深绿木霉中碳代谢抑制因子CRE1功能特性研究

木霉菌(Trichoderma spp.)是一种广泛存在于土壤及植物根系生境中的丝状真菌,具有拮抗植物病原真菌和促进植物生长的双重功效。碳代谢抑制子CRE1全局性调控细胞生长代谢过程,保障木霉在不同生境中的存活及拮抗病原菌特性。比较深绿木霉(T.atroviride)T23及其Cre1突变株(T23Δcre1)在不同培养基中的生长和代谢特性,结果表明:cre1基因沉默后,T23Δcre1较T23菌丝生长变慢,产孢滞后且降低一个数量级,cre1对菌丝生长和产孢的调控依赖于培养基组分。此外,cre1基因抑制几丁质酶、β-1,3-葡聚糖酶基因转录,区别性调控部分次级代谢合成的非核糖体肽合成酶NRPS和聚酮合成酶PKS基因的表达。综上,碳代谢因子CRE1作为一个多效性的转录调控因子,抑制细胞壁降解酶和代谢产物合成相关的基因表达,赋予木霉菌在环境中的适应性和竞争性,是深绿木霉T23生长的必要因子。     

出芽短梗霉菌株PA-2全基因组测序及分析

【背景】出芽短梗霉菌株PA-2是一株分离自青海省海东市平安区患病杨树叶片上的真菌,前期研究表明该菌株具有除草和抑菌能力,说明其在生物农药方面具有潜在的应用前景。【目的】了解菌株PA-2的基因组序列信息,挖掘其生防相关功能基因簇,为进一步研究解析该菌株生防机理及生防功能改造提供遗传背景信息。【方法】利用IlluminaHiSeq高通量测序平台对生防菌株PA-2进行全基因组测序,用生物信息学的方法对测序数据进行基因组组装、基因预测及功能注释、碳水化合物活性酶预测、次级代谢产物合成基因簇预测,利用刚果红染色等方法对水解酶活性进行衡量。【结果】菌株PA-2基因组序列全长28 932 793 bp,平均GC含量为50%,共编码10 839个基因,预测到该菌株具有4个已知的次级代谢产物合成基因簇,编码Melanin、Burnettramic Acid A、ACR-Toxin I、Abscisic Acid,该菌株能水解纤维素和果胶。【结论】有助于在基因组层面上解析菌株PA-2生防机制的内在原因,为深入了解出芽短梗霉菌次级代谢物合成途径提供参考,对菌株PA-2的下一步相关研究具有重要意义。     

三个木霉菌株防病促生效应

【背景】木霉(Trichoderma sp.)是重要的生防微生物资源,对不同的木霉菌株进行生物学特性比较,可为获得功能较广的复配型生防菌剂提供参考信息。【目的】通过对绿木霉(Trichoderma virens) T23、哈茨木霉(Trichoderma harzianum) T22、G30的防病效果及对作物的促生潜力等生物学特性进行比较分析,明确不同菌株的生物功能差异。【方法】用平板拮抗法比较分析菌株T23、T22、G30对植物病原真菌的拮抗效果;用乙酸乙酯萃取法对菌株T23、T22、G30培养液中的胶毒素进行提取并通过高效液相色谱法(high-performance liquid chromatography, HPLC)检测胶毒素;用打孔法比较分析培养液萃取物对植物病原真菌的拮抗效果;通过温室接种验证菌株T23、T22、G30的防病效果;分别以磷酸钙、卵磷脂为唯一磷源检测菌株T23、T22、G30对难溶性磷的转化利用潜力,用电感耦合等离子发射光谱法(inductively coupled plasma-atomic emission spectrometry, ICP-AES)检...     

非生物胁迫因子对棘孢木霉嗜铁素及钙调磷酸酶基因表达的影响

木霉菌(Trichoderma spp.)嗜铁素具有吸收、贮存和胞内运输铁的功能,对菌体自身的生长发育有重要作用,也是重要的生防促生因子,但嗜铁素的产量会受到环境中多种胁迫因子的影响。真菌的钙调磷酸酶基因能够调控外源离子的胁迫反应,但钙调磷酸酶信号通路与嗜铁素合成之间的关系并不明确。以棘孢木霉菌(Trichoderma asperellum)T6为材料,研究了NaCl、CaCl_2及pH对棘孢木霉菌T6嗜铁素产量以及嗜铁素合成关键基因(SidA)与钙调磷酸酶催化亚基基因(CnA)表达量的影响。结果表明,高浓度NaCl和CaCl_2以及高pH均能抑制嗜铁素的产生。利用半定量PCR分析得知,CnA基因与SidA基因之间存在一定的负调控关系,该结果与钙调磷酸酶专性抑制剂环孢菌素A能够提高嗜铁素产量的结果相一致。     

