亚稀褶黑菇和稀褶黑菇的ITS序列分析

对亚稀褶黑菇和稀褶黑菇的ITS全序列进行了测定和比较,首次报道了亚稀褶黑菇的ITS1和5.8S区域。在ITS区域中,不同采集地的亚稀褶黑菇与稀褶黑菇的5.8S rDNA具有100%的同源性,而两侧ITS之间表现出种内和种间的多态性,种内的差异均不超过5%,种间的差异达10%左右。ITS序列分析方法可以作为两者的鉴定方法。     

亚稀褶黑菇和稀褶黑菇的ITS序列分析

对亚稀褶黑菇和稀褶黑菇的ITS全序列进行了测定和比较,首次报道了亚稀褶黑菇的ITS1和5．8s区域。在ITS区域中,不同采集地的亚稀褶黑菇与稀褶黑菇的5．8S rDNA具有100％的同源性,而两侧ITS之间表现出种内和种间的多态性,种内的差异均不超过5％,种间的差异达10％左右。ITS序列分析方法可以作为两者的鉴定方法。     

亚稀褶黑菇对小白鼠肝肾超微结构的影响

对小白鼠用亚稀褶黑菇提取液进行毒性试验．测定了亚稀褶黑菇的半致死剂量LD50值;应用透射电镜观察了小白鼠亚稀褶黑菇中毒后的病理变化,结果表明．亚稀褶黑菇的LD50值为2852．1mg／kg,电镜检查发现。亚稀褶黑菇毒素主要损害肾脏,引起肾细胞核核膜肿胀．线粒体肿胀,溶酶体增多,肾细胞坏死．     

亚稀褶黑菇提取液诱导小白鼠肝肾细胞凋亡

采用琼脂糖凝胶电泳技术研究亚稀褶黑菇提取液诱导小打手势习肝肾细胞凋亡。结果显示,小白鼠亚稀褶黑菇抽提液中毒后,肝肾细胞DNA经琼脂糖凝胶电泳出现180－200bp整数倍的DNA梯形带,19.0-28.5g/L范围内,亚稀褶黑菇提取液诱导肝肾细胞凋亡表现出和剂量依赖性,结果表明,亚稀褶黑菇提取液能诱导小白鼠肝肾细胞凋亡。亚稀褶黑菇提取液诱导肝肾细胞凋亡是亚稀褶黑菇中毒后机体病变死亡的重要表现形式之一。     

亚稀褶黑菇中毒的临床表现和中毒机理探讨

本文通过对湖南省茶陵县亚稀褶黑菇中毒事件的调查,获得了尽的临床资料,并通过分析中毒病人的临床表现,首次探讨了亚稀褶黑菇的中毒机理, 结果表明,亚稀褶黑姑毒素作为一种外界信号分子与细胞膜受体结合,引起细胞内Ｃa^2 浓度上升,通过一系列生理生化反应过程,导致细胞死亡。     

亚稀褶黑菇的化学成分

从剧毒蘑菇─亚稀褶黑菇(Russula subnigricans Hongo)子实体中分离出5个已知化合物,经波谱分析鉴定为4个麦角甾醇(22E,24R)-ergosta-7,22-diene-3β,5α,6α,9α-tetraol(1)、(22E,24R)-ergosta-7,22-dien-3β,5α,9α-trihydroxy-6-one(2)、(22E,24R)-ergosta-7,22-dien-3β,5α,6β-triol(3)、(22E,24R)-5α,8α-epidioxyergosta-6,22-dien-3β-ol(4)和神经酰胺(2S,3S,4R,2′R)-2-(2′-hydroxytetracosanoylamino)octadecane-1,3,4-triol(5)。     

基于Taqman-MGB探针的亚稀褶红菇实时荧光定量PCR检测方法

本研究建立了一种基于Taqman-MGB探针的亚稀褶红菇Russula subnigricans实时荧光定量PCR检测方法。根据亚稀褶红菇与其近似种的内转录间隔区（internal transcribed spacers,ITS）序列差异,设计合成1对引物和1条特异性Taqman-MGB探针,并用常见有毒红菇种类进行验证。结果显示,引物特异性良好,仅亚稀褶红菇出现荧光信号,完成整个检测过程只需2h。该法能够为毒蘑菇中毒的快速检测提供技术支持。     

