白灵侧耳漆酶分离纯化及其酶学性质研究

为了探索白灵侧耳Pleurotus eryngii var.tuoliensis漆酶性质,以白灵侧耳菌株00485为试验材料,从发酵液中分离纯化得到胞外漆酶并对其酶学性质进行测定。纯化流程依次为DEAE-Cellulose阴离子交换层析,CM-Cellulose阳离子交换层析,SP-Sepharose强阳离子交换层析以及Superdex 75凝胶过滤层析,获得胞外白灵侧耳漆酶(Pn Lac)。SDS-PAGE检测结果表明Pn Lac为65k Da的单一蛋白。Pn Lac经过胰蛋白酶水解得到3种肽段,经过NBCI-BLAST后发现它们与糙皮侧耳、环柄韧伞、刺芹侧耳等的漆酶具有同源性。底物为2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)时该种漆酶的最适反应温度和p H分别为50℃和3.0,Ca2+和Hg2+能够抑制它的活性,相反地Cu2+和Mn2+能够提高它的活性,米氏常数Km和Vmax分别是0.17mmol/L和1.76OD/min/U。     

Enhancement of the antioxidants ergothioneine and selenium in Pleurotus eryngii var. eryngii basidiomata through cultural practices

Antioxidants are molecules that may reverse, prevent or slow cellular damage caused by free radicals. Increasing dietary intake of antioxidants is thought to reduce oxidative stress that may contribute to the development of several diseases. Mushrooms are known to contain antioxidants such as selenium, ergothioneine and phenolics that may serve this role. Here we sought to enhance selenium and ergothioneine concentration in Pleurotus eryngii var. eryngii basidiomata by modifying the techniques used for their commercial cultivation. To enhance selenium content in mushrooms, substrates were supplemented with sodium selenite (Na₂SeO₃) to reach selenium concentrations of 5 and 10 μg/g. Basidiomata of one commercial isolate (WC888) accumulated selenium up to 4.6 and 9.3 μg/g (d.w.), respectively. Therefore, a serving size (85 g) of fresh P. eryngii mushrooms produced on substrates supplemented with 5 and 10 μg/g of Na₂SeO₃ would supply 70.4 and 116.3% of the daily value of selenium (DV = 70 μg), respectively. Since selenium-enriched mushrooms would supply more than 20% of the DV, they could be considered an excellent source of selenium. Ergothioneine concentration was enhanced in mushrooms produced on low-moisture (55%) substrate compared to the commonly used 60% (high-moisture) in commercial cultivation. Mushrooms produced on low-moisture substrate had ergothioneine concentrations of 3.0 mg/g, while mushrooms produced on high-moisture substrate contained 2.2 mg/g or less. Use of a casing overlay for mushroom production resulted in significant yield increases on low-moisture substrate but not on high-moisture substrate.     

Interspecific Genetic Variability of the Oyster Mushroom Pleurotus ostreatusAs Revealed by Allozyme Gene Analysis

Allozyme variation in natural populations of basidiomycete fungus Pleurotus ostreatus (88 individuals) from three regions of central Russia was studied. The species was shown to have 92.86% of polymorphic allozyme loci and expected heterozygosity H _e = 0.49. The mean number of alleles per locus was 3.5. The genetic differences among populations were supported by F-statistics (F _ST = 0.750). The low level of inbreeding (F _IS = 0.018) suggests that the P. ostreatus populations are panmictic, and the main reproduction mode involves basidiospores dispersing at long distances. Using cluster analysis, geographically isolated populations and intersterile groups were differentiated within the complex P. ostreatus species.     

