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1.
Nat Commun ; 12(1): 2995, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016962

RESUMO

Studies along elevational gradients worldwide usually find the highest plant taxa richness in mid-elevation forest belts. Hence, an increase in upper elevation diversity is expected in the course of warming-related treeline rise. Here, we use a time-series approach to infer past taxa richness from sedimentary ancient DNA from the south-eastern Tibetan Plateau over the last ~18,000 years. We find the highest total plant taxa richness during the cool phase after glacier retreat when the area contained extensive and diverse alpine habitats (14-10 ka); followed by a decline when forests expanded during the warm early- to mid-Holocene (10-3.6 ka). Livestock grazing since 3.6 ka promoted plant taxa richness only weakly. Based on these inferred dependencies, our simulation yields a substantive decrease in plant taxa richness in response to warming-related alpine habitat loss over the next centuries. Accordingly, efforts of Tibetan biodiversity conservation should include conclusions from palaeoecological evidence.


Assuntos
Biodiversidade , DNA Antigo/análise , DNA de Plantas/análise , Aquecimento Global , Plantas/genética , Altitude , Código de Barras de DNA Taxonômico , Ecologia/métodos , Florestas , Paleontologia/métodos , Tibet
2.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800919

RESUMO

Trypsin inhibitors (TI), a common anti-nutritional factor in soybean, prevent animals' protein digestibility reducing animal growth performance. No commercial soybean cultivars with low or null concentration of TI are available. The availability of a high throughput genotyping assay will be beneficial to incorporate the low TI trait into elite breeding lines. The aim of this study is to develop and validate a breeder friendly Kompetitive Allele Specific PCR (KASP) assay linked to low Kunitz trypsin inhibitor (KTI) in soybean seeds. A total of 200 F3:5 lines derived from PI 547656 (low KTI) X Glenn (normal KTI) were genotyped using the BARCSoySNP6K_v2 Beadchip. F3:4 and F3:5 lines were grown in Blacksburg and Orange, Virginia in three years, respectively, and were measured for KTI content using a quantitative HPLC method. We identified three SNP markers tightly linked to the major QTL associated to low KTI in the mapping population. Based on these SNPs, we developed and validated the KASP assays in a set of 93 diverse germplasm accessions. The marker Gm08_44814503 has 86% selection efficiency for the accessions with low KTI and could be used in marker assisted breeding to facilitate the incorporation of low KTI content in soybean seeds.


Assuntos
Genes de Plantas , Melhoramento Vegetal , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Sementes/enzimologia , Soja/genética , Inibidor da Tripsina de Soja de Kunitz/genética , Alelos , Cromatografia Líquida de Alta Pressão/métodos , DNA de Plantas/análise , DNA de Plantas/genética , Ligação Genética , Fenótipo , Folhas de Planta/química , Soja/enzimologia , Inibidor da Tripsina de Soja de Kunitz/análise
3.
Methods Mol Biol ; 2250: 195-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33900606

RESUMO

Transposable elements (TEs) are ubiquitous repetitive components of eukaryotic organisms that show mobility in the genome against diverse stresses. TEs contribute considerably to the size, structure, and plasticity of genomes and also play an active role in genome evolution by helping their hosts adapt to novel conditions by conferring useful characteristics. We developed a simple and rapid method for investigation of genetic mobility and diversity among TEs in combination with a target region amplification polymorphism (TE-TRAP) marker system in gamma-irradiated sorghum mutants. The TE-TRAP marker system reveals a high level of genetic diversity, which provides a useful marker resource for genetic mobility research.


Assuntos
Elementos de DNA Transponíveis/genética , Variação Genética , Genoma de Planta/genética , Sorghum/genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , DNA de Plantas/análise , DNA de Plantas/genética , Eletroforese/métodos , Evolução Molecular , Tamanho do Genoma/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético
4.
Methods Mol Biol ; 2250: 207-218, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33900607

RESUMO

Transposable elements (TEs) are mobile, recurring DNA sequences scattered throughout genome and have a large impact on genome structure and function. Several genetic marker techniques were developed to exploit their ubiquitous nature. Sequence-specific amplified polymorphism (SSAP) is a TE-based genetic marker system that has been used in various purposes such as measuring genetic relatedness between species, deciphering the population structures, molecular tagging for agronomic development in marker-assisted breeding (MAS). In addition to SSAP, sequence characterized amplified region (SCAR) from the SSAP markers provides an added advantage in identifying qualitative traits. Once developed SCAR markers are efficient, fast, and reliable method for genetic evaluations. These methods can be useful especially for the crops which have no genetic sequence information. With improved discriminatory ability they offer access to dynamic and polymorphic regions of genome. These techniques can be useful in breeding programs to improve or develop high yielding crops.