亚稀褶黑菇菌丝体最适培养基的正交试验研究

以四因素三水平的正交试验设计培养条件,pH5．6,26．2℃,45d,对亚稀褶黑菇茵丝体进行固体培养。通过对菌丝体直径的测定及分析,得到该菌种生长最适培养基配方为：(NH4)2HPO4 1g／1000mL,甘露糖15g/1000mh,KH2PO4,0．5g／1000mL;维生素B1 100μg/1000mL,并证明氮源为其生长的最重要营养因素。     

裸盖菇和斑褶菇属真菌菌种的有效分离方法

【目的】探讨获取裸盖菇属及斑褶菇属真菌纯培养的有效分离方法。【方法】采用菌褶接种法和孢子弹射法进行分离,以形态鉴定为基础,通过ITS区测序并与DNA序列库中已知序列进行比对的分子鉴定方法鉴别分离培养物的真伪,以确定分离方法的可靠性。【结果】对采自云南的28个裸盖菇属和斑褶菇属菌株进行了分离,菌褶接种法有24个菌株分离纯化成功,成功率达86%,而孢子弹射法仅有7个菌株分离成功,成功率为25%。【结论】菌褶接种法对于裸盖菇属和斑褶菇属真菌是一种有效而简便易行的分离方法,该法利用菌褶为产孢组织的优势,无需对菌褶进行表面消毒,易于纯化成功,值得在其他类似的腐生小型薄盖伞菌类群的分离中尝试应用。     

中国的球盖菇科(五) 广义斑褶菇属

通过观察吉林农业大学菌物研究所标本馆(HMJAU)、中国科学院微生物研究所菌物标本馆(HMAS)、广东微生物研究所标本馆(GDGM)及作者野外采集的共180份标本的宏观形态和微观结构,对中国球盖菇科的广义斑褶菇属及其所含的灰斑褶菇属(Copelandia Bres.)、疣孢斑褶菇属(Panaeolina Maire)和狭义斑褶菇属[Panaeolus(Fr.)Qul(s.str.)]进行了分类学研究,对中国分布的16个种进行了形态学描述、显微线条图绘制,并编写了分种检索表。其中,包括1个新组合:栗褶疣孢斑褶菇[Panaeolina castaneifolia(Murr.)Sarentoya et Tolgor];2个中国新记录种:热带灰斑褶菇[Copelandia tropicalis(Ola′h)SingerR.A.Weeks]、小型斑褶菇[Panaeolus alcidisM.M.Moser];12个省级新记录种:北京新记录种硬腿斑褶菇[Panaeolus solidipes(Peck)Sacc.],内蒙古新记录种硬腿斑褶菇[Panaeolus solidipes(Peck)Sacc.]、锐顶斑褶菇[Panaeolus acuminatus(Schaeff.)Qul.]、黑斑褶菇[Panaeolus ater(J.E.Lange)KhnerRomagn.]、紧缩斑褶菇[Panaeolus sphinctrinus(Fr.)Qul.]、红褐斑褶菇[Panaeolus subbalteatus(Berk.Broome)Sacc.],吉林省新记录种半卵圆斑褶菇[Panaeolus semiovatus(Sowerby)S.LundellNannf.]、栗褶疣孢斑褶菇[Panaeolina castaneifolia(Murrill)Bon],黑龙江新记录种红褐斑褶菇[Panaeolus subbalteatus(Berk.Broome)Sacc.],宁夏新记录种变蓝灰斑褶菇[Copelandiacyanescens(Berk.Broome)Singer]、锐顶斑褶菇[Panaeolus acuminatus(Schaeff.)Qul.],西藏新记录种红褐斑褶菇[Panaeolus subbalteatus(Berk.Broome)Sacc.]。     

云南松幼苗上红菇类菌根真菌的物种多样性及其菌根形态

以滇中1～2年生云南松幼苗为研究对象,观察鉴定与其共生的红菇属真菌外生菌根。形态观察发现了6种形态型（morphotypes）。本文对这6种形态型的外观和显微特征进行了详细描述,尤其强调了菌套形态特征。对rDNAITS片段比对分析表明,6种形态型对应6种红菇属真菌,它们分别是与Russula li-vescens、R.violeipes、R.densifolia、R.nigricans、R.sanguinea及R.nauseosa相近的红菇种类。本研究表明,形成的菌根及其菌套和囊状体的特征在红菇属真菌的系统分支间表现出较为稳定的差异。这一研究与前人对该属真菌的菌根形态及分类学研究基本吻合。红菇属真菌是云南松1～2年幼苗期的主要共生真菌类群。     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

