DNA条形码与生物分类学研究

DNA条形码是一段特殊的、可用于物种鉴定的DNA序列.是近几年来国际上生物分类学研究的热点.该技术在动物分类学研究中已得到广泛的应用,所采用的DNA序列是线粒体细胞色素C氧化酶Ⅰ号基因(CO Ⅰ)的部分序列.在植物分类学研究中,DNA条形码的进展相对缓慢.目前尚处于对所提议的叶绿体DNA片段rpoB、rpoCl、matk、rbcL等进行比较和评价阶段.阐述了DNA条形码在生物分类学研究方面的应用现状、存在的问题及可能的发展趋势.     

DNA条形码及其在动物遗传多样性中的应用

DNA条形码技术是现今生物分类学中重要的分子技术之一,其利用线粒体细胞色素C氧化酶亚单位Ⅰ(COⅠ)等基因可以快速准确地鉴定新种和隐存种。COⅠ基因存在于所有动物中,呈母系遗传、比较保守、无内含子、进化速率比较快(是核基因进化速率的2～10倍)、具有明显的碱基偏好性,而且其DNA序列很少存在插入和缺失。DNA条形码技术不仅是生物形态学研究的一个重要补充,而且为研究生物分类与进化开辟了新的途径。本文就DNA条形码技术的技术原理、操作步骤及其在动物分类学及遗传多样性中的应用作一概述,旨在通过对其技术原理了解的同时,掌握其在当代生物分类学中的独特作用、现实意义及局限性,为进一步开展生物分类及进化研究提供思路。     

新疆野兔DNA条形码筛选

旨在筛选一种用于快速鉴定新疆兔属物种的DNA条形码。本研究以新疆4种野兔共计40例样本为研究对象,以CO1、ND4、16S rRNA和ITS2为候选基因,经PCR扩增和测序后,综合分析比较候选序列在种水平上的鉴定能力。结果表明,ITS2候选片段因未获得达到测序要求的PCR扩增产物最先被排除。相较于16S rRNA,CO1和ND4在序列特征、变异特征参数和秩和检验结果上均表现出更强的变异水平,遗传频率分布图有着更宽的gap,系统聚类树的鉴定结果有着更高的鉴定准确率。而从种间变异特征、种间秩和检验结果及遗传频率分布图来看,ND4均略优于CO1。本试验综合分析得到的最优DNA条形码为ND4,候补DNA条形码为CO1。该结论可为新疆兔属物种的分类分布研究和珍稀野兔资源的保护利用等提供一定的参考依据。     

DNA条形码技术在野生哺乳动物物种鉴定中的可行性

为新疆野生哺乳动物物种鉴定找出最好的方法并建立新疆哺乳动物DNA条形码数据库提供资料,本研究选取新疆野生哺乳动物的肌肉、血液、粪便作为研究材料,运用DNA条形码技术检测野生哺乳动物线粒体DNA COI基因序列。共检测片段长度为642～729 bp的52条COI基因序列,52条COI基因序列通过BLAST网站同源性比较,序列同源性达到98%～100%,相似度达到90%～99%。全部COI基因序列中A、T、C和G的平均含量为26. 3%、30. 3%、26. 0%和17. 4%,A+T的含量(56. 6%)高于C+G含量(43. 4%),有明显的碱基偏倚性。种内遗传距离为0%～2%,种间遗传距离为15%～42%,二者分离度高。邻接法和最小进化法构建的系统发育树,两种方法所得的系统进化树拓扑结构高度一致,分支明显,都分配在单个支上。说明DNA条形码技术可用于新疆野生哺乳动物物种鉴定并弥补其他物种鉴定方法的不足和缺点,在新疆濒危野生动物的管理与保护中有重要意义。     