Assuntos
Elementos de DNA Transponíveis/genética , Marcadores Genéticos/genética , Polimorfismo Genético , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Zea mays/genética , DNA de Plantas/análise , DNA de Plantas/genética , Eletroforese em Gel de Poliacrilamida/métodos , Variação Genética , Genoma de Planta , Melhoramento Vegetal/métodos
5.
Food Chem ; 356: 129684, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-33812194

RESUMO

In this study, we aim to develop a novel loop mediated isothermal amplification (LAMP) coupled with TaqMan (LAMP-TaqMan) method for quick qualitative detection of genetically modified organism (GMOs). We designed four LAMP primers and one TaqMan probe for the LAMP-TaqMan detection method to detect the nopaline synthase gene (NOS) terminator in GMOs. This assay enabled the amplification of DNA within ~20 min at a constant temperature of 65 °C. This assay detected as few as five copies of target sequences, which had a high specificity similar to the TaqMan qPCR method. Furthermore, the LAMP-TaqMan detection method was successfully used to amplify and detect DNA from food samples of the major crops (soybean, maize, rice, etc.). In summary, a novel LAMP-TaqMan assay has been developed, which has the similar sensitivity but takes less time than the TaqMan qPCR method. This method offers a novel approach for rapid detection of GMOs in foods.


Assuntos
Aminoácido Oxirredutases/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/enzimologia , Produtos Agrícolas/enzimologia , Produtos Agrícolas/genética , Primers do DNA/química , Primers do DNA/metabolismo , DNA de Plantas/análise , DNA de Plantas/metabolismo , Limite de Detecção , Plantas Geneticamente Modificadas/genética , Soja/enzimologia , Soja/genética , Zea mays/enzimologia , Zea mays/genética
6.
Molecules ; 26(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806424

RESUMO

The free online trading of herbal mixtures useful for various purposes facilitates the circulation of dangerous herbs or plant parts. This is the case, for example, of the illegal trade in seeds of Peganum harmala (Pgh), which contain alkaloids capable of inhibiting monoamine oxidase (MAO) and are therefore used in hallucinogenic preparations, such as the psychedelic drink ayahuasca. The precise identification of these seeds and their distinction from other very similar but not dangerous seeds are necessary for forensic purposes and represents an advance in avoiding the adulteration of mixtures. In this work, we show the qualitative identification of Pgh seeds by optical and electron microscopy and the parallel development of a real-time qPCR test, which reveals, in a species-specific manner, the presence of Pgh DNA up to quantities lower than 1 pg. In addition to the species specificity and high sensitivity, the reaction accurately quantifies the presence of seeds or parts of seeds of Pgh in complex herbal mixtures, thus giving an indication of the danger or otherwise of the product.


Assuntos
Alcaloides/análise , DNA de Plantas/análise , Suplementos Nutricionais/análise , Inibidores da Monoaminoxidase/análise , Peganum/química , Extratos Vegetais/análise , Sementes/química , Alcaloides/toxicidade , DNA de Plantas/genética , Suplementos Nutricionais/toxicidade , Inibidores da Monoaminoxidase/toxicidade , Peganum/classificação , Extratos Vegetais/toxicidade , Proteínas de Plantas/genética , Especificidade da Espécie
7.
Methods Mol Biol ; 2264: 47-53, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33263902

RESUMO

Isolating high-quality DNA is essential for several applications in molecular biology and genomics. Performing whole-genome sequencing in crops and development of reduced representation genomic libraries for genotyping require precise standard on DNA in terms of concentration and purity. For screening large populations it is essential to increase the extraction throughput at affordable costs. In this chapter a homemade protocol is provided that is able to isolate in 96-well plates 198 samples of DNA in a single extraction. The method has been validated in tomato and pepper and can be applied in several vegetable species.


