Diversity of laccase genes from basidiomycetes in a forest soil

Fungal oxidative exo-enzymes lacking substrate specificity play a central role in the cycling of soil organic matter. Due to their broad ecological impact and available knowledge of their gene structure, laccases appeared to be appropriate markers to monitor fungi with this kind of oxidative potential in soils. A degenerate PCR-primer pair Cu1F/Cu2R, specific for basidiomycetes, was designed to assess directly the diversity of laccase genes in soils. PCR amplification of mycelial cultures and fruit-bodies of a wide spectrum of basidiomycetes, covering all functional groups (saprophytes, symbionts, and pathogens), produced multiple DNA fragments around 200 bp. A neighbor-joining tree analysis of the PCR-amplified laccase sequences showed a clear species-specificity, but also revealed that most fungal taxa possess several laccase genes showing a large sequence divergence. This sequence diversity precluded the systematic attribution of amplified laccase of unknown origin to specific taxa. Amplification of laccase sequences from DNA, extracted from a brown (moder) forest soil, showed a specific distribution of laccase genes and of the corresponding fungal species in the various soil horizons (O_h, A_h, B_v). The most organic O_h-horizon displayed the highest gene diversity. Saprophytic fungi appeared to be less widespread through the soil horizons and displayed a higher diversity of laccase genes than the mycorrhizal ones.     

苇状羊茅EST-SSR标记在草甸羊茅和多年生黑麦草的通用分析

草甸羊茅(Festuca pratensis Huds)和多年生黑麦草(Lolium perenne Lam)均是禾本科、多年生草本植物,是优良的牧草和草坪草.本研究采用40对苇状羊茅(Festuca arundinacea Schred)的表达序列标签-简单序列重复(Expressed sequence tags-s...     

正交设计优化草地早熟禾SRAP-PCR反应体系及引物筛选

采用L9(34)正交试验设计方法,对草地早熟禾(Poa pratensis)基因组DNA SRAP PCR反应体系中的Taq DNA聚合酶、Mg2+、引物及dNTP四因素的用量进行优化,并比较不同模板DNA用量对扩增的影响,建立草地早熟禾SRAP PCR最佳反应体系,同时,利用该体系对SRAP引物进行筛选。结果表明,草地早熟禾SRAP PCR最佳反应体系为Taq DNA聚合酶1.0 U、Mg2+ 1.75 mmol·L-1、引物0.25 μmol·L-1、dNTP 220 μmol·L-1、40 ng模板DNA、2 μL 10×PCR buffer,总体积20 μL。运用该体系从100对SRAP引物中筛选出43对引物能够产生清晰稳定的扩增条带且多态性丰富。优化体系的建立及引物的筛选可为今后利用SRAP标记技术对草地早熟禾进行遗传多样性分析、图谱构建、种质资源鉴定奠定技术基础。     

中国鲎微卫星引物扩增圆尾鲎DNA的PCR反应体系优化

采用正交设计法,从dNTPs浓度、引物浓度、Mg2＋浓度、Taq DNA聚合酶用量4个因素3个水平出发,优化设计圆尾鲎DNA的PCR反应体系（引物为中国鲎微卫星引物）.并采用直观分析方法分析正交试验结果,最终建立了圆尾鲎SSR-PCR最佳反应体系：总体积20μL,Taq DNA聚合酶1.5 U、dNTPs 0.16 mmol/L、引物0.2μmol/L、Mg2＋2.0 mmol/L;并通过PCR梯度实验进一步优化模板DNA质量浓度、退火温度及退火时间,获得最佳反应条件：模板DNA质量浓度为30 ng/μL,退火温度为48℃,退火时间为20～25 s.对最佳反应体系和反应条件进行了检验,结果显示该反应体系稳定性高、重复性好.     

基于毛细管电泳技术的牡丹SRAP-PCR反应体系优化及引物筛选

以牡丹叶片DNA为模板,对SRAP-PCR反应程序进行研究,确定适合的反应程序,即94℃预变性5 min;94℃变性1 min,33℃退火1 min,72℃延伸1 min,共5个循环;随后94℃变性1 min,52℃退火1 min,72℃延伸1 min,共35个循环;最后72℃延伸10 min。基于毛细管电泳技术,采用正交设计L18(37)对牡丹SRAP-PCR反应体系的5因素(Taq酶,Mg2+,模板DNA,dNTP,引物)3个水平进行了优化,构建了牡丹SRAP最佳反应体系:模板DNA 50 ng,dNTP 0.25 mmol/L,Mg2+浓度2.5 mmol/L,引物浓度0.4μmol/L,TaqDNA聚合酶0.5 U,总体积为25μL。各因素对扩增结果影响程度均不同:dNTPs>引物>DNA模板>Mg2+>Taq酶。运用该体系从756个SRAP引物组合中筛选出多态性好、条带清晰的26个引物组合,并证明了该体系稳定可靠。该体系的建立以及引物组合的确定为今后利用SRAP分子技术进行牡丹的相关研究奠定了科学基础。     

DNA amplification fingerprinting and marker screening for pseudo-testcross mapping of flowering dogwood ( Cornus florida L.)