DNA条形码技术在中药领域的应用

DNA条形码技术可对物种进行快速自动鉴定,具有鉴定准确、结果稳定、操作简便、适用广泛等特点,目前在中药领域应用较多。从DNA条形码技术在中药材鉴定、种植、流通、市场监管、中成药鉴定和药用植物种质资源调查等中药领域的多个方面进行综述,并探讨DNA条形码技术在中药领域的优势和不足,以期为DNA条形码技术在中药领域的研究提供新的思路。     

线粒体DNA及蜜蜂线粒体DNA的研究现状与展望

线粒体DNA是真核细胞中核外唯一的遗传物质,是目前研究起源进化及群体遗传分析的理想研究对象。综述了线粒体DNA(mtDNA)基本特征、多态性及其分析方法的同时,介绍了蜜蜂线粒体DNA的组成结构和多态性,阐述了蜜蜂线粒体DNA研究的现状并对其未来的发展前景予以展望。     

西番莲DNA条形码遗传多样性分析

DNA条形码;西番莲;遗传多样性;聚类分析     

蜜蜂DNA甲基化与基因表达研究进展

在真核生物基因组中,DNA甲基化是一种重要的表观遗传学标记。DNA甲基化在有机体的正常生长发育过程中发挥重要作用,可以直接影响到基因的活性。目前,蜜蜂DNA甲基化的功能和生物学研究已取得了初步进展,越来越多蜜蜂甲基化基因和甲基化酶被报道。就近年研究人员对蜜蜂DNA甲基化与基因表达在蜜蜂级型分化、蜜蜂饮食和蜜蜂行为等方面的研究进展做一简要综述。     

国际生命条形码计划—DNA Barcoding

中国拥有数目庞大、种类繁多的动植物,也有很多珍贵、濒危生物,这些更是异常宝贵的科学资源。DNA条形码技术的出现可以更好的帮助鉴别这些物种,了解其分支来源,甚至可以预知其进化方向。笔者简述了DNA条形码的技术原理与操作步骤及这项技术的产生与发展情况;简要列出了DNA条形码发展过程中的重要进步与研究相对集中的物种;概述了DNA条形码的应用途径及目前DNA条形码研究中存在的主要问题、矛盾、研究思路与发展方向,并对其发展前景做出展望。     

DNA条形码技术在食品溯源中的应用

现在食品安全越来越关注食品的可追溯性.近年来,DNA条形码作为食品溯源中一个潜在有效的工具得到迅速发展.本综述介绍了DNA条形码在食品安全尤其是在食品溯源中的应用,回顾了DNA条形码从概念的提出,到食品检测、食品溯源及防止食品商业欺诈中的应用.DNA条形码能够保障食品安全、防止商业欺诈,但距离真正的大规模应用,还有较长的路要走.     

Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis

Biofilm formation where bacterial cells adhere to a surface and surround themselves in a polysaccharide matrix is thought to be an important factor in disease initiation and persistence for many bacterial species. We have examined biofilm formation by Mycoplasma mycoides subsp. mycoides small colony using a simple model without an air/liquid interface and have found that adherent Mmm SC was more resistant to many stresses, including heat, osmotic shock and oxidative stress. Biofilms of Mmm SC also exhibited remarkable persistence and were able to survive for up to 20 weeks in stationary phase. Significant variation was seen between Mmm SC strains in their ability to form a biofilm and the morphology of the biofilm produced with some strains unable to produce microcolonies. Proteomic analysis found that a number of proteins linked to adherence were over-expressed in biofilms compared with planktonic cells.     

Use of the Leptospira sp. ligB C-terminus coding region as a diagnostic tool of animal leptospirosis

Leptospirosis is the most widespread zoonosis worldwide, and it can cause reproductive failures in livestock, while in humans may vary from a mild fever to multi-organ failure and death. Due to this, in this study, we evaluated the usefulness of the segment encoding LigB C-terminus region, only present in pathogenic as target for a diagnostic PCR.This new PCR yielded a 100 % positivity for pathogenic Leptospira species and no cross-reactivity was found with intermediate or non-pathogenic species, or with other microorganisms, demostrating its high analytical specificity.The estimated analytical sensitivity was higher in serum samples than in blood or urine samples (6−9 × 10² lept/mL and 6−9 × 10⁵ and 6−9 × 10⁶ lept/mL, respectively). Multiple sequence alignment of the target region from different pathogenic Leptospira species confirmed that this gene region is highly conserved among these species, with few single nucleotide polymorphisms.The ligb-ct PCR here developed appears as a useful tool for the molecular diagnosis of leptospirosis.     