Assuntos
Produtos Agrícolas/genética , DNA de Plantas/análise , DNA de Plantas/isolamento & purificação , Genômica/métodos , Técnicas de Genotipagem/métodos , Ensaios de Triagem em Larga Escala/métodos , Verduras/genética
8.
Methods Mol Biol ; 2264: 55-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33263903

RESUMO

High-resolution melting (HRM) analysis is a cost-effective, specific, and rapid tool that allows distinguishing genetically related plants and other organisms based on the detection of small nucleotide variations, which are recognized from melting properties of the double-stranded DNA. It has been widely applied in several areas of research and diagnostics, including botanical authentication of several food commodities and herbal products. Generally, it consists of the main steps: (1) in silico sequence analysis and primer design; (2) DNA extraction from plant material; (3) amplification by real-time PCR with an enhanced fluorescent dye targeting a specific DNA barcode or other regions of taxonomic interest (100-200 bp); (4) melting curve analysis; and (5) statistical data analysis using a specific HRM software. This chapter presents an overview of HRM analysis and application, followed by the detailed description of all the required reagents, instruments, and protocols for the successful and easy implementation of a HRM method to differentiate closely related plant species.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA de Plantas/análise , DNA de Plantas/genética , Análise de Alimentos/métodos , Proteínas de Plantas/genética , Plantas Medicinais/genética , Reação em Cadeia da Polimerase/métodos , DNA de Plantas/isolamento & purificação , Plantas Medicinais/classificação , Especificidade da Espécie
9.
Methods Mol Biol ; 2264: 75-87, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33263904

RESUMO

Most plant agronomic traits are quantitatively inherited. Identification of quantitative trait loci (QTL) is a challenging target for most scientists and crop breeders as large-scale genotyping is difficult. Molecular marker technology has continuously evolved from hybridization-based technology to PCR-based technology, and finally, sequencing-based high-throughput single-nucleotide polymorphisms (SNPs). High-throughput sequencing technologies can provide strategies for sequence-based SNP genotyping. Here we describe the SLAF-seq that can be applied as the SNP genotyping approach. The high-throughput SNP genotyping methods will prove useful for the construction of high-density genetic maps and identification of QTLs for their deployment in plant breeding and facilitate genome-wide selection (GWS) and genome-wide association studies (GWAS).


Assuntos
Mapeamento Cromossômico/métodos , DNA de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Plantas/genética , Plantas/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , DNA de Plantas/análise , DNA de Plantas/isolamento & purificação , Estudo de Associação Genômica Ampla , Fenótipo
10.
Methods Mol Biol ; 2264: 89-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33263905

RESUMO

Forward genetic analysis remains as one of the most powerful tools for assessing gene functions, although the identification of the causal mutation responsible for a given phenotype has been a tedious and time-consuming task until recently. Advances in deep sequencing technologies have provided new approaches for the exploitation of natural and artificially induced genetic diversity, thus accelerating the discovery of novel allelic variants. In this chapter, a mapping-by-sequencing forward genetics approach is described to identify causal mutations in tomato (Solanum lycopersicum L.), a major crop species that is also a model species for plant biology and breeding.


Assuntos
Mapeamento Cromossômico/métodos , Cromossomos de Plantas/genética , DNA de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Lycopersicon esculentum/genética , Mutação , Proteínas de Plantas/genética , DNA de Plantas/análise , DNA de Plantas/isolamento & purificação , Genoma de Planta , Fenótipo
11.
Methods Mol Biol ; 2264: 105-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33263906

RESUMO

Quantitative trait loci mapping has become a common practice in crop plants and can be accomplished using either biparental populations following interval mapping or natural populations following the approach of association mapping. Because of its ability to use the natural diversity and to search for functional variants in a broader germplasm, association mapping is becoming popular among researchers. An overview of the different steps involved in association mapping in plants is provided in this chapter.


