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本文针对大豆内源基因Lectin和转基因大豆DAS81419品系的5′端插入位点序列,设计特异性引物及探针,建立了同时检测转基因大豆DAS81419品系和大豆内源基因Lectin的二重荧光定量PCR方法,运用15种转基因大豆、3种转基因玉米、1种转基因油菜、1种转基因水稻和非转基因大豆对该方法进行了特异性评价,并分析了该方法的灵敏度和稳定性。结果显示,该方法能准确从20种转基因样品和1种非转基因样品中检出靶目标,检测结果与待检样品信息一致,表明本方法具有良好的特异性;灵敏度高达0.01%;并具有良好的重复性。该方法特异性强、灵敏度高、稳定性强,适用于各口岸实验室进行转基因大豆DAS81419的快速、准确的检测。 相似文献
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转基因延熟番茄"华番一号"的品系特异性检测方法 总被引:3,自引:0,他引:3
本文使用反向PCR方法克隆了转基因延熟番茄"华番一号"(Bioscein)的外源基因和番茄基因组之间的一段边界序列,并依据此段序列设计了具有品系特异性的引物和荧光探针,以实时荧光PCR技术建立了华番一号的品系鉴定检测方法,扩增片段长108bp,横跨在Nos启动子和番茄基因组之间.以转基因大豆(RR)、转基因玉米(Mon810)、转基因抗草甘膦油菜、转基因棉花(保龄棉)、非转基因番茄、马铃薯、茄子、大椒、大米、小麦、烟草等为试材,证明本方法同其它转基因作物及其它蔬菜无非特异性反应.本方法在检测华番一号番茄时,相对检测灵敏度可达到0.1%,绝对灵敏度达到20个拷贝.由于本方法的PCR扩增产物长度只有108bp,因此该方法也可以用于检测加工产品中的转基因成分,或作为常规PCR定性检测后的确证实验方法. 相似文献
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多重PCR-DHPLC法检测转基因棉花品系 总被引:1,自引:0,他引:1
根据3种转基因棉花品系的边界序列设计品系检测引物,建立了一种特异性检测转基因棉花品系MON531、MON1445和MON15985的多重PCR-DHPLC方法。以20种不同的转基因及非转基因作物DNA验证该方法的特异性,结果只有MON531、MON15985和MON1445有特异的品系扩增片段峰,而其他转基因和非转基因作物无品系扩增片段峰,表明该方法特异性强。灵敏度实验结果表明3种转基因棉花的检测下限均为1 ng,灵敏度高。建立的方法可用于转基因棉花MON531、MON1445和MON15985品系及含有其成分产品的筛查或定性检测。 相似文献
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本文以具有抗二化螟性状的Csu-miR260转基因水稻为试验材料, 采用TaqMan探针实时荧光定量PCR (TaqMan RT-qPCR)和茎环实时荧光定量PCR(stem-loop RT-qPCR)分别对Csu-miR260转基因抗虫水稻中Csu-miR260插入序列和剪切形成的amiR260s的表达情况进行了定量检测。发现Csu-miR260插入序列和剪切形成的20 nt和21 nt两种长度amiR260s在转基因抗虫水稻苗期的根、茎、叶中表达, 且无组织表达特异性。该试验解决了人工miRNA作物中amiRNA及前体检测引物的特异性问题, 为人工miRNA植物干扰序列的表达分析提供技术参考, 并为miRNA转基因作物遗传表达相关安全评价提供了数据参考。 相似文献
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[目的] 明确武汉、荆州、孝感、随州、武穴5个棉花主产区烟粉虱生物型组成。[方法] 通过以线粒体细胞色素氧化酶I(mitochondrial cytochrome oxidase I, mtCOI)为基础的PCR RFLP和基因测序的方法,鉴定湖北5个棉花主产区烟粉虱地理种群的生物型组成,并分析5个地理种群与其他省种群的系统发育关系。[结果] Q型烟粉虱为湖北省棉花主产区的优势种群,而武汉温室种群则均为B型烟粉虱,土著生物型ZHJ1型在大田中略有分布;系统发育分析表明,湖北省棉花主产区B型和Q型烟粉虱种群均与中国其他多个省的烟粉虱种群亲缘关系较近。[结论] 本文研究结果可以为湖北省5个棉花主产区烟粉虱的有效防治提供参考。 相似文献
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c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)是MAPK家族(mitogen-activated protein kinases, 丝裂原活化蛋白激酶)的重要成员, 具有参与昆虫抗逆反应等多种功能。为明确JNK在棉铃虫 Helicoverpa armigera 中的表达特性及其对Bt杀虫蛋白的应激与免疫反应, 本研究通过PCR克隆得到2个棉铃虫 JNK 基因 HaJNK1 和 HaJNK2; 生物信息学分析结果显示: HaJNK1和 HaJNK2基因开放阅读框分别为1 191、1 143 bp, 分别编码397、381个氨基酸。系统进化树分析结果表明棉铃虫 HaJNK1 与黏虫 Mythimna separata 聚为一支, 亲缘关系较近, HaJNK2与 家蚕 Bombyx mori 聚为一支, 同源性较高。利用实时荧光定量PCR技术分析 HaJNK1 与 HaJNK2 在棉铃虫不同发育时期、不同组织中的表达量, 发现 HaJNK1 与 HaJNK2 在卵中表达量最高, 其次是雌成虫; HaJNK1 在性腺中表达量最高, 其次是唾液腺; HaJNK2 在头部表达量最高, 其次是性腺。取食Cry1Ac的4龄棉铃虫幼虫的中肠组织中, HaJNK1 与 HaJNK2 的表达量均显著升高。推测 HaJNKs 基因可能参与棉铃虫抵御Bt杀虫蛋白伤害的应激和抗逆反应。 相似文献
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Federica Bini Klaus Geider Carlo Bazzi 《European journal of plant pathology / European Foundation for Plant Pathology》2008,122(3):403-411
Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted
for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and
ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine
strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For
simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was
performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from
grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets
were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells
per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA
purification kit as a step for the isolation of nucleic acids. 相似文献
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C. M. Andrade M. L. P. Tinoco A. F. Rieth F. C. O. Maia F. J. L. Aragão 《Plant pathology》2016,65(4):626-632
Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens. 相似文献
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Mehrdad Madani Len J. Ward Solke H. De Boer 《European journal of plant pathology / European Foundation for Plant Pathology》2011,130(3):271-285
The heat-shock gene, Hsp90, was targeted as a new variable genomic region to supplement other DNA-based tests for identification and discrimination
