改良Chelex-100法快速提取转基因农产品DNA

旨在建立一种从转基因农产品中快速提取DNA的方法.分别采用改良Chelex-100法和常规CTAB法提取转基因大豆GTS40-3-2基因组DNA,测其浓度和纯度,PCR扩增其内源基因(Lectin)、启动子(CaMV35S)和品系特异性序列,对两种方法进行比较和评价,并研究两种方法提取的DNA在-20℃下保存一个月内的检测效果,以及改良Chelex-100法在玉米、小麦和水稻等其他转基因农产品的应用效果.结果表明,改良Chelex-100法能够快速在1.5h之内从样品中提取DNA,所提取的DNA直接用于PCR扩增反应,产物电泳条带清晰明亮.两种方法提取的DNA在-20℃下保存一个月内的检测效果未见明显差别.该方法在玉米、小麦和水稻等转基因农产品的应用效果稳定.因此,改良Chelex-100法提取的DNA可以作为PCR扩增模板用于转基因农产品检测.该方法具有经济、简便、快速、安全的特点,适合转基因农产品大规模筛选和鉴别.     

PCR法快速识别Actinobacteria的五种模板制备方法的比较

放线细菌的23S rRNA基因序列具有较好的保守性和相对的可变性,被认为是快速筛选放线细菌的合适部位。能否快速有效地扩增特异性区间,聚合酶链反应(PCR)扩增前的模板制备质量是关键,故比较了基因组DNA作为PCR模板的5种制备方法。旨在寻找准确、快速、简便、经济的放线细菌筛选技术,为普通和极端环境放线细菌资源的研究和开发利用创造条件。     

利用改进的酚-氯仿法从猪毛囊中提取基因组DNA

为提高从猪毛囊组织中提取基因组DNA的效率, 文章在借鉴从其他组织提取基因组DNA方法的基础上, 对经典的酚-氯仿法的反应体系和步骤进行了改进。利用改进的酚－氯仿抽提法, 从猪的毛囊组织中快速、高效地提取了高质量基因组DNA。利用该方法从1~6根猪毛囊中提取的基因组DNA可满足基于PCR技术的相关分子生物学实验需要。     

Chelex-100快速提取用于转基因检测DNA模板的研究

目的:建立从转基因作物中快速提取DNA的方法.方法 :采用Chelex-100法提取抗草甘膦大豆和非转基因大豆、转基因抗虫玉米Bt176和非转基因玉米中的DNA,使用PCR扩增大豆和玉米的内源基因(Lectin,zSSⅡb)及外源特异性序列(CaMV35S,Bt176)评价提取核酸的质量.结果 :Chelex-100法能够快速在1h之内从大豆和玉米中提取DNA,所提取的DNA可以直接用于PCR扩增反应,PCR扩增产物电泳条带清晰,转基因抗草甘膦大豆样品和转基因抗虫玉米Bt176检测均出现强阳性结果.结论 :Chelex-100法提取DNA可以作为转基因检测的模板,该方法具有经济、简便、快速的特点,适合于转基因检测工作.     

一种快速高效提取病原真菌DNA作为PCR模板的方法

真菌rDNA-ITS序列分析适合于较高等级水平的生物群体间的系统分析。真菌DNA的提取采用传统的方法,步骤繁琐,需要较长时间。采用Chelex-100法提取真菌DNA,使用PCR扩增rDNA-ITS序列评价提取核酸的质量。结果显示,该方法具有经济、简便、快速、高效的特点,是一种比较理想的提取真菌基因组DNA作为PCR模板的方法。     

一种提取蜘蛛基因组DNA的有效方法

介绍了用尿素法提取蜘蛛基因组DNA。通过与其他DNA提取方法相比较,证明尿素法具有可在室温条件下进行、DNA得率高、完整性好、简单快速等优点。以提取的DNA为模板进行PCR扩增,获得预期大小的、高重复、高GC含量的编码蜘蛛牵引丝蛋白基因的DNA片段。     

山蛭基因组DNA的提取及其PCR扩增产物的分析

利用PCR技术从海南山蛭体内分离山蛭素（抗凝血蛋白）基因,首先需获得不带有山蛭体表色素且完整的山蛭基因组DNA,本试验通过结合使用SDS-蛋白酶裂解法和CTAB法,有效的去除了山蛭基因组DNA提取过程中难以去除的色素,得到的基因组DNA保持完整,无降解,以之作为模板,进行PCR扩增,获得一个长度约为237bp的特异性片段,此片段与印度山蛭蛭素的cDNA大小几乎一致。初步表明,这个序列没有内含子,而仅是海南山蛭蛭素的编码序列。     

微波法快速提取放线菌基因组DNA

原有的放线菌基因组DNA提取方法费时、费力、费用高,且对极端环境放线菌成功率较低。利用微波热振惊提取固体培养基表面放线菌菌落基因组DNA,具有快速、简便、费用低廉等优点。所得的基因组DNA可作为PCR反应的模板进行16S rRNA基因有效扩增。这为大量放线菌菌株的快速鉴别和系统分类创造了条件。     