Assuntos
Mapeamento Cromossômico/métodos , Cromossomos de Plantas/genética , DNA de Plantas/genética , Proteínas de Plantas/genética , Plantas/genética , Polimorfismo de Nucleotídeo Único , DNA de Plantas/análise , DNA de Plantas/isolamento & purificação , Estudo de Associação Genômica Ampla , Genótipo , Desequilíbrio de Ligação , Fenótipo , Locos de Características Quantitativas
12.
Methods Mol Biol ; 2264: 119-135, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33263907

RESUMO

The global climate is changing, resulting in significant economic losses worldwide. It is thus necessary to speed up the plant selection process, especially for complex traits such as biotic and abiotic stresses. Nowadays, genomic selection (GS) is paving new ways to boost plant breeding, facilitating the rapid selection of superior genotypes based on the genomic estimated breeding value (GEBV). GEBVs consider all markers positioned throughout the genome, including those with minor effects. Indeed, although the effect of each marker may be very small, a large number of genome-wide markers retrieved by high-throughput genotyping (HTG) systems (mainly genotyping-by-sequencing, GBS) have the potential to explain all the genetic variance for a particular trait under selection. Although several workflows for GBS and GS data have been described, it is still hard for researchers without a bioinformatics background to carry out these analyses. This chapter has outlined some of the recently available bioinformatics resources that enable researchers to establish GBS applications for GS analysis in laboratories. Moreover, we provide useful scripts that could be used for this purpose and a description of key factors that need to be considered in these approaches.


Assuntos
Cromossomos de Plantas/genética , Biologia Computacional/métodos , Genoma de Planta , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Melhoramento Vegetal/métodos , Plantas/genética , DNA de Plantas/análise , DNA de Plantas/genética , Variação Genética , Fenótipo , Seleção Genética
13.
Food Chem ; 338: 127812, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32861133

RESUMO

Here, we describe DNA enrichment of the zein gene from maize using pyrrolidinyl peptide nucleic acid (PNA) immobilized on a magnetic solid support as a capture element. Magnetite nanoparticles (MNP) with a capacity of 373 pmolPNA/mg and coated with poly(N-acryloylglycine) (PNAG) showed a good response to magnetic field. The PNA probe immobilized on the MNP discriminated between non-complementary and complementary DNA using fluorophore-tagged DNA as a model. We applied this system for the enrichment of the zein gene from maize in eight cereal product samples. After DNA desorption from the MNP, and its amplification via polymerase chain reaction (PCR), gel electrophoresis indicated that only cereal samples containing the zein gene from maize yielded positive results, indicating a high binding specificity between the PNA used and the complementary DNA. This PNA-functionalized MNP is potentially useful as an effective nano-solid support for DNA enrichment from other samples.


Assuntos
DNA de Plantas/análise , Nanopartículas de Magnetita/química , Ácidos Nucleicos Peptídicos/química , Zea mays/genética , Zeína/genética , DNA Complementar/análise , Grão Comestível/genética , Eletroforese , Corantes Fluorescentes/química , Fenômenos Magnéticos , Reação em Cadeia da Polimerase , Espectrometria de Fluorescência
14.
J Vis Exp ; (160)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32628154

RESUMO

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.


Assuntos
Polinização , Prunus armeniaca/fisiologia , DNA de Plantas/análise , Flores/fisiologia , Genótipo , Microscopia de Fluorescência , Folhas de Planta/genética , Pólen/fisiologia , Reação em Cadeia da Polimerase , Prunus armeniaca/genética
15.
Food Chem ; 326: 126986, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32407998

RESUMO

In the present work, a barcode-DNA analysis method is described for the detection of plant oil adulteration in milk and dairy products. The method relies on the fact that plant DNA should not be present in readily detectable amounts in a dairy product unless it contains undeclared plant material. Thus, a universal plant barcode is chosen as the target to be amplified from dairy samples. Accordingly, barcode PCR-CE (PCR-capillary electrophoresis) assays are described, which do not require preliminary information on the species source of the adulterant oil type. Two PCR-CE assays, one operating on the plastid trnL (UAA) intron and the other targeting its inner P6 loop in nested format, were shown to detect corn, soybean, rapeseed and sunflower oils in clarified butter, milk and yogurt. Both barcodes are robustly amplified with extremely conserved primers. While the intron provides the species discrimination ability, the P6 loop provides superior detection sensitivity.