不同温度条件下白灵菇、杏鲍菇及其杂交菌株的生长情况

对5份白灵菇菌株、5份杏鲍菇菌株和3份杂交菌株进行了栽培出菇试验,比较分析了不同温度条件下白灵菇、杏鲍菇及其杂交菌株的生长情况。试验结果表明,在4~24℃范围内,白灵菇与杏鲍菇的菌丝体生长速度与温度的变化呈正相关,最适合菌丝体生长的温度为22℃。4~24℃范围内,白灵菇的原基形成周期与温度呈V型曲线变化,12~14℃原基形成周期最短;杏鲍菇的原基形成周期与温度呈负相关,随着温度的升高原基形成周期变短,22~24℃原基形成周期最短。3份杂交菌株11D、53D、70D的原基形成周期与其对应亲本白灵菇菌株的原基形成周期差异极显著(P0.01)。白灵菇子实体形成最短周期所需温度为12~14℃,而杏鲍菇子实体形成最短周期所需温度为22~24℃,3份杂交菌株11D、53D、70D的子实体形成周期与其对应亲本白灵菇菌株的子实体形成周期差异极显著(P0.01)。     

Molecular genetic analysis of two taxa of the Pleurotus eryngii complex: P. eryngii (DC.Fr.) Quèl. var. eryngii and P. eryngii (DC.Fr.) Quèl. var. ferulae

With the use of isozymes and PCR-fingerprinting analysis molecular markers were found between the varieties Pleurotus eryngii var. eryngii and P. eryngii var. ferulae within the Pleurotus eryngii complex, which allowed the identification of the fruitbodies collected in Southern and Central Italy. The study of sympatric localities has shown that there is no gene exchange between them in the field. The post-mating barriers between these taxa are not yet completely efficient. However, in the field the gene pools of the two taxa appear isolated and associated with specific host plants: Eryngium campestre and Ferula communis . On the basis of the genetic and ecological differences observed and given the absence of gene exchange in sympatric localities, P. eryngii and P. ferulae are to be considered distinct biological species. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 75 , 125–136.     

白灵侧耳(白灵菇)杂交育种研究进展

白灵菇是与杏鲍菇亲缘关系相近的侧耳属食用菌新品种,也是近年来开发栽培成功的集食用、药用、食疗于一体的大型珍稀食用菌品种。文中通过生物学特性、交配型基因、杂交育种技术3个方面综述了白灵侧耳杂交育种方面研究进展,讨论并分析了通过杂交育种综合优异性状,去除不利性状培育出更好的蘑菇新品种的可能性,为探讨及研发白灵菇杂交育种及其多倍化材料在生物学进化上的保守性提供理论参考,为解决我国食药用菌产业目前存在的瓶颈问题提供新的思路。     

白灵侧耳栽培种质对高温胁迫的反应研究

本文以“中农一号”、“华杂13”、“中农翅鲍”和“CCMSSC 00488”4个白灵侧耳菌株为材料,通过测定高温胁迫下菌丝体内硫代巴比妥酸反应物（thiobarbituric acid-reactive substances,TBARS）和蛋白质羰基（protein carbonyl,PCO）含量,观察适温培养和高温胁迫后恢复培养时菌落的生长速率、生长势和菌丝形态特征,研究不同白灵侧耳栽培种质对高温胁迫的反应。研究表明,白灵侧耳不同栽培种质对高温胁迫的反应在氧化损伤程度、菌落形态和菌丝形态特征上都有变化,不同材料的高温伤害程度有着较显著的差异。4个材料高温胁迫条件下TBARS和PCO含量均显著升高,但是升高程度不同,按照TBARS和PCO含量多少排序,依次为CCMSSC 00488>华杂13>中农翅鲍>中农一号。高温胁迫处理后恢复生长需要的时间依次为：CCMSSC 00488>华杂13>中农翅鲍、中农一号;菌落生长势：中农一号>中农翅鲍=华杂13>CCMSSC 00488;菌丝形态特征：菌丝表面增长率、菌丝顶端细胞表面积、菌丝直径和菌丝体分支频率均显著降低,但降低程度以中农一号为最大。菌丝体分支频率与菌落生长势相对应。     