植物叶片基因组DNA快速提取方法

为了满足分子生物学研究中大量植株基因需要PCR检测的需求,建立了一种无需研磨、无需有机试剂抽提的快速提取植物叶片基因组DNA的方法。通过比较基因组DNA的浓度及PCR检测结果,获得了最佳提取条件,即约50 mg叶片加入150μL含1%β-巯基乙醇的TE提取液,快速破碎1 min。破碎后的样品4℃13 500×g离心1 min,上清于-20℃冷冻后室温融解,4℃、13 500×g离心1 min,离心后收集的上清溶液即可用于PCR检测。整个提取过程仅需10-12 min,具有样品用量小、操作简单、廉价、高效等优点。使用此方法提取的烟草、水稻、大豆、玉米、油菜和花生的基因组DNA均可用于PCR扩增,并可成功扩增长度为3 244 bp的基因片段。此方法提取的基因组DNA也可用于对未知基因的扩增,获得4条新的花生actin基因序列。     

基于AFLP分析用吴茱萸叶高质量DNA的提取

目的：研究吴茱萸叶基因组DNA的提取方法,以用于扩增片段长度多态性（AFLP）分析。方法：设计了一种改良CTAB法：以石英砂代替液氮研磨;抽提前用不溶性PVP结合酚形成络合物,然后用缓冲液除去;抽提中加入Vc。将改良CTAB法所提吴茱萸叶DNA与传统的SDS法、CTAB法所提DNA进行比较。利用植物的核糖体DNA（rDNA）保守序列设计引物行PCR扩增鉴定吴茱萸DNA及其质量。并确定提取方法中最佳样本含量和β-巯基乙醇浓度。结果：改良CTAB法提取石虎、疏毛吴茱萸总DNA呈白色,A260/A280为1.721~1.886,DNA分子完整,约20kb左右,PCR扩增条带清晰、明亮,无杂带和脱尾。并确定0.10g为最佳样本量,2.0%为最佳β-ME浓度。结论：石英砂研磨简便、迅速、均匀,该实验所建立的改良CTAB法可有效避免次生代谢物的氧化褐变,是一种小量、快速提取吴茱萸叶DNA优化方法。     

Chelex-100快速提取放线菌DNA作为PCR扩增模板

旨在建立有效扩增16S rRNA基因序列的放线菌DNA快速提取的方法。采用Chelex-100法提取放线菌DNA,使用PCR扩增16S rRNA基因序列评价提取核酸的质量。结果显示,Chelex-100法能够在10 min之内从放线菌中快速提取DNA,所提取的DNA可以直接用于PCR扩增反应,PCR扩增产物电泳条带清晰,符合理论预期结果。因此,Chelex-100法提取放线菌DNA可以作为16S rRNA基因序列PCR扩增的模板,该方法具有经济、简便、快速的特点,适合于放线菌菌株大规模地筛选和分类鉴定。     

阴道毛滴虫染色体组型观察

采用火焰干燥法制备阴道毛滴虫染色体标本,按 Levan等方法,进行核型分析。阴道毛滴虫的染色体数目为n＝10,2n ＝20,染色体的组型为3m、3sm、1st和3T。 Abstract：　The chromosomes ofTrichomonas vaginaliswere prepared by flame-drying method. Data of chromosome were measured and calculated. According to Levan's method, analysis of karyotype was described. The results showed that the chromosome number ofT. vaginalisconsisted of twenty pairs(2n=20) and chromosome type was 3m, 3sm, 1st and 3T.     

Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.     

阴道毛滴虫铁氧还蛋白基因的原核表达

目的在原核细胞中表达阴道毛滴虫铁氧还蛋白(ferredoxin,Fd)基因。方法构建阴道毛滴虫Fd基因的原核表达重组质粒pUC19-Fd,转化大肠埃希菌JM109感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白质表达。结果经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blot)分析,重组质粒在大肠埃希菌中表达出Fd。结论在大肠埃希菌中表达出了Fd。     

Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences

The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.     

A potentially functional mariner transposable element in the protist Trichomonas vaginalis

Mariner transposable elements encoding a D,D34D motif-bearing transposase are characterized by their pervasiveness among, and exclusivity to, animal phyla. To date, several hundred sequences have been obtained from taxa ranging from cnidarians to humans, only two of which are known to be functional. Related transposons have been identified in plants and fungi, but their absence among protists is noticeable. Here, we identify and characterize Tvmar1, the first representative of the mariner family to be found in a species of protist, the human parasite Trichomonas vaginalis. This is the first D,D34D element to be found outside the animal kingdom, and its inclusion in the mariner family is supported by both structural and phylogenetic analyses. Remarkably, Tvmar1 has all the hallmarks of a functional element and has recently expanded to several hundred copies in the genome of T. vaginalis. Our results show that a new potentially active mariner has been found that belongs to a distinct mariner lineage and has successfully invaded a nonanimal, single-celled organism. The considerable genetic distance between Tvmar1 and other mariners may have valuable implications for the design of new, high-efficiency vectors to be used in transfection studies in protists.     

A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ～ 160 Mb) has been sequenced and is composed of ～65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.     

乳酸杆菌的代谢产物对体外培养的阴道毛滴虫的影响

目的研究乳酸杆菌代谢产物对体外培养的阴道毛滴虫的杀伤作用。方法检测不同浓度的乳酸杆菌代谢产物对体外培养的阴道毛滴虫在不同作用时间下的杀伤率。结果乳酸杆菌代谢产物浓度10%,作用时间2 h、4 h和6 h体外培养毛滴虫的杀伤率分别为26.43%、37.47%和46.35%;浓度25%时杀伤率分别为43.56%、74.65%和90.15%;浓度50%杀伤率分别为92.36%、95.23%和99.01%。结论乳酸杆菌代谢产物的浓度越高,对体外培养的毛滴虫的杀伤力越大,作用时间越长,效果越好。     

Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.