CN107338322A - Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis - Google Patents
Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis Download PDFInfo
- Publication number
- CN107338322A CN107338322A CN201710775490.6A CN201710775490A CN107338322A CN 107338322 A CN107338322 A CN 107338322A CN 201710775490 A CN201710775490 A CN 201710775490A CN 107338322 A CN107338322 A CN 107338322A
- Authority
- CN
- China
- Prior art keywords
- plant
- soybean
- gene
- cyst nematode
- heterodera glycines
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides the screening technique that a kind of soybean cyst nematode Heterodera glycines infect reference gene in lower wild soybean root tissue Real-time PCR Analysis, it is related to biology and genetic arts, using the anti-and high sense wild soybean germplasm of height as test material, detect expression conditions of 24 candidate's housekeeping genes in different soybean cyst nematode Heterodera glycines infect period wild soybean root system, evaluate its gene expression abundance and expression stability, to filter out the reference gene that suitable soybean cyst nematode Heterodera glycines infect gene expression research in lower wild soybean root system, for inquiring into resistance mechanism of the wild soybean to SCN.
Description
Technical field
The present invention relates to biology and genetic arts, more particularly to a kind of soybean cyst nematode Heterodera glycines to infect lower wild soybean root
Organize the screening technique of reference gene in Real-time PCR Analysis.
Background technology
Real-time quantitative PCR (Real-time quantitative PCR, qRT-PCR) is that current research gene expression has by force
The technological means of power, there is high sensitivity, reproducible, quantitative accurate, be widely used in crop molecular biology
With the field such as genetics research.Reference gene is that qRT-PCR analyzes whether accurate key factor, selects suitable reference gene
It is the important prerequisite of qRT-PCR researchs.The housekeeping gene of constructive expression is that plant sustains life necessary to activity in plant
The solvent of organelle skeleton, or basic biochemical metabolism process is participated in, such as actin gene ACT, α/β microtubule protein gene
TUA/TUB, glyceraldehyde-3-phosphate glyceraldehyde dehydrogenase gene GAPDH etc..In theory, these housekeeping genes in different cells or
The expression that can stablize under different physiological status.Therefore, in most plants gene expression research, using them as stablizing table
The reference gene reached.However, under the conditions of crop different tissues, different growth and development processes, varying environment etc. housekeeping gene table
It is obvious up to stability difference.Therefore, for the abiotic stress such as soybean high temperature, low-phosphorous, salt, aging, arid, aluminium, photoperiod and
The biotics such as soybean phytophthora induction, soybean mosaic virus, purple plague purpura germ, root-knot nematode, the infringement of velvet beans caterpillar, have carried out
Candidate housekeeping gene different tissues expression stability evaluation and be adapted to specified conditions reference gene screening study.
Soybean cyst nematode is the important disease in soybean in China.However, dependency basis of the soybean to SCN
Because expression study is using Actin or Actin11 as reference gene, there is not yet the cultivated soybean or wild soybean are in soybean cyst nematode Heterodera glycines
Infect the report of lower soybean housekeeping gene expression stability evaluation.The application is using the anti-and high sense wild soybean germplasm of height as experiment material
Material, gene expression feelings of the 24 candidate's housekeeping genes of detection in different soybean cyst nematode Heterodera glycines infect period wild soybean root system
Condition, its gene expression abundance and expression stability are evaluated, infected to filter out suitable soybean cyst nematode Heterodera glycines in lower wild soybean root system
The reference gene of gene expression research, for inquiring into resistance mechanism of the wild soybean to SCN.
The content of the invention
It is an object of the invention to provide a kind of soybean cyst nematode Heterodera glycines to infect lower wild soybean root tissue real-time quantitative PCR point
The screening technique of reference gene in analysis, to solve above-mentioned technical problem.