of Globodera pallida, G. rostochiensis and G. tabacum tabacum. Populations of the potato cyst nematodes, G. pallida and G. rostochiensis (PCN), originating from Canada, France, Belgium and USA, together with two populations of G. tabacum tabacum from the USA and France were used for the amplification of a fragment of the Hsp90 gene. General and specific primers and probes for each species were derived from the consensus and non-consensus regions
of the aligned sequences, respectively. A triplex conventional PCR assay, using a general forward and reverse or three specific
reverse primers, as well as a real-time PCR using general primers and specific TaqMan probes, were developed. Melting curve
analysis and restriction fragment polymorphisms using high resolution electrophoresis were explored for identifying PCR amplicons
that characterized and discriminated the three Globodera species in both pure and mixed samples. Results from the different molecular assay strategies confirmed the usefulness of
Hsp90 as a new additional gene target and showed that several different test options could be used for discrimination of PCN. 相似文献
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ABSTRACT Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB. 相似文献
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Alexandra Triantafyllopoulou Andriani Tzanaki Olga Iakovina Balomenou Rafael M. Jiménez-Díaz Aliki Tzima Epaminondas Paplomatas 《Plant pathology》2022,71(6):1404-1416
Verticillium dahliae is a soilborne fungus that causes Verticillium wilt disease in a plethora of crops. Based on symptoms that develop on cotton, olive and okra, V. dahliae isolates are categorized into two pathotypes, namely defoliating and nondefoliating, with the former showing increased virulence and causing severe defoliation. Reliable differentiation between V. dahliae pathotypes is crucial for the management of Verticillium wilt in cotton and olive. In the present study, a polymorphism was detected among isolates of defoliating and nondefoliating pathotypes in Southern blots using the VdNEP gene as a probe. The regions flanking this gene were isolated by inverse PCR and sequence differences in the 3′ untranslated region (3′-UTR) of the VdNEP gene were detected between the two pathotypes. Based on these sequences, primers were designed and assessed to develop a multiplex PCR detection assay. Using this assay, a collection of cotton and olive V. dahliae isolates from Greece and Cyprus was screened, revealing that the defoliating pathotype is present in several regional units of Greece. Thus, this work presents a new, sensitive molecular marker for the differentiation between V. dahliae pathotypes based on the VdNEP gene. Because the 3′-UTR is involved in the phenotypes displayed by the pathotypes, an expression experiment was conducted under conditions simulating the xylem of a host plant. Expression of the VdNEP gene was elevated at all time points in the defoliating compared to the nondefoliating strain, suggesting a possible involvement of VdNEP expression in the defoliation process. 相似文献
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Selected wild Dahlia species in their natural habitats from west‐central Mexico were tested for the presence of three caulimoviruses known to be associated with cultivated dahlia (Dahlia variabilis), viz. Dahlia mosaic virus (DMV), DMV‐D10 and Dahlia common mosaic virus. Virus species‐specific primers and PCR were used followed by cloning and sequencing of the amplicons. Results showed that the wild dahlia species in their natural habitat contained DMV‐D10, which is an endogenous plant pararetrovirus. Viral sequences were found in 91% of the samples (n = 56) representing four different wild species. The gene coding for the movement protein of DMV‐D10 from Dahlia coccinea and all other species was cloned and sequenced. Sequence comparisons showed divergence of this gene when compared to that of DMV‐D10 from cultivated dahlias. The discovery of plant pararetroviruses in wild dahlia species in their natural habitats suggests a possible emergence, co‐existence and co‐evolution of pararetroviruses and their host plants. 相似文献