DNA amplification fingerprinting (DAF) with arbitrary oligonucleotide primers was used to study genetic relationships between cultivars of flowering dogwood (Cornus florida L.), evaluate extent of plant hybridization, and generate markers in pseudo-testcross mapping at the intraspecific level. Modified Taguchi optimization methods defined a robust DAF system based on high annealing temperature (48–52 °C) and primer concentration (typically 8 μM) that was used to study genetic diversity of representative dogwood cultivars and hybrids. Phenetic analysis using cluster and numerical methods showed that: (1) cultivars were relatively conserved at the genetic level; (2) their hybridization could be identified in the F1 progeny in the absence of phenotypic or physiological markers; (3) several cultivars grouped according to their recorded ancestry; and (4) dogwood anthracnose-resistant lines originally selected in Catoctin Mountain Park (Maryland) grouped separately from those of southern origin. The DAF protocol was also tested in pseudo-testcross mapping of dogwood at the intraspecific level. A preliminary screening of parents ‘Pink Sachet’ and ‘Fragrant Cloud’ and 7 F1 segregants with 22 octamer primers produced 703 amplified loci, 30 and 39 of which were male and female markers segregating at 1:1 ratios with 98.6% confidence levels in pseudo-testcross configuration. Overall results show that DAF generated markers very efficiently (3 per primer) despite the close relatedness of parental dogwood cultivars. This study constitutes the basis for a future genetic linkage mapping and marker-assisted selection (MAS) effort initially targeted to control important fungal diseases in dogwood. This revised version was published online in July 2006 with corrections to the Cover Date.     

A quantitative method for detecting ammonia‐oxidizing bacteria in coastal aquaculture systems

There is a need to quantify autotrophic nitrifiers in coastal aquaculture systems for evolving a bioremediation strategy. Autotrophic nitrifiers are extremely slow‐growing organisms, which cannot be detected by traditional methods as they are notoriously difficult to culture. Molecular techniques based on functional genes could be deployed for the detection of nitrifiers. Ammonia monooxygenase (amoA), that catalyses the oxidation of ammonia to hydroxylamine in the rate‐determining step of nitrification is largely unique to ammonia‐oxidizing bacteria (AOB). In the present study, a quantitative real‐time polymerase chain reaction assay targeting amoA was developed to estimate AOB population size in coastal soil, ammonia‐removing bioaugmentors and the solid matrix. To achieve this objective, different set of primers and a dual labelled probe have been designed for SYBR Green and TaqMan real‐time assays. The abundance of AOB ranged from 10⁴ to 10⁶ order of magnitude in the samples. In the present study, biofilm formation of the consortium of nitrifying bacteria onto bagasse has also been quantified. The results demonstrate that the developed method is a rapid and sensitive tool for the quantitative detection of nitrifying bacteria in aquatic and related environment. This helps in making the bioremediation approach for ammonia removal by immobilization of nitrifying bacteria onto the natural substrate.     

狗牙根SRAP-PCR反应体系优化及引物筛选

利用正交设计,从Mg2 、dNTP、引物浓度和DNA聚合酶4种因素3个水平以及不同的模板DNA浓度来优化狗牙根SRAP-PCR反应体系,并对引物进行了全面筛选。狗牙根SRAP-PCR优化反应体系结果为:2μL 10×buffer4、0 ng模板DNA、Mg2 1.25 mmol/Ld、NTP 260μmol/L、引物0.2μmol/L、Taq DNA聚合酶1.0 U,总体积20μL。运用该结果从90对引物中共筛选出扩增条带清晰、多态性丰富的SRAP引物34对。优化体系的建立及其引物的筛选为今后利用SRAP标记技术进行狗牙根遗传分析、图谱构建、基因定位与种质资源鉴定奠定了技术基础。     

PRRSVNsp9蛋白基因的RT—LAMP检测方法的建立

根据GenBank中登录的PRRSVNsp9基因序列,应用PrimerExplorerV4软件,在该基因序列中选取保守区设计了4条RT—LAMP引物,旨在建立1种针对Nsp9蛋白基因的逆转录环介导等温扩增（RT-LAMP）的快速检测方法。采用引物分组扩增的方法对引物进行了检测,以确保引物的可靠性。对反应体系、温度以及时间进行了优化,检测了该方法的特异性和灵敏度。结果表明,该方法在等温条件下只需50min就能检测出结果。与RT-PCR方法相比,在判定检测结果时不需要借助昂贵仪器设备,具有高特异性、高灵敏度、操作简便快速等特点,适合于临床检测的应用。     

本氏针茅SRAP PCR反应体系的建立及引物筛选

以本氏针茅（Stipa bungeana）幼嫩叶片为材料,建立适合本氏针茅基因组DNA提取的改良CTAB法,在此基础上采用正交试验设计和单因素分析相结合的方法,对本氏针茅SRAP PCR反应体系中的5个主要因素（DNA、Taq酶、dNTPs、Mg2+和引物）进行优化,旨在建立适合本氏针茅SRAP分析的反应体系。结果表明,在20 μL总的反应体系中各组分的加入量分别为：DNA（20 ng·μL-1）3 μL、Taq DNA酶(5 U·μL-1)0.2 μL、dNTPs（2.5 mmol·L-1)1.4 μL、引物（10 μmol·L-1）1.0 μL、Mg2+ (25 mmol·L-1)2.0 μL、10×Buffer 2.5 μL、ddH2O 8.9 μL。体系验证和引物筛选试验表明,该体系适于本氏针茅遗传多样性分析,该体系的建立可为本氏针茅种质资源遗传多样性研究和黄土高原植被恢复及生态建设提供理论基础。