The present invention using following technical scheme in order to solve the above technical problems, realized：
Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis,
It is characterized in that：Material to be tested is high anti-each 30 of the wild soybean seed with high sense soybean cyst nematode Heterodera glycines respectively；
Method and step is：
(1) sample treatment of material to be tested
By each 30 of the seed of two parts of materials to be tested with after 0.5%NaClO solution disinfections 3 minutes, rushed with flowing running water
Wash 4 times；It is planted in after sterilized seed is carved into skin in the polypots for filling high-temperature sterilization perlite；At 27 DEG C, 9 small time
After being cultivated 7 days under conditions of/15 hours dark, first trifoliolate leaf expansion；Seedling is carefully extracted, and rinsed well；
The plant of every part of material to be tested is divided into 6 groups, and every group of 5 plant, wherein 3 groups are treatment group, 3 groups are control group, place
The inoculation worm's ovum processing of reason group, control group inoculation liquid is running water；
Every group of plant is moved on in the polypots for filling high-temperature sterilization perlite, perlite accounts for the 2% of polypots volume,
The root surface for the treatment of group is inoculated with the fresh worm egg suspensions of 5ml, 23000 ovum/ml；Control group is inoculated with 5ml running water, then
Cover the perlite of the thick high-temperature sterilizations of 2cm；27 DEG C are positioned over, is cultivated under conditions of 9 hours illumination/15 hour dark；
9 days after inoculation, every part of material to be tested randomly selects 1 control group and 1 treatment group, by the plant of each polypots
Gently extract, and root system is rinsed well；The root system of 3 plant is taken quickly to be positioned over liquid nitrogen at random from the root system of 5 plant
In, remaining 2 plants of observations for being used for soybean cyst nematode Heterodera glycines colonization status；The remaining 4 groups plant of material to be tested is gently extracted respectively, will
The worm's ovum of root surface and nematode are transplanted in the polypots for filling high-temperature sterilization perlite again after rinsing out, and are placed on 27 DEG C, and 9
Continue to cultivate under conditions of hour illumination/15 hour dark；
Difference 15 days and 20 days after inoculation, every part of material to be tested randomly selects 1 control group and 1 treatment group, will be every
The plant of individual polypots is gently extracted, and root system is rinsed well；Take the root system of 3 plant at random from the root system of 5 plant
Quickly it is positioned in liquid nitrogen, remaining 2 plants of observations for being used for soybean cyst nematode Heterodera glycines colonization status；
(2) RNA extraction and cDNA synthesis
The root system sample that will be preserved in liquid nitrogen, pulverizes last in liquid nitrogen, carries out RNA extraction, with 1.5% fine jade
Sepharose electrophoresis detection RNA integrality, detect RNA concentration and purity；CDNA synthesis is carried out, reverse transcription system is 40 μ
L, wherein containing 14 μ L RNA solutions；
(3) design of primers
Select 24 housekeeping genes；QRT-PCR primer sequences are designed, synthesize cDNA；
(4)qRT-PCR
The cDNA solution synthesized is diluted into 6 times of initial concentrations as qRT-PCR；20 μ L qRT-PCR reaction system bags
SYBR Premix Ex TaqTM solution containing 10 μ L, 8 μ L cDNA solution, 0.38 μM of sense primer and 0.38 μM of downstream
Primer；
(5) data analysis
Carry out data processing；Expression stability evaluation is carried out to candidate's housekeeping gene.
Reference gene is that whether accurate real-time quantitative PCR (Real-time quantitative PCR, qRT-PCR) analysis is
Key, select suitable reference gene be qRT-PCR research important prerequisite.In terms of soybean, for the photoperiod, arid,
The biotic such as the abiotic stress such as salt and soybean phytophthora, root-knot nematode, has carried out the sieve of the reference gene of suitable specified conditions
Choosing.However, there is not yet soybean cyst nematode Heterodera glycines infect the report of lower screening reference gene.The application is with the anti-and high sense HG0 soybean of height
The wild soybean germplasm of SCN is test material, using qRT-PCR technology for detection ACT11 (Glyma.18G290800),
24 candidate's housekeeping genes such as Tubulin_motif (Glyma.19G194800) infect period in different soybean cyst nematode Heterodera glycines (to be connect
9 days, 15 days and 20 days after kind) expression conditions in wild soybean root system, analyze the expression of wherein 23 housekeeping genes
Abundance, and its expression stability is evaluated using BestKeeper, geNorm and Normfinder.Between housekeeping gene
Gene expression abundance is not quite similar, and some housekeeping genes are between different cultivars or between different disposal (inoculation worm's ovum and inoculation water)
The difference of gene expression abundance be present.The expression stability of 23 housekeeping genes also differs, in general, the least stable base of expression
Because Cons9 (Glyma.10G152200), Cons2 (Glyma.17G138500), Cons1 (Glyma.15G270900) and
Tubulin_motif (Glyma.20G136000), they are not suitable for making reference gene under this experiment condition；Remaining housekeeping gene
It is relatively stable, Tubulin_motif (Glyma.15G132200), Cons6/SKIP16 may be selected under this experiment condition
(Glyma.12G051100) or Tubulin_motif (Glyma.05G110200) is used as reference gene, and their expression quantity are higher,
Expression is the most stable, and its Ct average value is respectively 25.7,26.5 and 25.0, and standard deviation is respectively 0.540,0.575 and
The M values of 0.490, geNorm software evaluation its stability are respectively 0.698,0.715 and 0.727.The research resists for wild soybean
The expression analysis of SCN related gene provides reference.
