宁南山区典型植物根际与非根际土壤微生物功能多样性

选择宁南山区9种典型植物的根际与非根际土壤为研究对象,采用Biolog方法对土壤微生物功能多样性进行了研究。结果表明:9种不同植物根际土壤与非根际土壤的微生物活性(AWCD)、微生物多样性指数和微生物均匀度指数均存在明显差异;除冰草外,其他各种植物的根际土壤的微生物活性AWCD、微生物多样性指数和微生物均匀度指数均比非根际土壤的高;9种典型植物根际土壤微生物主要碳源利用类型是羧酸类和氨基酸类,非根际土壤微生物主要碳源利用类型是羧酸类、胺类、氨基酸类;微生物活性、微生物多样性指数和微生物均匀度指数两两之间均达到了极显著相关,与土壤化学性质各指标之间均未达到显著相关水平。     

伊犁野生羊肚菌根际土壤微生物功能多样性分析

为研究羊肚菌根际土壤微生物的多样性,采用Biolog-ECO法分析羊肚菌不同深度(土层0～10 cm、土层10～20 cm、土层20～30 cm)土壤根际微生物的多样性;结果表明,培养72 h后,微生物的颜色平均变化率(AWCD)随培养时间的延长而增加,符合微生物培养生长曲线;微生物的代谢活性、对碳源的利用能力、AWCD、多样性指数U、多样性指数H′、丰富度指数S、优势度指数D等随土层加深而减弱,且上层土壤微生物的AWCD、U、H′、S、D等指数显著高于中、下层的;均匀度指数随土层加深而增强,均匀度指数越高,群落一致性越好,多样性则越低,因此羊肚菌根际土壤微生物功能多样性随土层加深而减小,上层具有较高的多样性。     

独山子区优势草本植物根际与非根际土壤微生物功能多样性

以独山子区3种优势草本植物的根际与非根际土壤为研究对象,采用Biolog-ECO微平板法对土壤微生物功能多样性进行了研究。结果表明:3种不同植物根际与非根际土壤微生物代谢活性(AWCD)、丰富度指数Shannon(H)和均匀度指数Mc Intosh(U)均存在不同差异,且博乐蒿根际土壤微生物功能多样性均优于非根际土壤及其他两种植物;根际土壤微生物对糖类、脂类、酸类和胺类碳源物质比较敏感,非根际土壤微生物敏感于酸类、氨基酸类以及糖类碳源,根际土壤微生物对碳源的利用能力更强,且不同植物根际环境微生物碳源利用特征不同;微生物活性、丰富度指数和微生物均匀度指数与土壤pH值、SOM、AP和NO-3-N存在显著正相关(P0.05);博乐蒿根际土壤养分含量与微生物活性均较高,对环境的适应性更强,在独山子区生态环境管理与建设中可对其进行关注。     

不同药用植物根际土壤原核微生物多样性研究

为研究不同药用植物根际土壤中的原核微生物多样性,分别采集白术(Atractylodes macrocephala)、白芍(Paeonia sterniana)、牡丹(Paeonia suffruticosa)、玄参(Scrophularia ningpoensis)四种药用植物的根际土壤以及非种植区的土壤,针对16S rRNA基因的V3～V4区进行测序,分析土壤细菌群落的组成。结果表明,药用植物根际土壤中的细菌群落多样性指数显著高于非种植区土壤。五组样本的优势类群差异不大,总体相对丰度较高的有变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、芽单胞菌门(Gemmatimonadetes)、绿弯菌门(Chloroflexi)等,药用植物根际中的放线菌相对丰度高于非种植区。属水平上四种药用植物根际细菌和非种植区的群落结构有较大差异,四种中药材的根际土壤中各自富集了特异性的有益细菌属。药用植物根际土壤中的NMD1、Dongia、Gaiella、Streptomyces等相对丰度高于非种植区,而非种植区土壤中Lysoba...     