Nitric oxide alleviates heat stress-induced oxidative damage in Pleurotus eryngii var. tuoliensis

High temperature is one of the major impediments limiting the growth and development of most edible fungi. While many efforts have been made in agricultural practice, the mechanism for resistance to high temperature remains elusive. Nitric oxide (NO) is considered as a signaling molecule involved in regulation of diverse physiological processes and stress responses in animals and plants. However, the role of NO in regulating fungal, particularly edible fungi, response to abiotic stresses, is unknown. The present study demonstrated that NO could effectively alleviate oxidative damage induced by heat stress in mycelia of Pleurotus eryngii var. tuoliensis. Heat stress induced increased thiobarbituric acid reactive substance (TBARS) content in mycelia, and the NO donor sodium nitroprusside (SNP) dramatically decreased TBARS content under high temperature. Moreover, the specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxyl-3-oxide (cPTIO), could arrest the SNP action under the stress. Heat stress induced an increase in endogenous NO production in mycelial cells. However, the effect was significantly blocked by the NO synthase (NOS) inhibitor l-N(G)-nitroarginine methyl ester (l-NAME). In contrast, nitrate reductase (NR) activities were not obviously altered during heat stress. The NR suppressor tungstate had no effect on intracellular NO abundance under heat stress. These results suggest that NO can effectively protect mycelia of edible fungi from heat stress-induced oxidative damage and the NOS-dependent NO production may participate in the response to heat stress.     

外源一氧化氮提高白灵侧耳菌丝耐热性生化途径分析

为了探索外源一氧化氮（NO）提高食用菌菌丝体耐热性的生化途径,以白灵侧耳Pleurotus eryngii var. tuoliensis菌株CCMSSC 00489为材料,通过测定高温胁迫下外源添加硝普钠（sodium nitroprusside,SNP,NO供体）后,菌丝体内超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）和过氧化物酶（POD）等4个抗氧化酶活性的变化,研究外源NO在高温胁迫响应中对抗氧化酶的影响。试验表明,高温胁迫致使菌丝体内TBARS含量升高,膜脂过氧化加剧。在正常温度培养（CK）下,外源添加SNP无显著缓解膜脂过氧化的效果,而高温胁迫条件下缓解效果显著,高温胁迫6h和12h,TBARS含量较对照（未添加）分别下降31.5%和25%。研究表明,抗氧化酶类对外源NO的响应不同。在有外源添加SNP的高温胁迫条件下,菌丝体内的SOD、CAT和GR活性随处理时间的延长而显著增强,在处理72h达到最高,分别是对照（0h）的1.73、7.29和4.95倍。其中CAT是高温胁迫响应的主要抗氧化酶类,其活力可以mmol/L·min^-1·mg^-1 of protein计量,而其他种类的活力均仅以μmol/L·min^-1·mg^-1 of protein计量。在试验条件下,这些抗氧化酶类活性的提高与TBARS含量的降低相呼应,表明外源NO通过提高SOD、CAT、GR的活性降低高温胁迫下的活性氧水平,缓解其氧化损伤,提高菌丝体耐热性。POD活性在外源添加SNP的高温胁迫条件下显著降低。     

刺芹侧耳自交S1代若干性状的遗传分化

本文选用刺芹侧耳Pleurotus eryngii24号(广杏)为自交材料,对其自交S1代群体的质量性状(颜色、畸形、生长特性)和数量性状(形状、产量、大小、数目)进行了综合分析。分析结果表明:自交后代的出菇率为70%,自交导致后代群体菌丝平均生长速度和平均产量都低于亲本,16%的后代菌株单产高于亲本,最高单产达204.0g,高出亲本45.7%。木屑培养基上菌丝日均生长速度与产量无显著相关性(R=-0.028)。均菇数目与单产成显著正相关性(R=0.543)。大部分菌株(52%)菌盖颜色为亲本颜色(灰色),自交后代出现20%非亲本形状(棒状)菌株,畸形菇占19%。后代群体盖径、柄径、柄长的变异程度相当,差异性不显著。可根据育种目标对自交子代的较优菌株进行定向选择利用。     