The beneficial effects of the invention are as follows：
The present invention is using the anti-and high sense wild soybean germplasm of height as test material, and 24 candidate's housekeeping genes of detection are different big
Beans SCN infects the expression conditions in period wild soybean root system, evaluates its gene expression abundance and expression stability, with
Phase filters out the reference gene that suitable soybean cyst nematode Heterodera glycines infect gene expression research in lower wild soybean root system, for inquiring into open country
Resistance mechanism of the raw soybean to SCN.
Embodiment
In order that the technical means, the inventive features, the objects and the advantages of the present invention are easy to understand, tie below
Specific embodiment is closed, the present invention is expanded on further, but following embodiments are only the preferred embodiments of the present invention, and it is not all.
Based on the embodiment in embodiment, those skilled in the art obtain other realities on the premise of creative work is not made
Example is applied, belongs to protection scope of the present invention.Experimental method in following embodiments, it is conventional method unless otherwise specified,
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis,
It is characterized in that：Material to be tested is high anti-each 30 of the wild soybean seed with high sense soybean cyst nematode Heterodera glycines respectively；
1st, method and step is：
(1) sample treatment of material to be tested
By each 30 of the seed of two parts of materials to be tested with after 0.5%NaClO solution disinfections 3 minutes, rushed with flowing running water
Wash 4 times；It is planted in after sterilized seed is carved into skin in the polypots for filling high-temperature sterilization perlite；At 27 DEG C, 9 small time
After being cultivated 7 days under conditions of/15 hours dark, first trifoliolate leaf expansion；Seedling is carefully extracted, and rinsed well；
The plant of every part of material to be tested is divided into 6 groups, and every group of 5 plant, wherein 3 groups are treatment group, 3 groups are control group, place
The inoculation worm's ovum processing of reason group, control group inoculation liquid is running water；
Every group of plant is moved on in the polypots for filling high-temperature sterilization perlite, perlite accounts for the 2% of polypots volume,
The root surface for the treatment of group is inoculated with the fresh worm egg suspensions of 5ml, 23000 ovum/ml；Control group is inoculated with 5ml running water, then
Cover the perlite of the thick high-temperature sterilizations of 2cm；27 DEG C are positioned over, is cultivated under conditions of 9 hours illumination/15 hour dark；
9 days after inoculation, every part of material to be tested randomly selects 1 control group and 1 treatment group, by the plant of each polypots
Gently extract, and root system is rinsed well；The root system of 3 plant is taken quickly to be positioned over liquid nitrogen at random from the root system of 5 plant
In, remaining 2 plants of observations for being used for soybean cyst nematode Heterodera glycines colonization status；The remaining 4 groups plant of material to be tested is gently extracted respectively, will
The worm's ovum of root surface and nematode are transplanted in the polypots for filling high-temperature sterilization perlite again after rinsing out, and are placed on 27 DEG C, and 9
Continue to cultivate under conditions of hour illumination/15 hour dark；
Difference 15 days and 20 days after inoculation, every part of material to be tested randomly selects 1 control group and 1 treatment group, will be every
The plant of individual polypots is gently extracted, and root system is rinsed well；Take the root system of 3 plant at random from the root system of 5 plant
Quickly it is positioned in liquid nitrogen, remaining 2 plants of observations for being used for soybean cyst nematode Heterodera glycines colonization status；
(2) RNA extraction and cDNA synthesis
The root system sample that will be preserved in liquid nitrogen, pulverizes last in liquid nitrogen, using TaKaRa MiniBEST Plant
RNA Extraction Kit carry out RNA extraction according to its specification, detect RNA's with 1.5% agarose gel electrophoresis
Integrality, RNA concentration and purity are detected with NanoDrop 2000；Use TaKaRa PrimeScriptTM RT reagent
Kit with gDNA Eraser carry out cDNA synthesis, and reverse transcription system is 40 μ L, wherein containing 14 μ L RNA solutions；
(3) design of primers
Select 24 housekeeping genes (table 1)；Using Oligo6.0 Software for Design qRT-PCR primer sequences, by Hua Da gene
(Beijing) synthesizes cDNA；
The housekeeping gene of table 1 and its qRT-PCR primer sequences
(4)qRT-PCR
The cDNA solution synthesized is diluted into 6 times of initial concentrations as qRT-PCR；QRT-PCR is in gene Co., Ltd
Carried out on EcoTM quantitative real time PCR Instruments, operation is with reference to (the Tli RNAaseH of TaKaRa SYBR Premix Ex TaqTM II
Plus kit specification) is carried out；The SYBR Premix Ex TaqTM that 20 μ L qRT-PCR reaction systems include 10 μ L are molten
Liquid, 8 μ L cDNA solution, 0.38 μM of sense primer and 0.38 μM of anti-sense primer；Response procedures are：50℃2min；95℃
30S；(95 DEG C of 5S, 60 DEG C of 30S) × 40 circulations；95℃15S；55℃15S；95℃15S；Each sample sets 2 repetitions；
(5) data analysis
Data processing is carried out using Excel 2016；Candidate is held using GeNorm, BestKeeper and Normfinder
Family's gene carries out expression stability evaluation.