不同种植年限香榧根际土壤微生物多样性

为探明不同种植年限对香榧根际土壤微生物群落特征的影响,采用高通量测序技术,分析种植5 a、10 a和15 a香榧根际土壤细菌、真菌的群落结构和多样性特征.结果表明: 在种植15 a的香榧土壤中细菌Chao1指数、ACE指数和Shannon指数显著降低,Simpson指数无显著变化.NMDS分析显示,种植年限对细菌群落结构变化有显著影响,而种植5 a和10 a香榧林地土壤具有相似的细菌群落.细菌相对丰度、多样性以及群落结构(基本上由变形菌、放线菌、酸杆菌和绿弯菌组成)的变化与有机质、C/N、全氮呈极显著相关.香榧根际土壤真菌Chao1指数和ACE指数随种植年限的增加显著降低,Shannon指数和Simpson指数在种植10 a香榧林地中较高.真菌NMDS分析显示,相同种植年限土壤真菌群落聚在一起,不同种植年限之间能明显分开.真菌优势菌群主要包括子囊菌门、担子菌门、接合菌门.有机质是影响真菌丰富度、多样性和群落结构变化的主要因子.综上,香榧根际土壤微生物群落随种植年限不同而发生明显变化,种植年限、C/N、土壤全氮和有机质含量是影响香榧根际土壤微生物群落结构的主要因子.     

不同生境马铃薯根际土壤细菌多样性分析

近年来,随着国家对马铃薯产业重视程度的增加,生产的专业化和规模化程度越来越高,化肥农药用量不断加大,加之连作等种植模式,致使以疮痂病为代表的土传病害普遍发生且逐年加重,个别地块发病率达90%以上,给种植业者带来巨大的经济损失。[目的] 为了探索马铃薯疮痂病发生与土壤环境的关系,分析区域性种植方式和施肥量对土壤细菌种群变化的影响,为实现土传病害有效防治提供借鉴。[方法] 本文分别从1年连作土传病害轻的宁夏西吉（西北）、3年连作土传病害严重的河北沽源（华北）、5年轮作未发现土传病害的内蒙古海拉尔（东北）大田马铃薯根际采集土壤,利用高通量测序技术,比较了样品间细菌群落结构差异。[结果] 3组样品共获得有效条带617558条,可分类操作单元（OTUs）3077个。各样品中变形菌门（Proteobacteria）数量最多,含量均在33%以上。与未发病土壤样品相比,土传病害发生严重的样品中细菌数量、物种数、细菌多样性、种类丰富度均有所降低,有害菌数量增加,益生菌数量减少。其中,放线菌（Actinobacteria）数量明显增多,变形菌（Proteobacteria）、绿弯菌（Chloroflexi）和酸杆菌（Acidobacteria）数量明显减少,组分及数量差异明显的细菌（尤其是放线菌门）大多与土壤全磷含量呈显著相关。[结论] 过量施用化肥和常年连作改变了土壤细菌群落结构,生态环境恶化,导致土传性病害发生。其中,磷可能是影响土壤微生物群落结构变化最主要的肥料元素。     

东海洋山港沿岸土壤、海水样本中Ⅰ型PKS基因的初步筛选鉴定与功能分析

为考察土壤和海水中Ⅰ型聚酮合酶(polyketide synthase,PKS)基因的多样性和差异,本研究自行设计了一套针对PKS基因中酮缩合酶(ketosynthase,KS)片段的简并引物,使用PCR方法直接克隆东海洋山港沿岸土壤和海水DNA样本中的KS片段,去除重复序列后,共获得了23条不同的KS片段(长度为630 bp～690 bp),提交GenBank皆获登录号,其中19条来自土壤(DQ640993,DQ640997、DQ641926、DQ641927、DQ673137～DQ673151),4条来自海水(DQ673151,EF554859～EF554861),由核苷酸序列推断出的氨基酸序列保守,与GenBank中已知KS基因片段的相似度在45%到85%之间,种系发生分析表明,其中14条KS片段(来源于海水的KS片段皆在其中)应来源于典型的KS群,而剩余9条则来源于杂合的PKSmRPS(Non-ribosomalpeptide synthetase.非核糖体多肽合成酶)群.另外,几条KS基因特征明显,可用于进一步的研究.     

用SRAP标记研究根际土壤微生物的遗传多样性

将近年来新建立的分子标记技术——相关序列扩增多态性(sequence-related amplified polymorphism,SRAP)应用于土壤微生物遗传多样性研究.采用22对引物组合对20种植物根际土壤微生物进行了分析,共获得237个扩增位点,其中多态性位点221个,占93.2％.平均每对引物组合的多态位点比...     