刺芹侧耳Pleurotus eryngii自交S1代若干性状的遗传分化

本文选用刺芹侧耳Pleurotus eryngii 24号(广杏)为自交材料,对其自交S1代群体的质量性状(颜色、畸形、生长特性)和数量性状(形状、产量、大小、数目)进行了综合分析.分析结果表明:自交后代的出菇率为70%,自交导致后代群体菌丝平均生长速度和平均产量都低于亲本,16%的后代菌株单产高于亲本,最高单产达204.0g,高出亲本45.7%.木屑培养基上菌丝日均生长速度与产量无显著相关性(R=-0.028).均菇数目与单产成显著正相关性(R=0.543).大部分菌株(52%)菌盖颜色为亲本颜色(灰色),自交后代出现20%非亲本形状(棒状)菌株,畸形菇占19%.后代群体盖径、柄径、柄长的变异程度相当,差异性不显著.可根据育种目标对自交子代的较优菌株进行定向选择利用.     

中国白灵侧耳自然群体的交配型因子分析

为了研究我国新疆白灵侧耳自然种群的遗传多样性,估测自然种群的遗传丰度,对51株新疆野生白灵侧耳的84个原生质体单核体的交配型因子进行分析。在84个单核体中存在54个不同的A因子和59个不同的B因子。x2检测表明交配型因子A和B的系列因子均为等概率分布。据此估算我国白灵侧耳自然群体中有79个A因子,100个B因子,自然群体中的单核交配型总数79×100=7,900个,双核交配型总数31,201,050个。可见,新疆白灵侧耳遗传多样性丰富,新疆是我国白灵侧耳的野生种质资源宝库。     

The Genetic Structure of Natural Populations of DROSOPHILA MELANOGASTER. Xi. Genetic Variability in a Local Population

Six hundred and ninety-one second chromosomes were extracted from a Raleigh, North Carolina population, and the following experimental results were obtained: (1) Salivary gland chromosomes of all lines were observed and the number of inversion-carrying chromosomes was 130, among which 76 carried In(2R)NS, 36 carried In(2L)t, 4 carried In(2L)t and In(2R)NS, and 14 carried different kinds of rare inversions. (2) Viabilities of homozygotes and heterozygotes were examined. The frequency of lethal-carrying chromosomes was 275/691 (or 0.398):70/130 (or 0.538) in inversion-carrying chromosomes and 205/561 (or 0.365) in inversion-free chromosomes. The former is significantly higher than the latter. The average homozygote viability was 0.4342 including lethal lines and 0.7163 excluding those, the average heterozygote viability being 1.0000. The detrimental load to lethal load ratio (D:L ratio) was 0.334/0.501 = 0.67. The average viability of lethal heterozygotes was less than that of lethal-free heterozygotes, significantly in inversion-free individuals but not significantly so in inversion-carrying individuals. Inversion heterozygotes seem to have slightly better viability than the inversion-free heterozygotes on the average, but not significantly so. (3) The average degree of dominance of viability polygenes was estimated to be 0.293 +/- 0.071 for all heterozygotes whose component chromosomes had better viabilities than 0.6 of the average heterozygote viability, 0.177 +/- 0.077 for inversion-free heterozygotes and 0.489 +/- 0.082 for inversion heterozygotes. (4) Mutation rates of viability polygenes and lethal genes were estimated on the basis of genetic loads and average degrees of dominance of lethal genes and viability polygenes. Estimates were very close to those obtained by direct estimation. (5) Possible overdominance and epistasis were detected, but the magnitude must be very small. (6) The effective size of the population was estimated to be much greater than 10,000 by using the allelism rate of lethal-carrying chromosomes (0.0040) and their frequency.-On the basis of these findings and the comparison with the predicted result (Mukai and Maruyama 1971), the mechanisms of the maintenance of genetic variability in the population are discussed.     