Assuntos
DNA de Plantas/análise , Laticínios/análise , Eletroforese Capilar/métodos , Leite/química , Óleos Vegetais/química , Animais , Código de Barras de DNA Taxonômico , DNA de Plantas/genética , DNA de Plantas/metabolismo , Óleos Vegetais/metabolismo , Plastídeos/genética , Reação em Cadeia da Polimerase , Soja/genética , Iogurte/análise , Zea mays/genética
16.
Shokuhin Eiseigaku Zasshi ; 61(1): 22-30, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32336715

RESUMO

An identification method for testing contamination in products was assessed using various vegetables and fruits (70 types in total). DNA was extracted from plant fragments which are 1 to several millimeters long and the plastid rpl16-rpl14 linker sequence (approximately 550 base pairs) was amplified by PCR. The DNA nucleotide sequence was determined, and homology and SNP (single nucleotide polymorphism) analyses were carried out. Consequently, the test plants were difficult to distinguish between closely related species, but could be divided into 38 groups at the genus level or the species level. Although problems such as the accuracy of discrimination among some closely related plants and DNA stability under an acidic condition remain to be resolved, this method is considered to be expected to identify plant fragments mixed in products or raw materials.


Assuntos
DNA de Plantas/análise , Frutas/química , Plantas Comestíveis/genética , Plastídeos/genética , Verduras/química , Polimorfismo de Nucleotídeo Único
17.
Pharmaceut Med ; 34(1): 49-61, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32048209

RESUMO

INTRODUCTION: Methods for assessing the quality of herbal medicine preparations have advanced significantly in recent years in conjunction with increases in herbal medicine use and reports of adulteration and contamination. OBJECTIVE: This study examined the quality of analgesic and anti-inflammatory herbal medicine preparations available on the Australian market by detecting the presence of listed ingredients, adulterants and contaminants. METHODS: Forty-nine analgesic and anti-inflammatory herbal medicine preparations were randomly sourced from Australian capital cities. They were audited using a dual approach of liquid chromatography-mass spectrometry (LC-MS) combined with next-generation DNA sequencing. Once screened, a comparison of listed ingredients with verified ingredients was conducted to determine the accuracy of labelling, and the extent of adulteration and contamination. RESULTS: Twenty-six of 49 (53%) herbal medicines were adulterated or contaminated with undeclared ingredients. LC-MS revealed the presence of pharmaceutical adulterants including atropine and ephedrine. DNA sequencing uncovered concerning levels of herbal substitution, adulteration and contamination, including the use of fillers (alfalfa, wheat and soy), as well as pharmacologically relevant species (Centella asiatica, Panax ginseng, Bupleurum and Passiflora). Pig/boar and bird DNA was found in some preparations, inferring substandard manufacturing practices. Of the 26 contaminated samples, 19 (73%) were manufactured in Australia, and 7 (27%) were imported from other countries (6 from China, 1 from New Zealand). In 23 of 49 (47%) herbal medicine samples, no biological ingredients were detected at all. These were predominantly pain and anti-inflammatory preparations such as glucosamine and eicosapentaenoic and docosahexaenoic acids found in krill and fish oils, so DNA would not be expected to survive the manufacturing process. CONCLUSION: The high level of contamination and substitution of herbal medicine preparations sourced from Australian dispensaries supports the need for more stringent pharmacovigilance measures in Australia and abroad.


Assuntos
Analgésicos/análise , Anti-Inflamatórios/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Preparações de Plantas/análise , Austrália , China , Cromatografia Líquida , DNA de Plantas/análise , Contaminação de Medicamentos , Espectrometria de Massas , Nova Zelândia , Plantas , Análise de Sequência de DNA
18.
PLoS One ; 15(2): e0228624, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101546

RESUMO

We report a rapid and accurate quantitative detection method using droplet digital PCR (ddPCR) technology to identify cassava adulteration in starch products. The ddPCR analysis showed that the weight of cassava (M) and cassava-extracted DNA content had a significant linear relationship-the correlation coefficient was R2 = 0.995, and the maximum coefficient of variation of replicates was 7.48%. The DNA content and DNA copy number (C) measured by ddPCR also had a linear relationship with R2 = 0.992; the maximum coefficient of variation of replicates was 8.85%. The range of cassava ddPCR DNA content was 25 ng/µL, and the formula M = (C + 32.409)/350.579 was obtained by converting DNA content into the median signal. The accuracy and application potential of the method were verified using the constructed adulteration model.


