CN104075983B - A kind of assay method being applicable to Gesneriaceae Genome Size - Google Patents
A kind of assay method being applicable to Gesneriaceae Genome Size Download PDFInfo
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Abstract
The invention discloses a kind of assay method being applicable to Gesneriaceae Genome Size.The fresh tender leaf of Gesneriaceae; Select tomato to be main internal reference, paddy rice is the second internal reference, and is that standard is proofreaded again to rice genome size according to tomato dna group size; According to the amount adding 20mg tender leaf in 1ml core Extraction buffer LB01, respectively the tender leaf of object plant and internal reference species is added in core Extraction buffer LB01, then tender leaf is shredded, obtain nucleus suspension by the filter screen filtration in 50 μm of apertures, be placed in for subsequent use on ice; To use the RNA in RNA digestive ferment vitellophag core suspension again, then add propidium iodide fluorescence dyeing, does is final concentration 100 μ g? ml
-1, lucifuge dyes, and dyeing time is 40min; And then apply the Genome Size of cells were tested by flow cytometry object plant.The present invention's can be multiple hare's-lettuce section determination of plant Genome Size accurately, coefficient of variation CV is lower than 5%, and the method is reproducible, simple to operate, meets the research of hare's-lettuce section botany aspect.
Description
Technical field:
The invention belongs to molecular genetic and Study on Evolution field, be specifically related to a kind of assay method being applicable to Gesneriaceae Genome Size.
Background technology:
Genome Size (C-value) refers to the DNA content of haploid cell core, Genome Size variation is one of important motive power of species formation and evolution, increasing research shows the biological research fields of Genome Size in many keys, as aspects such as ecology, cytology, molecular biology, systematics and evolution biologies, there is important theory and practical application meaning.Theory expectation, the complexity of genomic size and species is directly proportional, i.e. simpler species, and its genome is less; More complicated species, its genome is larger.Even increasing research shows the species belonging to same class, the change of their Genome Size also may be very large, and the complicacy between species is more or less the same, i.e. so-called " C value antinomy ".In order to better understand the relation between the complicacy of species and its Genome Size, the accurate estimation of Genome Size is just seemed more and more important.
Flow cytometry (Flowcytometry, FCM) measures Plant Genome size effective method fast.But, in plant, the existence (as anthocyanidin etc.) of a large amount of secondary metabolites can reduce PI fluorescent dye intensity greatly, have a strong impact on accuracy and validity that FCM method measures Genome Size, and then cause the subsequent analysis of Genome Size and application to produce great deviation, there is result and the conclusion of mistake in the research in Plant Taxonomy, evolution biology, molecular genetics etc.Therefore, before mensuration Genome Size, determine the optimal FCM assay method of object species, comprise the technical conditions such as nuclear extraction, dyeing most important.
Gesneriaceae (Gesneriaceae) plant is the characteristic monoid of China karst, about has 150 genus 3700 kinds in the whole world, and China is distributed with 58 genus more than 470 and plants.This section at the distribution of China and peculiar center in south China karst, due to the height heterogeneity in habitat, karst, the functional trait of Gesneriaceae has strong differentiation, show that this platymiscium is one and carries out the idealized model system of karst plant to environmental adaptation, significant for the responses and adaptation of research karst bio-diversity to environmental change.Due to the low water holding capacity of karst soil, periodically water resource lacks, and the features such as nutrition is barren create stronger selection pressure to this area's plant species.Research before shows, soil nutrient lack and abiotic selection pressure as arid, high light, low temperature etc., can the synthesis of the secondary metabolites such as inducer blade anthocyanidin.Therefore, the selection pressure of karst environment may affect the accuracy that FCM measures this region Plant Genome size.Although this phyto-group has critical role in China's karst environment adaptive evolution and conservation of resources research, its optimal Genome Size assay method is not yet clear and definite.
Visible, optimize this monoid Genome Size measure FCM method for its follow-up biological study as systematics, evolutionary genetics have important theory and practical significance.Specify its optimal assay method, the accuracy that guarantee Genome Size measures, and follow-up study is as genealogical classification, the validity analyzed with correlativity etc. that is ecological and grade of fit feature, has important impetus to the research of the adaptive evolution mechanism of China's karst special habitats regional characteristic phyto-group.
Summary of the invention:
In order to the practical application that the technology barrier and follow-up Plant Taxonomy, evolution biology, molecular genetics etc. that solve Gesneriaceae Genome Size Accurate Determining are studied, the object of this invention is to provide a kind of assay method being applicable to Gesneriaceae Genome Size.