Genetic Diversity of the Edible Mushroom Pleurotus sp. by Amplified Fragment Length Polymorphism

Pleurotus strains are the most important fungi used in the agricultural industry. The exact characterization and identification of Pleurotus species is fundamental for correct identification of the individuals and exploiting their full potential in food industry. The amplified fragment length polymorphism (AFLP) method was applied for genomic fingerprinting of 21 Pleurotus isolates of Asian and European origin. Using one PstI restriction endonuclease and four selective primers in an AFLP assay, 371 DNA fragments were generated, including 308 polymorphic bands. The AFLP profiles were found to be highly specific for each strain and they unambiguously distinguished 21 Pleurotus sp. fungi. The coefficient of Jaccard's genome profile similarity between the analyzed strains ranged from 0.0 (Pleurotus sp. I vs. P. sajor-caju 237 and P. eryngii 238) to 0.750 (P. ostreatus 246 vs. P. ostreatus 248), and the average was 0.378. The AFLP-based dendrogram generated by the UPGMA method grouped all the Pleurotus fungi studied into two major clusters and one independent lineage located on the outskirt of the tree occupied by naturally growing Pleurotus species strain I. The results of the present study suggest the possible applicability of the AFLP-PstI method in effective identification and molecular characterization of Pleurotus sp. strains.     

The Genetic Structure of Natural Populations of DROSOPHILA MELANOGASTER. Xvii. a Population Carrying Genetic Variability Explicable by the Classical Hypothesis

About 400 second chromosomes were extracted from the Aomori population, a northernmost population of D. melanogaster on Honshu in Japan, and the following experimental results were obtained. (1) The frequency of lethal chromosomes was 0.23. (2) The effective size of the population was estimated to be about 3000, from the allelism rate of lethal chromosomes and their frequency. (3) The detrimental and lethal loads for viability were 0.243 and 0.242, respectively, and the D/L ratio became 1.00. (4) The average degree of dominance for mildly deleterious genes was estimated to be 0.178 ± 0.056. (5) Additive (σ²_A) and dominance (σ²_D) variances of viability were estimated to be 0.00276 ± 0.00090 and 0.00011 ± 0.00014, respectively. (6) There was no significant difference in environmental variances between homozygotes and heterozygotes. Using these estimates, we discuss the maintenance mechanisms of genetic variability of viability in the population. The mutation-selection balance explained these experimental results.     

新疆阿魏菇与杏鲍菇中氨基酸含量的分析研究

对新疆阿魏菇与杏鲍菇的氨基酸成分进行系统分析,结果显示新疆本地的阿魏菇和杏鲍菇中17种氨基酸含量丰富,种类齐全,阿魏菇中氨基酸总量为16.80%高于杏鲍菇中氨基酸总量(14.34%);两种菇中必需氨基酸含量(EAA)占总氨基酸含量的比例分别为34.72%和37.92%。新疆阿魏菇与杏鲍菇种富含人体必需的氨基酸,可作为饮食氨基酸来源的重要补充。     