木霉菌对芸芥生长相关生理指标的影响

芸芥(Eruca sativa)是当今我国蔬菜市场中一种颇具开发价值的新特芳香蔬菜。为了研制促进芸芥生长和提高品质的生防木霉菌剂,本研究以芸芥为试材,在大田条件下,采用本实验室分离鉴定的哈茨木霉T8进行浸种和浇根处理,分析木霉菌对一个生长季内连续栽培三茬30 d龄芸芥的生长相关生理指标的影响。结果表明,施用木霉菌能显著提高芸芥的生物量,改善其光合特性,增强其防御酶活性和脯氨酸含量,提高其产量和营养价值。     

链霉菌次级代谢产物高产菌株的构建策略

随着基因测序技术的发展,人类获得了大量有关链霉菌次级代谢产物合成基因簇的信息,通过合理的构建策略可以激活其中的隐性基因簇或提高基因簇的表达水平,从而获得新的链霉菌次级代谢产物,或显著提高已知次级代谢产物的发酵水平。从基因表达调控、转座子突变、合成生物学方法、组学方法等四个方面,综述了提高链霉菌次级代谢产物产量的构建策略及其研究进展。     

Streptomyces sahachiroi ATCC 33158中孢子色素合成基因簇(sah)的鉴定

【目的】Streptomyces sahachiroi ATCC 33158全基因组测序后,通过生物信息学分析找到一个II型聚酮合酶(polyketide synthase,PKS)基因簇sah。我们通过基因敲除和异源表达的方法对sah的生物学功能进行了研究。【方法和结果】对sah基因簇ORF(open reading frame)进行分析后发现,除了一个额外的氧甲基转移酶基因sahI以外,该基因簇与天蓝色链霉菌中负责孢子色素合成的基因簇whiE具有很高的相似性。将sah中负责后修饰的3个基因sahG、sahH和sahI分别敲除后,发现孢子色素的颜色随之发生明显的变化。将sah-minimal PKS基因和whiE-minimal PKS基因分别导入变铅青链霉菌ZX1中进行异源表达,高效液相色谱及液质联用分析证实它们产生相同的水溶性红色色素物质。【结论】sah与whiE基因簇具有相似的生物学功能,负责链霉菌孢子色素的合成。这两个基因簇所合成的孢子色素具有相同的母核,差别在于sah基因簇中多了一个编码氧甲基转移酶的后修饰基因,这可能是导致两种链霉菌孢子在颜色上有细微差别的原因。     

基因组挖掘默诺霉素生物合成基因簇的比较分析

通过对NCBI数据库的检索,使用次级代谢基因挖掘的方法对整个基因组数据进行扫描,发现含有潜在的默诺霉素家族磷酸糖脂类抗生素合成途径基因簇的6株链霉菌:Streptomyces ghanaensis、Streptomyces bambergiensis、 Streptomyces prasinus、 Streptomyces lincolnensis、 Streptomyces. sp. SAT1和Streptomyces clavuligerus。将搜寻获取的候选基因簇与S. ghanaensis中的默诺霉素合成相关基因簇进行比较基因组学分析,从共线性比对、合成基因的进化水平和同源性比对结果的分析,说明默诺霉素合成基因簇存在两种不同的进化来源与途径。其中5株链霉菌的合成基因簇位于染色体的两臂区,该区域常发生染色体插入或缺失的水平转移。通过对移动元件的基因岛序列的预测,发现默诺霉素合成基因簇是由20 kb大小的基因组岛实现了种间水平转移。基因组岛的插入是链霉菌获得新性状的重要途径,可以阐述基因簇在种间转移的途径和进化方向,以生物信息学基因组分析的角度为高产菌株的构建提供数据支持和改造。     

