金针菇功能性蛋白的研究进展

主要综述了近年关于金针菇功能性蛋白,即核糖体失活蛋白、真菌免疫调节蛋白、金针菇毒素(一种成孔溶细胞素)的分离纯化及其性质,它们的生物学作用与作用机制和它们可能的应用及展望。     

金针菇FV908菌株液体培养工艺的研究

通过单因子试验统计分析 ,优化筛选了适于金针菇 (Flammulinavelutipes)FV90 8的适宜培养基和摇瓶培养条件 ,结果表明 ,其适宜的液体培养基组成为玉米粉 5 .0 % ,麸皮 2 .0 % ,KH2 PO4 0 .1% ,MgSO4 ·7H2 O 0 .0 5 % ,10 μgVB1/ 10 0mL ,5 0 μgVB2 / 10 0mL ;适宜的摇瓶培养条件为 :培养基的起始pH 6 .0～ 7.0 ,5 0 0mL摇瓶装量为15 0mL ,接种量为 10 % ,培养温度 2 5℃ ,摇床转速为 12 0r/min ,菌丝干收率 39g/L。     

A possible role of the key enzymes of the glyoxylate and gluconeogenesis pathways for fruit-body formation of the wood-rotting basidiomycete Flammulina velutipes

 Biochemical roles of the representative enzymes involved in carbon metabolism of glucose were investigated in relation to the fruit-body formation of the basidiomycete Flammulina velutipes. Changes in specific activities of the enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate (GLOX) and gluconeogenesis pathways were measured at different stages of development of the fungus. The enzyme activities of malate synthase (MS) and fructose bisphosphatase (FBP) as the key enzymes for the GLOX-gluconeogenesis pathways increased in mycelia during the fruit-body formation. The activities of isocitrate dehydrogenase (IDH) for the TCA cycle and NADP-linked glutamate dehydrogenase (GLTDH (NADP)) for glutamate synthesis increased more markedly. Moreover, the mycelial mat of the cultures producing fruit bodies yielded greater enzyme activities of isocitrate lyase (ICL), MS, FBP, and IDH than that of the cultures that did not produce fruit bodies. These results suggest that the GLOX-gluconeogenesis pathways as well as the glutamate synthesis have a strong correlation with the fruit-body formation of F. velutipes. Received: January 22, 2002 / Accepted: May 10, 2002     

火菇素蛋白的免疫印迹检测*

从金针菇子实体浸提液中提取了抗癌活性物质火菇素,将纯化的火菇素用Freund佐剂乳化后注射到新西兰白兔体内,经数次加强免疫后采血并分离了抗血清,并以抗血清为探针建立了火菇素蛋白的免疫印迹法定性检测方法。应用免疫学方法检测金针菇菌丝体和子实体,结果显示,只在金针菇子实体中发现与火菇素抗体相结合的抗原信号。     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

金针菇基因组测序与组装策略分析

采用二代和三代测序技术分别对金针菇单核体菌株“6-3”进行测序,应用4种组装策略进行基因组的de novo组装,对比组装效果。基因组组装的参数方面,仅使用二代测序组装的效果最差,长度大于10kb的Contig全长只有24.6Mb,Contig N50只有23kb,组装率只有59.27%。采用三代组装二代校正的组装策略效果最好,长度大于10kb的Contig全长为38.3Mb,Contig N50为2.8Mb,组装率高达92.16%。保守单拷贝基因拼接效果方面,4种组装策略获得基因组序列与BUSCO数据库里的担子菌的保守单拷贝基因比对,基因完整性均大于94%。在组装准确性方面,经过PCR扩增、Sanger测序验证,三代组装二代校正的基因组序列完整并且连续,同时序列上碱基的SNP、InDel数量最少。综上所述,三代组装二代校正得到的基因组序列具有Contig N50值大、组装率高、碱基准确性高的特点,是食用菌基因组测序较为理想的方案。     