The assay method being applicable to Gesneriaceae Genome Size of the present invention, is characterized in that, comprise the following steps:
Material prepares: the fresh tender leaf of the object plant of Gesneriaceae;
Internal reference is selected: the prediction (1C is about 0.5-1.1pg) carrying out fluorescent intensity and Genome Size after instrument testing, tomato (Solanumlycopersicumcv. ' Stupick é polniran é ' is selected according to predicted gene group size, 1C=0.98pg) be main internal reference, paddy rice (Oryzasativassp.japonica, 1C=0.43-0.45pg) be the second internal reference, and be that standard is proofreaded again to rice genome size according to tomato dna group size;
Prepare nucleus suspension: object plant and internal reference species, according to the amount adding 20mg tender leaf in 1ml core Extraction buffer LB01, respectively the tender leaf of object plant and internal reference species is added in core Extraction buffer LB01, then tender leaf is shredded, nucleus suspension is obtained by the filter screen filtration in 50 μm of apertures, the formula being placed in core Extraction buffer LB01 for subsequent use, described is on ice: 15mMTris, 2mMNa
2eDTA, 0.5mMspermine.4HCl, 80mMKCl, 20mMNaCl, 15mM β-mercaptoethanol, 0.1% (v/v) TritonX-100, pH7.5;
Use the RNA in RNA digestive ferment vitellophag core suspension again, RNA digestive ferment final concentration is 50 μ gml
-1; Then the dyeing of propidium iodide fluorescence lucifuge is added; Finally apply the Genome Size of cells were tested by flow cytometry object plant.
Preferably, the final concentration of described propidium iodide is 100 μ gml
-1, dyeing time is 40min.
The present invention is according to the character of hare's-lettuce section plant, start with from the factor affecting Flow cytometry Genome Size, the Genome Size accurately for multiple hare's-lettuce section determination of plant, coefficient of variation CV (coefficientofvariation) is lower than 5%, the method is reproducible, simple to operate, meet the research of hare's-lettuce section botany aspect.
Accompanying drawing illustrates:
Fig. 1 adopts core Extraction buffer LB01 of the present invention and other buffer (deLaat ' s, Galbraith ' s, GeneralPurpose, MgSO
4, Partec, Tris.MgCl
2and WoodyPlant) peak figure and CV that measure line leaf primulina tabacum be worth comparing;
Fig. 2 is that 24h dark processing and non-dark processing measure the comparison of (ungated does not establish door) to Huaiji primulina tabacum and tongue post primulina tabacum Genome Size.
Fig. 3 is the peak figure and the CV value thereof that adopt method of the present invention to measure line leaf primulina tabacum.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
The experimental technique used in following embodiment if no special instructions, is conventional method.
In following embodiment use material, reagent etc. if no special instructions, all can obtain from commercial channels.
Embodiment 1:
For line leaf primulina tabacum, gathering line leaf primulina tabacum, tomato (Solanumlycopersicumcv. ' Stupick é polniran é ') and paddy rice (Oryzasativassp.japonica, 1C=0.43-0.45pg) each 20mg of tender leaf, be main internal reference with tomato, paddy rice is the second internal reference, and is that standard is proofreaded again to rice genome size according to tomato dna group size, have in the double dish of 1ml core Extraction buffer LB01 dripping respectively, with sharp cutter fast respectively by line leaf primulina tabacum, tomato and paddy rice chopping, with the filter screen filtration acquisition nucleus suspension of 50 μm, be placed in for subsequent use on ice, add RNA digestive ferment, final concentration is 50 μ gml
-1, digest half an hour, add propidium iodide (PI) dyeing, final concentration is 100 μ gml
-1, lucifuge dyes, and dyeing time is 40 minutes, the flow cytometer (model C yFlowSpace) of German Partec company is adopted to measure its genome, adopt 488nm laser excitation, by FL2 Air conduct measurement fluorescence intensity, the nucleus of each sample determination more than 5000, accompanying software FloMax2.80 is used to carry out data acquisition and analysis, nucleus amount and the CV value thereof at each peak directly obtain by this software, by the polygon door of hand drawn (Polygongates) on the scatter diagram of PIvs.SSC, unnecessary fragment (debris) can be removed further, obtain higher-quality peak figure.Can obtain the Genome Size of line leaf primulina tabacum thus, be about 0.66pg, its peak figure is shown in Fig. 3, and CV value is 3.51%.