不同植物对黄顶菊根际土壤微生物和土壤养分的影响

[目的] 不同植物对外来入侵植物的抵御能力不同,研究不同植物对入侵植物根际土壤生态的影响可为筛选入侵植物的竞争替代植物提供科学依据。[方法] 利用同质园试验,以入侵植物黄顶菊为研究对象,设置黄顶菊单种、黄顶菊与不同植物（地肤、苘麻、苏丹草、反枝苋）混种处理,采用磷脂脂肪酸分析方法来研究不同植物对黄顶菊根际土壤微生物群落结构的影响,并结合土壤养分的变化探究不同植物对黄顶菊根际土壤生态的影响。[结果] 与黄顶菊单种相比,地肤和苘麻降低了黄顶菊根际微生物的总含量,改变了黄顶菊根际微生物群落结构。地肤、苘麻能竞争性抑制黄顶菊对铵态氮的吸收,从而抑制黄顶菊的生长。[结论] 不同植物的抵御能力与其土壤生态有关,替代植物通过改变黄顶菊根际土壤微生物,抑制黄顶菊对氮的吸收,从而抑制黄顶菊的生长,实现对黄顶菊的替代控制。     

西宁南山4种灌木根际和非根际土壤微生物、酶活性和养分特征

西宁南山区植被退化情况严重,人工造林植被恢复被看作是最有效的恢复手段,其中选择合适造林树种尤为关键。选择人工种植的唐古特白刺Nitraria tangutorum、柠条Caragana korshinskii、西北小蘗Berberis vernae和短叶锦鸡儿Caragana brevifolia共4种灌木树种造林试验区为研究对象,通过测定根际和非根际土壤微生物数量、酶活性及养分含量,综合比较种植4种灌木树种根际和非根际土壤肥力差异,科学评价其对土壤的改善效果。研究表明:(1)土壤微生物数量和酶活性总体呈现出根际高于非根际的规律,仅放线菌数量和脲酶活性出现了根际低于非根际现象。(2)土壤养分方面,4种灌木根际土壤和非根际土壤p H值、全N、全P、全K含量差异不显著,有机质、有效P、速效K含量均呈现出根际非根际,而碱解N则是根际非根际。(3)土壤酶活性与土壤微生物数量相关性不显著,土壤有机质含量与土壤细菌、真菌数量呈极显著正相关,有效P含量与土壤细菌、真菌和放线菌数量呈极显著正相关,速效K含量与过氧化氢酶、酸性磷酸酶活性呈显著正相关,全N、碱解N含量均与脲酶活性呈显著正相关。(4)从土壤肥力综合水平来看,根际非根际,其中根际土壤中西北小蘗柠条短叶锦鸡儿唐古特白刺,研究结果表明西北小蘗和柠条能大幅提高土壤肥力,改良土壤效果较好。     

Phylogenetic analysis of type I polyketide synthase and nonribosomal peptide synthetase genes in Antarctic sediment

The modular polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) have been found to be involved in natural product synthesis in many microorganisms. Study on their diversities in natural environment may provide important ecological insights, in addition to opportunities for antibacterial drugs development. In this study, the PKS and NRPS gene diversities in two coast sediments near China Zhongshan Station were studied. The phylogenetic analysis of amino acid (AA) sequences indicated that the identified ketosynthase (KS) domains were clustered with those from diverse bacterial groups, including Proteobacteria, Firmicutes, Planctomycetes, Cyanobacteria, Actinobacteria, and some uncultured symbiotic bacteria. One new branch belonging to hybrid PKS/NRPS enzyme complexes and five independent clades were found on the phylogenetic tree. The obtained adenylation (A) domains were mainly clustered within the Cyanobacteria and Proteobacteria group. Most of the identified KS and A domains showed below 80 and 60% identities at the AA level to their closest matches in GenBank, respectively. The diversities of both KS and A domains in natural environmental sample were different from those in sewage-contaminated sample. These results revealed the great diversity and novelty of both PKS and NRPS genes in Antarctic sediment.     

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.     

Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067

Fungal type I polyketide (PK) compounds are highly valuable for medical treatment and extremely diverse in structure, partly because of the enzymatic activities of reducing domains in polyketide synthases (PKSs). We have cloned several PKS genes from the fungus Xylaria sp. BCC 1067, which produces two polyketides: depudecin (reduced PK) and 19,20-epoxycytochalasin Q (PK-nonribosomal peptide (NRP) hybrid). Two new degenerate primer sets, KA-series and XKS, were designed to amplify reducing PKS and PKS-NRP synthetase hybrid genes, respectively. Five putative PKS genes were amplified in Xylaria using KA-series primers and two more with the XKS primers. All seven are predicted to encode proteins homologous to highly reduced (HR)-type PKSs. Previously designed primers in LC-, KS-, and MT-series identified four additional PKS gene fragments. Selected PKS fragments were used as probes to identify PKS genes from the genomic library of this fungus. Full-length sequences for five PKS genes were obtained: pks12, pks3, pksKA1, pksMT, and pksX1. They are structurally diverse with 1-9 putative introns and products ranging from 2162 to 3654 amino acids in length. The finding of 11 distinct PKS genes solely by means of PCR cloning supports that PKS genes are highly diverse in fungi. It also indicates that our KA-series primers can serve as powerful tools to reveal the genetic potential of fungi in production of multiple types of HR PKs, which the conventional compound screening could underestimate.     