Assuntos
Análise de Alimentos/métodos , Contaminação de Alimentos , Manihot/química , Reação em Cadeia da Polimerase/métodos , Amido/química , DNA de Plantas/análise , DNA de Plantas/genética , Análise de Alimentos/normas , Manihot/genética , Reação em Cadeia da Polimerase/normas , Amido/normas
19.
Anal Bioanal Chem ; 412(7): 1701-1707, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31965247

RESUMO

Two "mass ratio-DNA copy concentration ratio" formulas were established respectively on droplet digital PCR (ddPCR) and chip-based digital PCR (cdPCR) to determine the mass ratio of kidney bean, a common alternative plant-derived ingredient in lotus seed paste. The limit of detection for DNA copy concentration of kidney bean and lotus seed was 6 copies/µL. Quantitative detection range was set from 5 to 80%, and the limit of quantification for mass ratio of kidney bean in lotus seed paste was defined as 5%. Results of 6 simulated samples and 16 prepackaged pastes in this work offer compelling evidence that an innovative scheme for quantitative detection of kidney bean in lotus seed paste was available, and provide technical support for the identification of suspicious ingredients from fraudulent substitution or adventitious contamination. Graphical abstract Two "mass ratio-DNA copy concentration ratio" formulas were established respectively on droplet digital PCR (ddPCR) and chip digital PCR (cdPCR) to determine the mass ratio of kidney bean in adulterated lotus seed paste. It was the first time to quantify adulterate food by directly converting DNA copy concentration ratio obtained from digital PCR to mass ratio, which could provide strong technical support for quantitative detection of adulterated food.


Assuntos
Contaminação de Alimentos/análise , Lotus/embriologia , Phaseolus , Reação em Cadeia da Polimerase/métodos , Sementes , Variações do Número de Cópias de DNA , DNA de Plantas/análise , Genes de Plantas , Limite de Detecção , Lotus/genética , Phaseolus/genética , Reprodutibilidade dos Testes
20.
Mol Biol Rep ; 47(1): 639-654, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31781917

RESUMO

3-Hydroxy-3-methylglutaryl-coenzymeA reductase (HMGR), the first rate-limiting enzyme of Mevalonate (MVA) pathway was isolated from Andrographis paniculata (ApHMGR) and expressed in bacterial cells. Full length ApHMGR (1937 bp) was submitted to NCBI with accession number MG271748.1. The open reading frame (ORF) was flanked by a 31-bp 5'-UTR, 118-bp 3'-UTR and ApHMGR contained a 1787 bp ORF encoding protein of 595 amino acids. ApHMGR protein was approximately 64 kDa, with isoelectric point of 5.75. Isolated ApHMGR was cloned into pET102 vector and expressed in E. coli BL21 (DE 3) cells, and characterized by SDS-PAGE. HPLC analysis for andrographolide content in leaf, stem and root of A. paniculata revealed highest in leaf tissue. The expression patterns of ApHMGR in different plant tissues using qRT-PCR revealed high in root tissue correlating with HPLC data. Three dimensional (3D) structural model of ApHMGR displayed 90% of the amino acids in most favored regions of the Ramachandran plot with 93% overall quality factor. ApHMGR was highly conserved with plant specific N-terminal membrane domains and C-terminal catalytic regions. Phylogenetic analysis showed A. paniculata sharing common ancestor with Handroanthus impetiginosus. 3D model of ApHMGR was screened for the interaction with substrates NADPH, HMG CoA and inhibitor using Auto Dock Vina. In silico analysis revealed that full length ApHMGR had extensive similarities to other plant HMGRs. The present communication reports the isolation of full length HMGR from A. paniculata, its heterologous expression in bacterial cells and in silico structural and functional characterization providing valuable genomic information for future molecular interventions.


Assuntos
Andrographis , Hidroximetilglutaril-CoA Redutases , Proteínas de Plantas , Andrographis/classificação , Andrographis/enzimologia , Andrographis/genética , Andrographis/metabolismo , DNA de Plantas/análise , DNA de Plantas/genética , Hidroximetilglutaril-CoA Redutases/química , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/isolamento & purificação , Hidroximetilglutaril-CoA Redutases/metabolismo , Ácido Mevalônico/metabolismo , Simulação de Acoplamento Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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