1株产纤维素酶木霉菌种的鉴定

对自行筛选分离的1株木霉菌进行形态学及分子生物学鉴定。采用CTAB法抽提其基因组总DNA,利用真菌通用引物ITS1和ITS4扩增菌株rDNA ITS区序列,扩增产物纯化后进行测序。测序结果在GenBank中进行同源性搜索,并下载部分具有代表性种的ITS序列,利用软件MEGA4构建分子系统发育树,通过序列分析,并结合形态学鉴定该菌属于半知菌亚门,丝孢纲,丛梗孢目,木霉属,康宁木霉(Trichoderma koningii)。     

侧耳木霉T2-1植酸酶基因的序列分析及蛋白结构预测

利用GenBank发表的植酸酶A编码序列设计的引物,通过PCR的方法对侧耳木霉(Trichoderma pleuroticola)T2-1基因组DNA进行扩增,获得了一条长约1.7 kb的特异性DNA片段.序列测定结果表明,该DNA片段含有植酸酶编码基因的完整序列和3段内舍子序列,其中植酸酶基因全长1 443 bp,编码480个氨基酸,5'端有一段编码23个氨基酸的信号肤序列,其余的457个氨基酸残基为成熟植酸酶的氨基酸序列.对该基因编码的氨基酸序列进行三级结构预测,发现它为磷酸单酯酶.已将侧耳木霉T2-1植酸酶基因序列在GenBank中注册(登录号:GQ325590).这是目前中国在GenBank注册的第一个完整的木霉植酸酶编码基因.     

核糖体基因ITS作为苎麻疫霉、恶疫霉分类辅助性状的研究

以2个雄器大多围生、少数侧生的苎麻疫霉菌株与1个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ITS）通用引物,PCR扩增3个供试菌株核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果是苎麻疫霉的ITS1和ITS2分别由206和453个碱基组成,而恶疫霉则分别由218和415个碱基组成。2个供试苎麻疫霉菌株的ITS1和ITS2的碱基序列同源性均分别为100%。苎麻疫霉和恶疫霉ITS1同源性为74.9%,其中中间区域40bp-164bp之间在两种间变异丰富,同源性只有59.4%,而1bp-39bp和165bp-239bp两区域的同源性分别为92.3%和92.1%;ITS2在两种疫霉菌间的同源性为71.0%。结果表明苎麻疫霉和恶疫霉ITS的碱基序列有明显差异。上述结果提示,ITS区域碱基序列可区分苎麻疫霉和恶疫霉。     

核糖体基因ITS作为苎麻疫霉、恶疫霉分类辅助性状的研究*

以2个雄器大多围生、少数侧生的苎麻疫霉菌株与1个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ITS）通用引物,PCR扩增3个供试菌株核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果是苎麻疫霉的ITS1和ITS2分别由206和453个碱基组成, 而恶疫霉则分别由218和415个碱基组成。2个供试苎麻疫霉菌株的ITS1和ITS2的碱基序列同源性均分别为100%。苎麻疫霉和恶疫霉ITS1同源性为74.9%,其中中间区域40bp-164bp之间在两种间变异丰富,同源性只有59.4%,而1bp-39bp和165bp-239bp两区域的同源性分别为92.3%和92.1%; ITS2在两种疫霉菌间的同源性为71.0%。结果表明苎麻疫霉和恶疫霉ITS的碱基序列有明显差异。上述结果提示,ITS区域碱基序列可区分苎麻疫霉和恶疫霉。     

长梗木霉纤维素酶基因的克隆及序列分析

从富含纤维素环境筛选获得一株纤维素降解菌株FU05,通过形态学特征及ITS序列分析确定其为长梗木霉(Trichoderma longibrachiatum)。PCR扩增获得该菌株的bgl2、cbh2和eg1。序列分析表明,这3种纤维素酶基因与GenBank上其他木霉同种纤维素酶基因具有较高同源性:bgl2基因与里氏木霉bgl2基因(AB003110)同源性达91%;cbh2基因与康宁木霉cbh2基因(DQ504304)同源性达99%;eg1基因与长梗木霉eg1基因(X60652)同源性达95%。3种纤维素酶基因编码的相应氨基酸序列与其他木霉纤维素酶的氨基酸序列相似性也非常高。对上述纤维素酶基因编码的相应蛋白进行PROSITE motif search,对其N端糖基化位点、纤维素结合区、糖基水解酶家族特征结构区等进行了定位。     