Cloning and characterization of a bacterial iterative type I polyketide synthase gene encoding the 6-methylsalicyclic acid synthase

Unusual polyketide synthases (PKSs), that are structurally type I but act in an iterative manner for aromatic polyketide biosynthesis, are a new family found in bacteria. Here we report the cloning of the iterative type I PKS gene chlB1 from the chlorothricin (CHL) producer Streptomyces antibioticus DSM 40725 by a rapid PCR approach, and characterization of the function of the gene product as a 6-methylsalicyclic acid synthase (6-MSAS). Sequence analysis of various iterative type I PKSs suggests that the resulting aromatic or aliphatic structure of the products might be intrinsically determined by a catalytic feature of the paired KR-DH domains in the control of the double bond geometry. The finding of ChlB1 as a 6-MSAS not only enriches the current knowledge of aromatic polyketide biosynthesis in bacteria, but will also contribute to the generation of novel polyketide analogs via combinatorial biosynthesis with engineered PKSs.     

Engineering biodiversity with type II polyketide synthase genes

A very important task in the ongoing search for new clinically useful drugs is the generation of large numbers of structurally diverse compounds. The emerging field of combinatorial biosynthesis, in which nature's chemical capabilities are exploited in a combinatorial 'mix-and-match' fashion, has generated libraries of novel molecules representing great structural diversity which are not available naturally or readily generated through (combinatorial) synthesis. Novel polyketides have been generated by manipulating type II iterative polyketide synthase (PKS) systems that express a variety of combinations of a minimal PKS with ketoreductases, cyclases, and other tailoring enzymes, resulting in a set of design rules to rationally engineer new metabolites. Engineering studies with the Streptomyces coelicolor whiE (spore pigment) and the 'Streptomyces maritimus' enterocin type II PKS provide additional insight on designing diverse assemblies of aromatic, as well as nonaromatic, polyketides.     

Identification of unique type II polyketide synthase genes in soil

Many bacteria, particularly actinomycetes, are known to produce secondary metabolites synthesized by polyketide synthases (PKS). Bacterial polyketides are a particularly rich source of bioactive molecules, many of which are of potential pharmaceutical relevance. To directly access PKS gene diversity from soil, we developed degenerate PCR primers for actinomycete type II KS(alpha) (ketosynthase) genes. Twenty-one soil samples were collected from diverse sources in New Jersey, and their bacterial communities were compared by terminal restriction fragment length polymorphism (TRFLP) analysis of PCR products generated using bacterial 16S rRNA gene primers (27F and 1525R) as well as an actinomycete-specific forward primer. The distribution of actinomycetes was highly variable but correlated with the overall bacterial species composition as determined by TRFLP. Two samples were identified to contain a particularly rich and unique actinomycete community based on their TRFLP patterns. The same samples also contained the greatest diversity of KS(alpha) genes as determined by TRFLP analysis of KS(alpha) PCR products. KS(alpha) PCR products from these and three additional samples with interesting TRFLP pattern were cloned, and seven novel clades of KS(alpha) genes were identified. Greatest sequence diversity was observed in a sample containing a moderate number of peaks in its KS(alpha) TRFLP. The nucleotide sequences were between 74 and 81% identical to known sequences in GenBank. One cluster of sequences was most similar to the KS(alpha) involved in ardacin (glycopeptide antibiotic) production by Kibdelosporangium aridum. The remaining sequences showed greatest similarity to the KS(alpha) genes in pathways producing the angucycline-derived antibiotics simocyclinone, pradimicin, and jasomycin.     