金针菇HMG-box转录因子鉴定及一个候选基因的差异表达分析

食用菌的原基形成受到环境因子和内源基因的共同调控,转录因子在其生长发育过程中能够起到关键作用。本研究在已测序金针菇单核菌株L11全基因组中鉴定到29个高速泳动蛋白（high-mobility-group box,HMG-box）转录因子编码基因。基于金针菇双核菌株H1123各发育时期转录组数据,筛选到一个与金针菇原基形成相关的HMG-box转录因子基因Fv-hmg。通过同源比对发现Fv-hmg的DNA序列在6个不同金针菇菌株中十分保守,一致性为98.38%,有92个SNP位点。克隆分析表明,该基因长度为1 517bp,含有一个内含子。该基因编码489个氨基酸残基的蛋白序列,含有一个HMG-box结构域。通过实时荧光定量PCR和转录组测序共同分析表明,该基因在金针菇原基时期的表达量比双核菌丝时期高达4倍以上,具有显著性差异（P<0.01）,由此推测该转录因子参与调控金针菇的原基形成。HMG-box转录因子Fv-Hmg在金针菇原基形成过程中的作用仍需进一步研究。     

Cloning and sequencing analysis of Trp1 gene of Flammulina velutipes

The genomic TRP1 gene from basidiomycete Flammulina velutipes was cloned by complementation of yeast Saccharomyces cerevisiae trp1 mutation. Sequencing analysis revealed that the TRP1 gene encoded a single protein consisting of three catalytic functional domains; glutamine amidotransferase, indole-3-glycerol phosphate synthase ) and N-(5'-phosphoribosyl) anthranilate isomerase, in order of NH2-glutamine amidotransferase-indole-3-glycerol phosphate synthase N-(5'-phosphoribosyl) anthranilate isomerase-COOH. The coding sequence of the TRP1 gene was interrupted by a single intron of 48 bases, the position and flanking sequences of which were highly homologous to those of basidiomycete Phanerochaete chrysosporium trpC.     

Comparison of plant and fungal gravitropic responses using imitational modelling

The mechanisms of gravity perception are still hypothetical, but there are sufficient data from experiments with plants to enable mathematical modelling to imitate the behaviour of gravitropic response systems. We have a much less complete picture of gravitropic kinetics in agaric mushrooms. However, some existing mathematical models which imitate plant responses are in principle universal because their conceptual components are not limited to any specific cellular entities. In this work we have used such models to compare plant and fungal gravitropism, using recently acquired kinetic data from the agarics Coprinus cinereius and Flammulina velutipes. The results show striking similarities between plants and fungi. First, it is evident that the basic assumptions of the plant models are logically applicable to fungi. Secondly, the mechanism of bending is the same (differential growth of opposite flanks of the growing organ). Thirdly, the distribution of growth seems very similar: in both plants and fungi growth of the organ is most intensive just behind the apex and is almost absent at the apex and at the base. Fourthly, in both fungi and plants the gravitropic response exhibits a substantial time delay suggesting that many time-consuming processes are involved in reception, transduction and realization of gravitropic stimuli. Important differences in plant and fungal gravitropism kinetics were: (i) the agaric stem apex always returned to the vertical, whereas some plant organs show stable plagiogravitropic growth; (ii) inflections were usually seen in C. cinereus stem gravitropism time courses suggesting that a curvature compensation process delayed bending for a time; (iii) C. cinercus stems very rarely overshot or oscillated around the vertical although many plant subjects oscillate and the (limited) data for F. velutipes showed a single, exaggerated overshoot and oscillation. In this latter case, experimental modelling with parameters characteristic of a low level of perception improved the fit to the F. velutipes data, indicating that the two fungi may differ in this factor. Application of the plant models focused future research attention on the urgent need for data bearing on angle-response and acceleration–response relationships in fungi, and their detection–level thresholds for gravitational acceleration. Since the modelling also highlighted some fundamental kinetic differences between the only two fungi for which sufficient data are available at the moment, it is also clear that detailed observations need to be made of gravitropism kinetics in a larger number and wider range of fungi.     

一种金针菇核糖体失活蛋白的分离纯化研究

获得一种为研究其他菌类核糖体失活蛋白的对照品,并论述一种金针菇核糖体失活蛋白的分离纯化及其活性的研究结果。实验中采用了DEAE和CM-离子交换纤维素与Bio-Gel 100柱层析方法。从1 000 g新鲜金针菇中得到5.58 mg的核糖体失活蛋白-Velutin,并证明其具有明显的抑制蛋白质的翻译作用,同时简要介绍了它的应用及展望。分离纯化到具有活性的Velutin,分子量为13.8 ku。