无绿藻PCR ITS1-ITS2基因分型法的建立及应用

目的建立无绿藻(Prototheca)PCR ITS1-ITS2基因鉴定分型方法,并通过系统发育树以研究各型别无绿藻菌株间的亲缘关系。方法抽提5株无绿藻临床分离菌株的DNA,通过特异性引物扩增ITS1及ITS2区后,根据该区域核苷酸片段的差异进行基因分型,借助MEGA6.0软件建立系统发育树,并通过SSU rDNA测序加以验证。结果 5株菌株中,1株鉴定为P.zopfii hydrocarbonea,2株鉴定为P.wickerhamii。虽然由于数据库中缺乏P.zopfii portoricensis菌株ITS序列导致1、2号菌株不能被鉴定,但ITS区系统发育树中这两株菌仍被归为P.zopfii中独立的一簇,与SSU rDNA系统发育树相近,证明此分型方法无误。结论 PCR ITS1-ITS2基因分型法可作为一种有效基因分型方法用于鉴定和监测无绿藻感染。     

长梗木霉内切葡聚糖酶I基因的克隆及其在毕赤酵母中的表达

一株纤维素酶高产菌株经ITS序列鉴定并命名为长梗木霉SSL (Trichoderma longibrachiatum, SSL)。利用RT-PCR的方法从该菌株中克隆出内切-1-4-β-D-葡聚糖酶I的基因 (eg1), 该基因全长1386 bp, 编码461个氨基酸。序列分析表明：该基因序列与T. longibrachiatum egl1基因具有90％以上的同源性。将该基因的成熟肽编码序列插入到Pichia pastoris表达载体ppic9k中, 构建重组表达质粒ppic9k-eg1, 转化P. pa     

木霉属中国新纪录种Trichoderma pleuroticola和T. pleurotum

【目的】对蔬菜大棚土壤中和阿魏菇腐烂的菌盖上分离的两株木霉菌进行分类鉴定。【方法】结合形态学分类特征和ITS序列分析的方法进行鉴定。【结果】从蔬菜大棚的土壤中和阿魏菇腐烂的菌盖上分离的两株木霉菌分别为Trichoderma pleuroticola和T.pleurotum。T.pleuroticola的形态特征与T.harzianum相似,但其分生孢子显著大于T.harzianum的分生孢子,且在PDA上产生黑褐色的色素以及黄色的结晶物。T.pleurotum典型特征是分生孢子梗单生,有时匍匐,分枝散生,初级分枝和分生孢子梗顶端聚生,类似粘帚霉。【结论】分离的两株木霉分别是T.pleuroticola和T.pleurotum,为木霉菌中国新纪录种。     

木霉属2中国新记录种

[目的]明确我国南部地区土壤中分离得到的2株木霉菌的分类地位.[方法]采用PDAm培养基分离木霉菌菌株,通过形态学鉴定和内转录间隔区(ITS)序列、翻译延长因子1-alpha (Tef1-α)部分序列相似性分析对菌株进行初步鉴定;选取MEGA 4.0软件用Bootstrap最大简约法、水平3、重复1 000次来构建ITS、Tef1-α序列系统进化树并分析其亲缘关系.[结果]菌株CM01的ITS序列与GenBank基因库中Trichoderma intricatum strain GJS 02-78 ITS序列同源性高达99％,在ITS序列系统进化树中与模式菌株T.intricatum GJS 02-78、Trichoderma atroviride DAOM 179514亲缘关系最近;其Tef1-α序列与GenBank基因库中Hypocrea intricate strain GJS 02-78 Tef1-α序列同源性高达99％,在Tef1-α序列系统进化树中与模式菌株H.intricata GJS 02-78亲缘关系最近;其形态描述和模式菌株一致.菌株SCGA5003的ITS序列与GenBank基因库中Trichoderma stromaticum strain GJS 97-181 ITS序列同源性高达99％,在ITS序列系统进化树中与模式菌株T.stromaticum GJS 97-179、GJS 97-180、GJS 97-181、GJS 97-182、GJS 97-183亲缘关系最近;其Tef1-α序列与GenBank基因库中T.stromatieum strain GJS 97-183 Tef1-α序列同源性高达94％,在Tef1-α序列系统进化树中与模式菌株T.stromaticum CQSQ1032、GXNN7006亲缘关系最近;其形态描述和模式菌株一致.[结论]结合形态学特征与分子鉴定结果,判定菌株CM01为交织木霉(Trichoderma intricatum Samuels&Dodd/Hypocrea intricata Samuels et Dodd)、SCGA5003 为子座木霉(Trichoderma stromaticum Samuels & Pardo-Schulth/Hypocrea stromatica Bezerra,Costa & Bastos),即发现木霉属2个中国新记录种.     