Nonactin biosynthesis: the potential nonactin biosynthesis gene cluster contains type II polyketide synthase-like genes

Nonactin is the parent compound of a group of highly atypical polyketide metabolites produced by Streptomyces griseus subsp. griseus ETH A7796. In this paper we describe the isolation, sequencing, and analysis of 15? omitted?559 bp of chromosomal DNA, containing the potential nonactin biosynthesis gene cluster, from S. griseus subsp. griseus ETH A7796. Fourteen open reading frames were observed in the DNA sequence. Significantly, type II polyketide synthase (PKS) homologues were discovered in an apparent operon structure, which also contained the nonactate synthase gene (nonS), clustered with the tetranactin resistance gene. The deduced products of two of the genes (nonK and nonJ) are quite unusual ketoacyl synthase (KAS) alpha and KASbeta homologues. We speculate that nonactic acid, the polyketide precursor of nonactin, is synthesized by a type II PKS system.     

A family of polyketide synthase genes expressed in ripening Rubus fruits

Quality traits of raspberry fruits such as aroma and color derive in part from the polyketide derivatives, benzalacetone and dihydrochalcone, respectively. The formation of these metabolites during fruit ripening is the result of the activity of polyketide synthases (PKS), benzalcetone synthase and chalcone synthase (CHS), during fruit development. To gain an understanding of the regulation of these multiple PKSs during fruit ripening, we have characterized the repertoire of Rubus PKS genes and studied their expression patterns during fruit ripening. Using a PCR-based homology search, a family of ten PKS genes (Ripks1-10) sharing 82-98% nucleotide sequence identity was identified in the Rubus idaeus genome. Low stringency screening of a ripening fruit-specific cDNA library, identified three groups of PKS cDNAs. Group 1 and 2 cDNAs were also represented in the PCR amplified products, while group 3 represented a new class of Rubus PKS gene. The Rubus PKS gene-family thus consists of at least eleven members. The three cDNAs exhibit distinct tissue-specific and developmentally regulated patterns of expression. RiPKS5 has high constitutive levels of expression in all organs, including developing flowers and fruits, while RiPKS6 and RiPKS11 expression is consistent with developmental and tissue-specific regulation in various organs. The recombinant proteins encoded by the three RiPKS cDNAs showed a typical CHS-type PKS activity. While phylogenetic analysis placed the three Rubus PKSs in one cluster, suggesting a recent duplication event, their distinct expression patterns suggest that their regulation, and thus function(s), has evolved independently of the structural genes themselves.     

A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and phenylphenalenone biosynthesis

Chalcone synthase (CHS) related type III plant polyketide synthases (PKSs) are likely to be involved in the biosynthesis of diarylheptanoids (e.g. curcumin and polycyclic phenylphenalenones), but no such activity has been reported. Root cultures from Wachendorfia thyrsiflora (Haemodoraceae) are a suitable source to search for such enzymes because they synthesize large amounts of phenylphenalenones, but no other products that are known to require CHSs or related enzymes (e.g. flavonoids or stilbenes). A homology-based RT-PCR strategy led to the identification of cDNAs for a type III PKS sharing only approximately 60% identity with typical CHSs. It was named WtPKS1 (W. thyrsiflora polyketide synthase 1). The purified recombinant protein accepted a large variety of aromatic and aliphatic starter CoA esters, including phenylpropionyl- and side-chain unsaturated phenylpropanoid-CoAs. The simplest model for the initial reaction in diarylheptanoid biosynthesis predicts a phenylpropanoid-CoA as starter and a single condensation reaction to a diketide. Benzalacetones, the expected release products, were observed only with unsaturated phenylpropanoid-CoAs, and the best results were obtained with 4-coumaroyl-CoA (80% of the products). With all other substrates, WtPKS1 performed two condensation reactions and released pyrones. We propose that WtPKS1 catalyses the first step in diarylheptanoid biosynthesis and that the observed pyrones are derailment products in the absence of downstream processing proteins.     

Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei

The 54-kbp Type I polyketide synthase gene cluster, most probably involved in rifamycin biosynthesis by Amycolatopsis mediterranei, was cloned in E. coli and completely sequenced. The DNA encodes five closely packed, very large open reading frames reading in one direction. As expected from the chemical structure of rifamycins, ten polyketide synthase modules and a CoA ligase domain were identified in the five open reading frames which contain one to three polyketide synthase modules each. The order of the functional domains on the DNA probably reflects the order in which they are used because each of the modules contains the predicted acetate or propionate transferase, dehydratase, and β-ketoacyl-ACP reductase functions, required for the respective step in rifamycin biosynthesis.