贝莱斯芽孢杆菌生防次级代谢产物研究进展

贝莱斯芽孢杆菌（Bacillus velezensis）是生防芽孢杆菌中的重要代表,作为微生物农药的核心菌种,已被广泛应用于作物病害生物防治。贝莱斯芽孢杆菌具有植物内生性,其生防作用机制主要包括产生次级代谢产物对抗植物病原物;改善宿主植物根际微生物群落,促进宿主营养和生长;激发宿主植物产生防御反应,诱导植物获得系统抗性。其中,产生次级代谢产物是其最重要的生防作用机制。贝莱斯芽孢杆菌含有多个编码生物合成次级代谢产物的基因簇,其中包括编码聚酮化合物合酶（PKS）和非核糖体肽合成酶（NRPS）的基因簇,同时存在核糖体途径合成次级代谢产物基因簇。通过非核糖体途径可产生脂肽类化合物、聚酮类化合物、二肽和铁载体;通过核糖体途径产生小菌素、细菌素、羊毛硫抗生素。这些具有生物活性的次级代谢产物成为了天然新药和候选抗生素的储存库,对于解析生防菌作用机制具有重要意义。本文综述了贝莱斯芽孢杆菌的命名与更迭,产生次级代谢产物的类型、合成与调控基因以及靶标病原菌,以期为生防菌株的改良和生物农药的研发提供参考。     

链格孢菌毒素合成相关基因研究进展

链格孢属真菌(Alternaria Nees)是世界上分布最广泛的真菌之一,可产生9种寄主选择性毒素(host-selective toxins,HST)。根据寄主不同,将链格孢属的7个生理小种称为链格孢菌(Alternaria alternata)的7种致病型。每种致病型产生的HST都是低分子质量的次级代谢产物,只对敏感品种有毒,对抗性品种无毒。参与HST生物合成的基因都是多拷贝的,这些共同表达的基因组成基因簇。各致病型的基因簇位于一条小于2.0Mb的conditionally dispensable(CD)染色体上,CD染色体的不稳定性不影响菌丝的生长和孢子的萌发,但与菌株的致病力有关。从链格孢菌6种致病型得到毒素合成基因,日本梨致病型、草莓致病型、橘致病型有共同的EDA结构域,这3种致病型有很多EDA生物合成需要的同源基因。HST的分子遗传学研究为链格孢菌不同致病型的演化提供了新的见解。     

Identification of loci and functional characterization of trichothecene biosynthesis genes in filamentous fungi of the genus Trichoderma

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function.     

Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene cluster of Aspergillus fumigatus

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic animal pathogen Aspergillus fumigatus. It is a member of the epipolythiodioxopiperazine (ETP) class of toxins characterised by a disulphide bridged cyclic dipeptide. A putative cluster of 12 genes involved in gliotoxin biosynthesis has been identified in A. fumigatus by a comparative genomics approach based on homology to genes from the sirodesmin (another ETP) biosynthetic gene cluster of Leptosphaeria maculans. The physical limits of the cluster in A. fumigatus have been defined by bioinformatics and by identifying the genes that are co-regulated and whose timing of expression correlates with the production of gliotoxin in culture.     

Differential expression of iron-sulfur cluster biosynthesis genes during peach flowering

Iron is required for the Fe-S cluster assembly which occurs in chloroplasts, mitochondria, and cytosol and here we characterized 44 Fe-S cluster biosynthesis genes and investigated their expression profiles during different peach flowering stages. Quantitative real-time PCR analysis shows that the highest expression of most peach Fe-S cluster biosynthesis genes appeared in the full bloom stage. Also, the highest Fe accumulation occurred in the full bloom stage followed by beginning bloom, petal fall, and bud swell stages. Activities of nitrite reductase (NiR) and succinate dehydrogenase (SDH) were closely correlated to the flower Fe content, whereas the aconitase (ACO) activity kept steady during the whole flowering process. Moreover, shading treatment significantly reduced Fe accumulation and NiR, SDH, and ACO activities of the full blooming flowers. Seventeen Fe-S cluster biosynthesis genes were down-regulated in response to a shading treatment. In particular, plastid sulfur mobilization genes were sensitive to the shading treatment.     

Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production

The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in DeltagliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the DeltagliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the DeltagliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.     