2nd, result and analysis
2.1RNA mass and purity detecting
By the RNA solution of 12 parts of samples of extraction carried out 1.5% agarose gel electrophoresis detection, find 28SrRNA and
18SrRNA bands of a spectrum are clear, and RNA integralities are good；RNA purity is detected with NanoDrop 2000 and concentration shows, nucleic acid is maximum
Absorbing wavelength light absorption value A260/ albumen and aldehydes matter maximum absorption wavelength light absorption value A280Between 1.70-2.01, purity compared with
Good, change in concentration scope is 55.0-99.7ng μ L-1；Therefore, the RNA of extraction synthesizes available for cDNA.
The expanding effect and specific amplification of 2.2 primers
Using the primer of 24 housekeeping genes of synthesis, using the cDNA for diluting 6 times as template, qRT-PCR inspections have been carried out
Survey.In addition to non-specific amplification occurs in housekeeping gene Actin (Glyma.08G182200), the qRT-PCR of other housekeeping genes expands
Synergy fruit is preferable, and specific amplification is high.
The gene expression abundance analysis of 2.3 housekeeping genes
Utilize the expression of Ct values evaluation housekeeping gene.Ct values are higher, and its expression quantity is lower, and its gene expression abundance is lower；
Conversely, then gene expression abundance is higher.Ct values reflect difference of the gene in sample room expression quantity in the change of sample room.23 are held
The gene expression abundance analysis of family's gene finds that the gene expression abundance between housekeeping gene is not quite similar.GAPDH and ACT11 in this experiment
(Glyma.18G290800) gene expression abundance is higher, and Average Ct values are respectively 21.6 and 22.7；And Tubulin_motif
(Glyma.20G136000) and UKN2 (Glyma.06G038500) gene expression abundance is relatively low, and Average Ct values are respectively 31.4 Hes
29.2；The gene expression abundance of other genes is between them.There is also table between different cultivars or between different disposal for housekeeping gene
Difference degree up to the difference of abundance, and different housekeeping gene gene expression abundances is also not quite similar.Such as in anti-, sense wild soybean
Between, Cons9 (Glyma.10G152200) Ct values difference is maximum, up to 8.1, TUA5 (Glyma.05G157300) differences 1.2, and
Cons10 (Glyma.05G207500), Cons6/SKIP16 (Glyma.12G051100) and Cons2 (Glyma17g14750) phase
Difference is 0；Between different disposal (inoculation worm's ovum and inoculation running water), Cons2 (Glyma.17G138500) Ct values differ most
Greatly, for 1.9, Cons17 (Glyma.04G047900) difference 0.7, and Tubulin_motif (Glyma.15G132200) and
Tubulin_motif (Glyma.19G194800) phase difference is 0.It can be seen that housekeeping gene is in different wild soybean kinds, no
Under the conditions of processing (inoculation worm's ovum and inoculation running water), the performance of its gene expression abundance is different, and its stability is relative.
The expression stability evaluation of 2.4 housekeeping genes
In order to which clear and definite soybean cyst nematode Heterodera glycines infect lower 23 housekeeping genes in the expression stability of wild soybean root system, this Shen
The please housekeeping gene Ct values by BestKeeper, GeNorm and Normfinder software analysis, and according to software requirement, by Ct
Value.