Embodiment 2:
The present embodiment is substantially the same manner as Example 1, and just material is more than 100 species that Gesneriaceae primulina tabacum belongs to, and after measured, the outcome quality obtained is all better, and its CV value is all lower than 5%.
Comparative example 1:
This comparative example is substantially the same manner as Example 1, just changes core Extraction buffer LB01 into deLaat ' s, Galbraith ' s, GeneralPurpose, MgSO
4, Partec, Tris.MgCl
2with WoodyPlant seven kinds of damping fluids, other steps are identical with embodiment 1, and the peak figure that test obtains and CV value thereof are shown in Fig. 1, as can be seen from Figure 1, be 3.51%, and the CV value of other damping fluids are all higher with the CV of core Extraction buffer LB01.
Comparative example 2:
This comparative example is substantially the same manner as Example 1, just has 2 differences:
1, species are different: the material of this comparative example is Huaiji primulina tabacum and tongue post primulina tabacum;
2, lucifuge dyeing, dyeing time is 24 hours or the dyeing of non-lucifuge, and dyeing time is 24 hours, and other steps are identical with embodiment 1.
The peak figure that test obtains and CV value thereof are shown in Fig. 2, and as can be seen from Figure 2, the lucifuge dyeing of 24 hours and the dyeing of non-lucifuge, its CV value is all higher, particularly dark processing, and its CV value is higher.
Claims (2)
1. be applicable to an assay method for Gesneriaceae Genome Size, it is characterized in that, comprise the following steps:
Material prepares: the fresh tender leaf of the object plant of Gesneriaceae;
Internal reference is selected: the prediction carrying out fluorescent intensity and Genome Size after instrument testing, tomato is selected to be main internal reference according to predicted gene group size, paddy rice is the second internal reference, and is that standard is proofreaded again to rice genome size according to tomato dna group size;
Prepare nucleus suspension: object plant and internal reference species, according to the amount adding 20mg tender leaf in 1ml core Extraction buffer LB01, respectively the tender leaf of object plant and internal reference species is added in core Extraction buffer LB01, then tender leaf is shredded, nucleus suspension is obtained by the filter screen filtration in 50 μm of apertures, the formula being placed in core Extraction buffer LB01 for subsequent use, described is on ice: 15mMTris, 2mMNa
2eDTA, 0.5mM spermine four hydrochloric acid, 80mMKCl, 20mMNaCl, 15mM beta-mercaptoethanol, 0.1% volume ratio TritonX-100, pH7.5;
Use the RNA in RNA digestive ferment vitellophag core suspension again, RNA digestive ferment final concentration is 50 μ gml
-1; Then the dyeing of propidium iodide fluorescence lucifuge is added; Finally apply the Genome Size of cells were tested by flow cytometry object plant.
2. assay method according to claim 1, is characterized in that, the final concentration of described propidium iodide is 100 μ gml
-1, dyeing time is 40min.
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| CN104316373A (en) * | 2014-10-22 | 2015-01-28 | 江苏省农业科学院 | Extraction method for cell nucleuses of eggplant leaves suitable for flow cytometry |
| CN105259097B (en) * | 2015-10-28 | 2018-03-06 | 中国科学院植物研究所 | A kind of method that blueberry ploidy is identified with flow cytometry |
| CN107449717A (en) * | 2017-08-01 | 2017-12-08 | 中国科学院昆明植物研究所 | A kind of method for determining nymphaeaceae plant Genome Size |
| CN109295185B (en) * | 2018-09-05 | 2022-03-22 | 暨南大学 | Method for determining genome size of unicellular eukaryotic algae |
| CN110274865B (en) * | 2019-06-21 | 2021-09-17 | 中南民族大学 | Method for calculating genome size of Chrysosplenium plant |
| CN110514495B (en) * | 2019-08-30 | 2022-06-10 | 中国热带农业科学院香料饮料研究所 | Cell nucleus extraction buffer solution and method for determining genome size of pepper plant |
| CN111139214B (en) * | 2020-01-06 | 2022-03-15 | 中国科学院植物研究所 | Liquid for extracting cell nucleus and application thereof |
| CN114277093B (en) * | 2021-12-24 | 2024-02-27 | 中国农业科学院生物技术研究所 | Method for extracting plant cell nucleus |
| CN115326686B (en) * | 2022-08-15 | 2024-01-19 | 江苏省林业科学研究院 | Quick identification method for sex of holly based on genome size difference |
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| CN100541178C (en) * | 2007-09-18 | 2009-09-16 | 南京农业大学 | A kind of methods for ploidy determination of Chinese cabbage isolated microspore regeneration plant |
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