木霉属补充DNA条形码筛选

木霉属真菌是一类重要的生物资源,在工农业、环境保护等方面具有较高经济价值,对其进行快速、准确的物种鉴定兼具理论意义和应用前景。以木霉属35个概念清晰的种为材料,选择ITS、rpb2和 tef1作为候选基因序列,利用TaxonGap对231个序列片段进行分析,将种内与种间序列差异以及序列获取难易程度作为评价指标,筛选该属的补充条形码片段。结果表明,rpb2具有适宜的种内与种间序列差异,其最小的种间差异（2.48%）,大于最大种内差异（1.8%）,种内、种间遗传距离存在明显的间隔区,并且该基因序列具有较高的PCR扩增与测序成功率（94.4%）;ITS和tef1基因序列的种内与种间序列差异之间存在交叉重叠。因此建议rpb2作为木霉属的补充DNA条形码序列。     

Molecular characterization and identification of biocontrol isolates of Trichoderma spp

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.     

焦磷酸测序检测食品中鼠伤寒沙门氏菌

根据鼠伤寒沙门氏菌的特异序列,分别设计扩增引物和测序引物,建立焦磷酸测序检测鼠伤寒沙门氏菌的方法。针对鼠伤寒沙门氏菌设计特异性扩增引物,对目标片段进行PCR扩增,然后制备单链模板,并利用测序引物进行焦磷酸测序。测序结果表明,6株不同来源的鼠伤寒沙门氏菌均可以扩增出碱基序列为TACAACCGGA GTGCACATTA ATCCCGCAGC的基因片段,而30株阴性对照菌株均未得到扩增。进行BLAST比对表明,该序列与GenBank中鼠伤寒沙门氏菌的碱基序列100%匹配。焦磷酸测序法是一种快速、准确的检测方法,可用于食品中鼠伤寒沙门氏菌的快速检测。     

Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches

Molecular approaches including internal transcribed spacer (ITS) sequences of ribosomal DNA, universal primer polymerase chain reaction (UP-PCR) fingerprinting, and DNA-DNA hybridization were used to study the genetic relatedness of species within Trichoderma sect. Pachybasium. In the analysis of ITS and 5.8S sequences of ribosomal DNA, parsimony analysis demonstrated that forty-one strains were distributed into five main groups supported by high bootstrap values. The species of Trichoderma sect. Pachybasium were clustered into groups I, II, and IV, with the strains of Trichoderma fasculatum and Trichoderma strictipile forming a separate branch, an independent group V. Some species within each group showed nearly identical sequence differences (fewer than 1-3 bp). UP-PCR and DNA-DNA hybridization were further used to clarify the genetic relatedness of these species with highly similar ITS sequences. Highly similar or identical UP-PCR profiles and high values of DNA complementarity (>70%) were observed among some species, Trichoderma hamatum and Trichoderma pubescens; Trichoderma croceum, Trichoderma polysporum and Trichoderma album, Trichoderma crassum and Trichoderma flavofuscum; and Trichoderma strictipile and Trichoderma fasciculatum. Although every species can be differentiated morphologically, the species showed highly similar molecular characteristics in the above cases, indicating that they could be conspecific. However, in some cases (Trithoderma longipile, T. crassum and T. flavofuscum; Trichoderma fertile and Trichoderma minutisporum; Trichoderma tomentosum, Trichoderma inhamatum and Trichoderma harzianum) there were discriminative patterns of UP-PCR and (or) low levels (<50%) of DNA-DNA hybridization; even their ITS sequences were similar, suggesting a closely phylogenetic relationship.     