Evaluation of PetrifilmsTM as a diagnostic test to detect bovine mastitis organisms in Kenya

The study purpose was to validate Petrifilms^TM (3M Microbiology, 2005) against standard culture methods in the diagnosis of bovine mastitis organisms in Kenya. On 128 smallholder dairy cattle farms in Kenya, between June 21, 2010 and August 31, 2010, milk samples from 269 cows that were positive on California Mastitis Test (CMT) were cultured using standard laboratory culture methods and Petrifilms^TM (Aerobic Count and Coliform Count –3M Microbiology, 2005), and results were compared. Staphylococcus aureus was the most common bacterium isolated (73 % of samples). Clinical mastitis was found in only three cows, and there were only two Gram-negative isolates, making it impossible to examine the agreement between the two tests for Gram-negative- or clinical mastitis samples. The observed agreement between the standard culture and Petrifilm^TM (3M Microbiology, 2005) results for Gram-positive isolates was 85 %, and there was fair agreement beyond that expected due to chance alone, with a kappa (κ) of 0.38. Using culture results as a gold standard, the Petrifilms^TM had a sensitivity of 90 % for Gram-positive samples and specificity of 51 %. With 87 % of CMT-positive samples resulting in Gram-positive pathogens cultured, there was a positive predictive value of 93 % and a negative predictive value of 43 %. Petrifilms^TM should be considered for culture of mastitis organisms in developing countries, especially when Gram-positive bacteria are expected.     

Detection of Brucella abortus in mammalian tissue, using biotinylated, whole genomic DNA as a molecular probe

A method has been developed for the detection of Brucella abortus in complex tissue homogenates. The technique uses tissue homogenization in the presence of sucrose and Triton X-100 and subsequent filtration through a 5-microns pore size filter to remove mammalian nuclei and cellular debris. The DNA from the bacteria is then extracted, dot blotted onto nitrocellulose, and hybridized with a biotinylated probe of B abortus strain 19 DNA. In the present study, BALB/C mice were inoculated intraperitoneally with either 10(9) or 10(11) B abortus strain 2308S organisms. After 6 days, the mice were euthanatized by cervical dislocation and the livers were removed, weighed, and the appearance of each was noted. The tissues were homogenized, and a viable cell count was performed to determine the number of bacteria in each organ. The DNA was extracted, blotted onto nitrocellulose, and hybridized with the Brucella probe. The biotin label was detected by use of a commercially available streptavidin/alkaline phosphatase system. In control experiments, the technique detected 10(5) organisms in a mixture of bacteria and 1 g of rat liver. The technique also detected 10(7) B abortus organisms/g of tissue from experimentally inoculated mice. The probe was specific for Brucella and had no affinity for contaminating bovine or bacterial DNA.     

Telomerase enzyme activity as a diagnostic tool to distinguish effusions of malignant and benign origin

Telomerase enzyme activity is high in populations of cells that are dividing, and is low or undetectable in quiescent cell populations. Activation of telomerase in tissues that normally lack the capacity for self-renewal is strongly correlated with neoplasia. Telomerase activity can be detected in samples containing very small numbers of cells and studies of human patients suggest that measurement of telomerase activity may be useful for the evaluation of samples that can be obtained in a minimally invasive manner. This study compares the presence or absence of telomerase activity with cytologic evaluation of body cavity effusions, to determine if neoplasia is the underlying cause for the effusion in dogs and cats. Detection of telomerase in effusions was no more sensitive than cytologic evaluation for the identification of underlying neoplasia, and was less specific (telomerase assay: sensitivity = 50%, specificity = 83%; cytology: sensitivity = 50%, specificity = 100%). We conclude that although the telomerase assay may constitute a useful adjunctive test for the diagnosis of neoplasia in some dogs and cats with body cavity effusions, the results of this assay are not sufficiently reliable to be used as a sole diagnostic test.     