Reconstitution of the early steps of gliotoxin biosynthesis in Aspergillus nidulans reveals the role of the monooxygenase GliC

The gliotoxin, a member of the epipolythiodioxopiperazine (ETP), has received considerable attention from the scientific community for its wide range of biological activity. Despite the identification of gliotoxin cluster, however, the sequence of steps in the gliotoxin biosynthesis has remained elusive. As an alternative to the gene knock-out and biochemical approaches used so far, here we report using a heterologous expression approach to determine the sequence of the early steps of gliotoxin biosynthesis in Aspergillus nidulans. We identified the GliC, a monooxygenases that involved in the second step of gliotoxin biosynthesis pathway through the catalyzing the hydroxylation at the α-position of l-Phe.     

Characterization of Cellulase Secretion and Cre1-Mediated Carbon Source Repression in the Potential Lignocellulose-Degrading Strain Trichoderma asperellum T-1

Trichoderma asperellum, a traditional bio-control species, was demonstrated to be an excellent candidate for lignocellulose degradation in this work. Comparing to the representatively industrial strain of Trichoderma reeseiQM6a, T. asperellum T-1 showed more robust growth, stronger spore production, faster secretion of lignocellulose-decomposing enzymes and better pH tolerance. The reducing sugar released by strain T-1 on the second day of fermentation was 87% higher than that of strain QM6a, although the maximum reducing sugar yield and the cellulase production persistence of the strain T-1 were lower. Our experiment found that the cellulase secretion was strongly inhibited by glucose, suggesting the existence of carbon source repression pathway in T. asperellum T-1. The inhibiting effect was enhanced with an increase in glucose concentration and was closely related to mycelium growth. SDS-PAGE and secondary mass-spectrum identification confirmed that the expression of endo-1,4-β-xylanase I in T. asperellum T-1 was down-regulated when glucose was added. The factor Cre1, which plays an important role in the down-regulation of the endo-1,4-β-xylanase I gene, was investigated by bioinformatics methods. The protein structure of Cre1, analyzed using multiple protein sequence alignment, indicates the existence of the Zn-fingers domain. Then, the binding sites of Cre1 on the endo-1,4-β-xylanase I gene promoter were further elucidated. This study is the first report about Cre1-mediated carbon repression in the bio-control strain T. asperellum T-1. All of the above results provided good references for better understanding T. asperellum T-1 and improving its application for lignocellulose degradation.     

Expression profiling analysis for genes related to meat quality and carcass traits during postnatal development of backfat in two pig breeds

The competitive equilibrium of fatty acid biosynthesis and oxidation in vivo determines porcine sub-cutaneous fat thickness(SFT) and intramuscular fat(IMF) content.Obese and lean-type pig breeds show obvious differences in adipose deposition;however, the molecular mechanism underlying this phenotypic variation remains unclear.We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with meat quality and carcass traits in backfat at five growth stages(1―5 months) of Landrace(a leaner, Western breed) and Taihu pigs(a fatty, indigenous, Chinese breed).Variance analysis(ANOVA) revealed that differences in the expression of 25 genes in Landrace pigs were significant(FDR adjusted permutation, P<0.05) among 5 growth stages.Gene class test(GCT) indicated that a gene-group was very significant between 2 pig breeds across 5 growth stages(PErmineJ<0.01), which consisted of 23 genes encoding enzymes and regulatory proteins associ-ated with lipid and steroid metabolism.These findings suggest that the distinct differences in fat deposition ability between Landrace and Taihu pigs may closely correlate with the expression changes of these genes.Clustering analysis revealed a very high level of significance(FDR adjusted, P<0.01) for 2 gene expression patterns in Landrace pigs and a high level of significance(FDR adjusted, P<0.05) for 2 gene expression patterns in Taihu pigs.Also, expression patterns of genes were more diversified in Taihu pigs than those in Landrace pigs, which suggests that the regulatory mechanism of micro-effect polygenes in adipocytes may be more complex in Taihu pigs than in Landrace pigs.Based on a dy-namic Bayesian network(DBN) model, gene regulatory networks(GRNs) were reconstructed from time-series data for each pig breed.These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of adipose metabolism between the two pig breeds;from these results, some potential key genes could be identified.Quantitative, real-time RT-PCR(QRT-PCR) was used to verify the microarray data for five modulated genes, and a good correlation between the two measures of expression was observed for both 2 pig breeds at different growth stages(R=0.874±0.071).These results highlight some possible candidate genes for porcine fat characteristics and provide some data on which to base further study of the molecular basis of adipose metabolism.     

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co-regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.