Phytase activity in some groups of bacteria. Search for and cloning of genes for bacterial phytases

A search for phytase genes in 9 Bacillus strains from the collection of IMGAN was implemented. The growth optimum of strains IX-22, IX-12B, K17-2, K18, IMG I, IMG II, M4 and M8 was 50-60 degrees C; the optimal growth temperature for Bacillus sp. 790 was 45-47 degrees C. According to the sequence data of 16S RNA genes, Bacillus sp. 790 belongs to the B. subtilis/amyloliquefaciens group. The other 8 strains were identified as B. licheniformis. Selection of Bacillus strains, potentially containing the phytase genes, was performed via PCR with primers designed on the basis of the conserved sequence regions of the phyA gene from B. amyloliquefaciens FZB45 with chromosomal DNA being used as the template. The nucleotide sequences of all PCR fragments showed a high level of homology to the known Bacillus phytase genes. The gene libraries of B. licheniformis M8 and B. amyloliquefaciens 790 in E. coli were constructed and phytase-containing clones were selected from them. Twenty-four Pseudomonas strains of different species, 5 Xanthomonas maltophilia strains and 1 Xanthomonas malvacearum (all from the mentioned collection) were tested for phytase activity. Such activity was found in 13 Pseudomonas strains and in 6 Xanthomonas strains. The accumulation of phytase in Pseudomonas was shown to take place at later (over 2 days') growth stages. The optimum pH for phytase from 3 Pseudomonas strains were established. The enzymes were found to be most active at pH 5.5.     

Endochitinase gene-based phylogenetic analysis of Trichoderma

DNA sequences of the single-copy gene coding for the 42 kDa endochitinase enzyme (EC 3.2.1.14) were used for phylogenetic analysis in Trichoderma. A set of 12 primers was developed and the entire gene was sequenced for 16 strains, and nucleotide and deduced amino acid sequences were compared to data from GenBank for additional Trichoderma strains. Analysis of the sequences revealed parsimony informative variation from 2.4 to 43.6% depending on the part of the gene (exons/introns) and the taxonomic level considered. Results are discussed in comparison to previous data from ITS-1 and ITS-2 rDNA sequencing and suggest the 42 kDa endochitinase gene as a potential molecular marker for reconstructing phylogenetic relationships in the genus Trichoderma at species level.     

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。     

平菇产植酸酶菌株的筛选及植酸酶基因区域的克隆

从平菇（Pleurotus ostreatus）8个菌株中筛选出3株高产植酸酶菌株,并根据GenBank中植酸酶基因的保守区设计并合成一对特异性引物,以平菇菌丝的总DNA为模板,通过PCR扩增,获得了一条长约920 bp的片段.DNA序列测定结果表明,该片段长度为919 bp.采用blast进行序列比对,结果表明：该片段与曾报道的源于Trametes pubescens的植酸酶phyA（GenBank Accession：AJ310700）基因相比较,其DNA序列同源性为93%.该片段含有3个内含子,含有植酸酶基因的活性位点保守序列（Active-site sequence）RHGARYPT.     

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.     

木霉属Trichoderma组和Pachybasium组的分子系统学研究

对来源不同的木霉及其有性型Longibrachiatum组、Trichoderma 组和Pachybasium组的81个菌株进行了ITS序列测定,并对ITS1序列用PHYLIP程序包中的DNAPARS程序进行系统发育分析。结果表明Trichoderma 组和Pachybasium组的所有菌株可分成两个群(A,B),B群进一步分为4个分支(B1,B2,B3,B4);A群由Trichoderma 组的H. aureoviridis和未鉴定到种的3个Hypocrea菌株构成;B1,B2,B4群均由Pachybasium组菌株构成;B3群由Pachybasium组的T. hamatum、T. strigosum和Trichoderma 组的T. asperellum、T. atroviride、T. koningii、T. viride和Hypocrea菌株T261构成。2个组相互交叉,组间没有明确的区分,进一步证明Pachybasium组是多系的。建议将Trichoderma 组中的T. viride aggr.、T. atroviride、T. koningii归并入Pachybasium组,对Trichoderma 组重新定义。