Species Diversity of Russuloid Mycorrhizae forming Fungi on Pinus yunnanensis Seedlings and the Mycorrhizal Morphology

Russuloid ectomycorrhizae on 1-2 years old seedlings of Pinus yunnanensis collected from the central Yunnan, China were investigated. Six morphotypes were recognized by macro and anatomical morphological approaches as well as molecular analyses. The six morphotypes were confirmed to represent six phylotypes by matching rDNA ITS sequences. The fungal partners in the six morpho phylotypes are those closely related with Russula livescens, Rvioleipes ,Rdensifolia ,Rnigricans ,Rsanguinea and Rnauseosa respectively. The correlation between the morphology of mantles and cystidia and the phylogenetic clades is further supported in our work. The framework, in which this report is included, shows that russuloid mycorrhizae are one of the most dominant representative ectomycorrhizae formed on 1-2 years seedlings of Pinus yunnanensis.     

枇杷属内栽培种和部分野生种ITS序列分析

对不同栽培区的25种普通枇杷品种以及7种枇杷属野生种的ITS序列进行扩增并测序,采用邻接法和最大简约法进行系统发育树的构建并对枇杷属内不同种间的遗传关系进行了分析。结果表明:枇杷属植物ITS序列ITS1+5.8S rDNA+ITS2总长度为592 bp或594 bp,长度变化发生在ITS2。所有样本的ITS1和5.8S rDNA长度一样,都是223 bp和168 bp;而ITS2为201 bp或203 bp。5种枇杷属野生种的ITS序列长度为594 bp,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;其余2种枇杷属野生种(麻栗坡枇杷、小叶枇杷)和普通枇杷栽培种的ITS序列长度都为592 bp。所有样本ITS序列的GC含量为64.2%~64.5%,其中ITS1为64.1%~65.5%,ITS2为68.1%~72.6%。对所有样本的ITS序列比对产生44个可变位点,其中38个为简约信息位点,其中11个位于ITS1,5个位于5.8S rDNA,22个位于ITS2。最大的种间序列差异为7.7%,最小的种间差异发生在麻栗坡枇杷和小叶枇杷之间,仅为0.2%。普通枇杷种内的ITS序列差异很低,25种普通枇杷栽培种之间的序列差异为0~1.5%。所研究的枇杷属植物可分为3个分支。分支Ⅰ包括所有普通枇杷品种,分支Ⅱ包含5种野生枇杷种,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;分支Ⅲ由2个野生枇杷种(麻栗坡枇杷、小叶枇杷)组成。该研究结果表明ITS序列对枇杷种间鉴定和系统发育分析具有一定意义,但对普通枇杷栽培种间的鉴定作用不大。     

福建青梅rDNA ITS区克隆与序列分析

利用PCR法对青梅ITS1、5.8S、ITS2序列扩增后克隆测序,用软件DNAMAN和MEGA3.1分析测序结果,研究18个福建青梅样品的核糖体ITS碱基序列差异。获得青梅18个样品rDNA中的ITS和5.8S完全序列,ITS1、5.8S和ITS2序列长度分别为223~224bp、164bp和241~246bp,5.8S较为保守。根据测序结果,以UPGMA法建立系统发生树,从分子水平说明18个样品间的变异程度,并将福建青梅差异较大的14条rDNA ITS序列登录GenBank,获得登录号:EF523482-EF523493、EF529435和EF529436。     

Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I-VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type alpha and type beta) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata

The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.     

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。     

基于香菇菌株rDNA-ITS序列的系统发育分析

根据真菌核糖体通用引物ITS1和ITS4扩增出13个福建袋栽香菇主要菌株的5.8S rDNA、ITS序列,对该序列进行测序后,得到完整的5.8S rDNA、ITS序列,将该序列提交NCBI并获得登录号,对该序列进行比对分析并构建了系统发育树,从分子水平对香菇菌株进行了区分鉴定,结果显示13个菌株可以明显的分成2丛,而其他菌株又可以从一丛中延伸出几个亚丛。