IGS2-RFLP、SCoT和ISSR在白灵侧耳遗传多样性分析中的比较研究

应用IGS2-RFLP、SCoT和ISSR标记分析新疆5个采集地点的28个白灵侧耳野生样本的遗传多样性,比较这3种分子标记在白灵侧耳种质资源遗传多样性研究中的效用。结果显示,IGS2-RFLP的3个内切酶、5个SCoT引物、5个ISSR引物分别检测到42、59和77条多态性条带,多态性比率分别为91.3%、92.4%和92.8%。3种标记检测出的有效等位基因数(ne)和位点平均的预期杂合度(Hep)没有显著性差异。表明3种标记都适宜作为遗传学标记对白灵侧耳野生种质进行多样性分析。多态性检测效率最高的标记为ISSR,E=15.4,Ai=23.4,其次为IGS2-RFLP,E=14.0,Ai=22.4,SCoT则为最低,E=11.8,Ai=18.2。3种标记中,SCoT和ISSR标记的聚类结果极其相似,且均能较为准确地反映样本的地理来源,虽然二者的聚类图不完全相同。IGS2-RFLP的标记效率较高,准确性和可重复性最好,可用于菌株标准图谱的制作,更适用于品种权保护中的菌株鉴定鉴别;SCoT和ISSR标记的多态性高,信息量大,评价范围广,则更适用于白灵侧耳种质资源的遗传多样性研究。     

Genetic Variability and Population Structure of Disanthus cercidifolius subsp. longipes (Hamamelidaceae) Based on AFLP Analysis

Disanthus cercidifolius subsp. longipes is an endangered species in China. Genetic diversity and structure analysis of this species was investigated using amplified fragments length polymorphism (AFLP) fingerprinting. Nei''s gene diversity ranged from 0.1290 to 0.1394. The AMOVA indicated that 75.06% of variation was distributed within populations, while the between-group component 5.04% was smaller than the between populations-within-group component 19.90%. Significant genetic differentiation was detected between populations. Genetic and geographical distances were not correlated. PCA and genetic structure analysis showed that populations from East China were together with those of the Nanling Range. These patterns of genetic diversity and levels of genetic variation may be the result of D. c. subsp. longipes restricted to several isolated habitats and “excess flowers production, but little fruit set”. It is necessary to protect all existing populations of D. c. subsp. longipes in order to preserve as much genetic variation as possible.     

Genetic Variability among Strains of the Entomopathogenic Nematode Steinernema feltiae

A systematic program of genetic improvement was initiated by assessing the phenotypic variation of Steinernema feltiae strains for two traits assumed to limit efficacy: ultraviolet tolerance and host-finding ability. All of the strains assayed showed both low ultraviolet tolerance and poor host-finding ability, indicating that the likelihood of improving these traits through more extensive population sampling is remote. Limited genetic variation was detected among the strains for tolerance to ultraviolet, suggesting that selective breeding for increased tolerance would be inefficient. By contrast, highly significant phenotypic differences were found with regard to host-finding ability, suggesting that this trait would be responsive to selection. A genetically heterogeneous population was constructed by round-robin mating of 10 strains; it will serve as the foundation population for selective breeding.     

Genetic Diversity and Population Genetic Structure Analysis of Echinococcus granulosus sensu stricto Complex Based on Mitochondrial DNA Signature

The genetic diversity and population genetics of the Echinococcus granulosus sensu stricto complex were investigated based on sequencing of mitochondrial DNA (mtDNA). Total 81 isolates of hydatid cyst collected from ungulate animals from different geographical areas of North India were identified by sequencing of cytochrome c oxidase subunit1 (coxi) gene. Three genotypes belonging to E. granulosus sensu stricto complex were identified (G1, G2 and G3 genotypes). Further the nucleotide sequences (retrieved from GenBank) for the coxi gene from seven populations of E. granulosus sensu stricto complex covering 6 continents, were compared with sequences of isolates analysed in this study. Molecular diversity indices represent overall high mitochondrial DNA diversity for these populations, but low nucleotide diversity between haplotypes. The neutrality tests were used to analyze signatures of historical demographic events. The Tajima’s D test and Fu’s FS test showed negative value, indicating deviations from neutrality and both suggested recent population expansion for the populations. Pairwise fixation index was significant for pairwise comparison of different populations (except between South America and East Asia, Middle East and Europe, South America and Europe, Africa and Australia), indicating genetic differentiation among populations. Based on the findings of the present study and those from earlier studies, we hypothesize that demographic expansion occurred in E. granulosus after the introduction of founder haplotype particular by anthropogenic movements.