柑橘修剪废弃物的堆肥基质化特性分析

为促进柑橘修剪废弃物的资源化利用,以柑橘修剪废弃物与菜籽饼混合物为原料,添加尿素、过磷酸钙于室外进行条垛式覆膜高温好氧发酵处理,研究其发酵进程及堆肥产品的基质特性。结果表明：经过35d处理,夏季覆膜堆肥能使堆体温度迅速上升至70℃左右,并维持在55℃以上超过一周。堆肥结束时,pH值为8.27,EC值为1.90 mS.cm-1,E4/E6为2.3,C/N为16.36,GI为102.69%,达到腐熟标准。堆肥后TN、TP、TK分别增加23.71%、75.42%、26.45%,金属元素Fe、Cu、Zn总量分别为3200.15 mg.kg-1、149.85 mg.kg-1、561.45 mg.kg-1,养分含量丰富。从基质化利用的角度来看,堆肥产品的容重、持水孔隙度、通气孔隙度、气水比等指标均达到理想基质的要求。由此表明,柑橘修剪废弃物经过高温好氧堆制发酵后,堆肥达到腐熟,堆肥产品具有作为植物栽培基质的特性。     

Morphological and molecular data on the dermal microfilariae of a species of Cercopithifilaria from a dog in Sicily

Dermal microfilariae found in a dog from Sicily, Italy, were characterized morphologically and genetically and differentiated from those of all the other blood microfilariae commonly found in dogs. In particular, the microfilariae were short (mean length of 186.7 μm), presented a body flattened dorso-ventrally and a rounded head, bearing a tiny cephalic hook. The genetic identity of microfilariae herein studied was also assessed by molecular amplification, sequencing and analyzing of multiple ribosomal ITS-2 and mitochondrial (cox1 and 12S) target genes. Both morphologic and genetic characterization as well as the molecular phylogenetic history inferred using sequences of a barcoding dataset were concordant in supporting the identification of Cercopithifilaria at the genus level. Surprisingly, microfilariae here examined were well distinct from Cercopithifilaria grassii (Noè, 1907), from northern Italy, and resembled those of a species described in Brazil, Cercopithifilaria bainae Almeida & Vicente, 1984. This paper provides evidence for the existence of a Cercopithifilaria species infesting a dog from Sicily and also presents a PCR protocol on skin samples as a tool for further epidemiological studies, which could provide evidence on the aetiology and the natural history of this filarial species.     

Application of Lineweaver-Burk data transformation to explain animal and plant performance as a function of nutrient supply

This study evaluates the effect of dry-season concentrate supplementation on growing cattle performance grazing tropical pasture and the impact of nitrogen fertilization on the growth rate of tropical pasture (tons of dry herbage mass/ha/110 days) and on the subsequent stocking rate and cattle performance during the rainy season (kg body weight gain/ha/110 days). The animal and plant responses were curvilinear to the increasing amount of nutrient supply and followed the typical saturation kinetics of enzyme systems, a Michaelis-Menten relationship. The Lineweaver-Burk data transformation explained efficiently the animal and plant responses to the nutrient supply. This methodology consists in evaluating the linear regressions of the reciprocal of animal and plant responses as a function of the reciprocal of nutrient supply. The half maximum growth rates for plant and animal to nutrient supply were verified with the proportions from .048 to .056 of the amount needed to cause .95 of theoretical maximum responses. From the curvilinear response, it can be verified that the marginal increase in animal and plant growth rate reduces as the amount of nutrient supply increases.