysaccharide-enriched fraction displayed a more prominent effect than that of C. sinensis polysaccharide-enriched fraction which in turn was more potent than that of O. lapidescens and T. mongolicum polysaccharide-enriched fractions. The hypoglycemic effect of C. militaris polysaccharide-enriched fraction was dose-dependent.     

基于农业废弃物培育优质蛹虫草的研究

利用农业废弃物甘薯藤及蛹虫草培养基废弃物作为培养基的主要原料进行蛹虫草菌种驯化。蛹虫草子实体培养基中添加不同比例蛹虫草培养基废弃物及甘薯藤粒,通过适宜的培养条件,废弃物中淀粉、蛋白质、糖类、氨基酸等营养物质及蛹虫草胞外酶酶解产生的小分子物质被充分利用,以培育优质蛹虫草子实体。当一级种子中加入蛹虫草培养基废弃物20 g/L和甘薯藤粉10 g/L,二级种中加入蛹虫草培养基废弃物20 g/L、甘薯藤粉10 g/L,使蛹虫草子实体栽培料中蛹虫草培养基废弃物占32%～45%,甘薯藤粒占10%～15%,二者比例为(3～4)：1时,栽培效果最好。本研究蛹虫草培养基替代原料资源丰富易得,并可节约生产用粮,降低原料成本,从而实现农用废弃物再利用,减少环境污染,也符合绿色环保可持续发展的理念。     

Improvement of fruiting body production in Cordyceps militaris by molecular assessment

Cordyceps militaris is a heterothallic ascomycetous fungus that has been cultivated as a medicinal mushroom. This study was conducted to improve fruiting body production by PCR assessment. Based on single-ascospore isolates selected from wild and cultivated populations, the conserved sequences of α-BOX in MAT1-1 and HMG-BOX in MAT1-2 were used as markers for the detection of mating types by PCR. PCR results indicated that the ratio of mating types is consistent with a theoretical ratio of 1:1 (MAT1-1:MAT1-2) in wild (66:70) and cultivated (71:60) populations. Cross-mating between the opposite mating types produced over fivefold more well-developed fruiting bodies than self- or cross-mating between strains within the same mating type. This study may serve as a valuable reference for artificial culturing of C. militaris and other edible and medicinal mushrooms and may be useful to develop an efficient process for the selection, domestication, and management of strains for industrial-scale production.     

生物磁效应对蛹虫草活性物质含量的影响

利用场强为0.1、0.25、0.4T恒定磁场,以流速为1 m·s^-1,分别对普通水进行9次处理,串联（SC）磁场处理3次的磁处理水,进而用于栽培蛹虫草,并研究了生物磁效应对蛹虫草虫草素、虫草酸、多糖含量及生物学转化效率的影响。结果表明,0.4T处理有利于蛹虫草虫草酸含量及生物学效率的提高,相比对照组分别提高了7.29%和15.37%;0.25T处理组有利于虫草素的积累,比对照组提高了11.31%;0.1T处理有利于多糖的积累,相比对照组提高了14.80%。方差分析表明,经不同场强处理的磁化水对蛹虫草子实体生长和活性物的代谢影响差异显著。但磁处理水对于不同物质含量的影响规律存在一定的差异性。     

冬虫夏草MYB家族基因鉴定及其在生长发育中的表达分析

MYB蛋白是一类广泛存在于真核生物中的转录因子,在真菌的生长发育中发挥调控作用。本研究对冬虫夏草菌中的潜在MYB转录因子家族基因进行了全基因组鉴定和生物信息学分析,并研究了MYB家族成员在冬虫夏草生长发育不同阶段和不同部位的表达模式。结果表明,冬虫夏草MYB转录因子家族包含3个1R-MYB和3个2R-MYB;6个MYB转录因子蛋白均包含近50个保守氨基酸序列,形成螺旋-转角-螺旋的结构。通过分析MYB转录因子基因在不同发育阶段（MYB-1-6）和子实体不同部位（MYB-1、3、6）的相对表达量,发现MYB-1在冬虫夏草整个发育阶段表达较为稳定,MYB-3在子实体成熟阶段（MF）表达量最高,远高于其他阶段,且在MF的顶部可育部分MF-3高表达,表明其可能参与冬虫夏草子实体的有性发育;MYB-6在子座发育初期（YF）的中部YF-2表达量最高,为菌丝阶段（HY）的5倍,表明MYB-6可能参与子座柄部的伸长。     

Optimization of a solid culture medium based on Monochamus alternatus for Cordyceps militaris fruiting body formation

Monochamus alternatus (Coleoptera: Cerambycidae; M. alternatus), popularly known as the Japanese pine sawyer, is a vector of pinewood nematode (Bursaphelenchus xylophilus) that causes pine wilt disease. A solid medium culture with M. alternatus produced Cordyceps militaris fruiting bodies with the longest strips and the highest biological efficiency. Supplementing the original form of M. alternatus with oats resulted in slightly enhanced fruiting body production. The original form of M. alternatus showed higher production than its powder form. The solid culture medium was optimized using a response surface methodology, and the optimal medium contained the following: 8·5 g per bottle of M. alternatus and 11·5 g per bottle of oats mixed with 22·4 ml of water in a 300-ml cylindrical plastic bottle. The optimal culturing period for the fruiting body formation was 37·1 days. Under these conditions, a fruiting body dry weight of 38·0 g per bottle (actual value) was attained. The fruiting body produced using a solid culture medium based on M. alternatus had a cordycepin content of about 25 µg g⁻¹. The solid culture medium containing M. alternatus is highly efficient and eco-friendly, and its effectiveness in large-scale fruiting body production from C. militaris has been demonstrated.     

不同组织分离期对大圆头蛹虫草菌种性能的影响

为明确大圆头蛹虫草组织分离最佳分离时期,以大圆头蛹虫草优良母种为母本,采用组织分离法制备子代母种,以优良母种为对照,通过对不同组织分离期（35、40、45、50 d）子代母种平板培养、液体培养及栽培实验,考察不同组织分离期对子代母种培养性状及子实体形成能力的影响。结果表明,不同组织分离期大圆头蛹虫草子代母种培养性状存在较大的差异,其子实体形成能力与子代母种培养性状具有显著相关性,以组织分离期为40 d的子代母种性能表现最优,优良菌种筛选率最高（48%）,45 d次之（44%）,50 d最低（32%）。 明确大圆头蛹虫草优良菌种选育组织分离最佳时期为40 d,试验结果为大圆头蛹虫草优质菌种选育、复壮提供参考。     

The medicinal fungus Cordyceps militaris: research and development

Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a highly valued medicinal fungus. This entomopathogen has a limited distribution, has been overharvested in the wild, and its stromata have not been artificially cultivated. Another entomopathogenic fungus, Cordyceps militaris (commonly known as orange caterpillar fungus), has chemical capacities similar to those of O. sinensis, but unlike O. sinensis, its stromata can be easily cultivated. Consequently, C. militaris is being studied as an alternative to O. sinensis, and the large-scale production of stromata is receiving substantial attention. Significant research has been conducted on the genetic resources, nutritional and environmental requirements, mating behavior, and biochemical and pharmacological properties of C. militaris. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biology. The current paper reviews the biological aspects of the fungus including host range, mating system, cytology and genetics, insect- and non-insect nutritional requirements, environmental influence on stroma development, and commercial development.