2.4.1BestKeeper the stability of housekeeping gene is evaluated
BestKeeper softwares are the mark in different cultivars, different disposal and different Proper Sampling Period Ct values using housekeeping gene
When quasi- difference and the coefficient of variation are to evaluate the stability of gene expression, standard deviation and the smaller coefficient of variation, the expression of gene is stable
Property is preferable, and standard deviation and the coefficient of variation are larger, and the expression stability of gene is poor.Usually, when standard deviation is more than 1, this is illustrated
The stability of gene is poor.In 23 housekeeping genes, 20 gene expressions are relatively stable, and the standard deviation of its Ct value is 0.405-0.863,
The coefficient of variation is 1.43%-3.05%.Express most stable of 2 housekeeping genes for HDC (Glyma.08G050200) and
Tubulin_motif (Glyma.19G194800), its Ct standard deviation is respectively 0.405 and 0.440, and the coefficient of variation is respectively
1.43% and 1.52%；And least stable housekeeping gene is Cons9 (Glyma.10G152200), Cons2
(Glyma.17G138500) and Cons1 (Glyma.15G270900), its Ct standard deviation are all higher than 1, respectively 4.029,1.400
The coefficient of variation with 1.309, Ct is respectively 13.78%, 5.53% and 4.69%.
2.4.2geNorm the stability of housekeeping gene is evaluated
GeNorm evaluates the stability of gene according to the size of M values.M values are smaller, and gene expression is more stable.In this research
3 least stable housekeeping genes of expression for Cons9 (Glyma.10G152200), Cons2 (Glyma.17G138500) and
Cons1 (Glyma.15G270900), its M value are respectively 1.061,0.769 and 0.703.Expressing most stable of 2 is
Tubulin_motif (Glyma.15G132200) and Tubulin_motif (Glyma.05G110200), M values are 0.223；Its
Secondary is Cons6/SKIP16 (Glyma.12G051100) (M values are 0.240).
More accurate real-time quantitative PCR data can be obtained by carrying out calibration using multiple reference genes.Utilize geNorm
The normalization factor pairing difference (V that software calculatesn/n+1) number of reference gene can be determined.Under this experiment condition, simultaneously
2 housekeeping genes are selected as reference gene, its normalization factor pairing difference V2/3For 0.074, less than 0.15, therefore, simultaneously
Housekeeping gene Tubulin_motif (Glyma.15G132200) and Tubulin_motif (Glyma.05G110200) is selected to make
Accurate real-time quantitative PCR data can be obtained for reference gene.
2.4.3Normfinder the stability of housekeeping gene is evaluated
Similar to geNorm, the stability of Normfinder evaluation housekeeping genes needs the Ct values of gene being converted into relatively
Expression quantity, the stability of gene is then evaluated according to gene expression index of stability M.M values are smaller, and gene expression is more stable.This
It is Cons6/SKIP16 (Glyma.12G051100) (M values are 0.050) that most stable of gene is expressed in research, is secondly
Tubulin_motif (Glyma.15G132200) (M values are 0.051) and Tubulin_motif (Glyma.05G110200) (M
It is worth for 0.064).The least stable housekeeping gene of expression is that (M values are Tubulin_motif (Glyma.20G136000)
0.315), (M values are by Cons2 (Glyma.17G138500) (M values be 0.285) and Cons9 (Glyma.10G152200)
0.268)。
Candidate's housekeeping gene that the application selects actin mainly related to eucaryotic cell structure (ACT11, Gm β-
Actin, ACT2/7 and Actin), tubulin (Tubulin_motif, TUA5 etc.), ATP binding members (Cons4) and F-box
Albumen (Cons6/SKIP16) etc..Although they are the housekeeping genes of constructive expression, bar is infected in soybean cyst nematode Heterodera glycines
Under part, there is obvious difference in its gene expression abundance and expression stability in wild soybean root tissue.Forefathers study and also indicated that,
The expression stability difference of housekeeping gene is obvious under the conditions of soybean different tissues, different growth and development processes, varying environment etc..Can
See, to obtain reliable real-time quantitative PCR result, it is necessary to carry out the sieve of stable expression reference gene for special experiment condition
Choosing.
The application selected 3 conventional analysis reference gene stability software BestKeeper, NormFinder and
GeNorm, the expression stability of 23 soybean candidate's housekeeping genes is evaluated.Although this 3 softwares are to candidate's housekeeping gene
Expression stability sequence it is inconsistent, but its expression stability evaluation result correlation be up to 0.75 (BestKeeper with
NormFinder), 0.86 (BestKeeper and geNorm) and 0.98 (NormFinder and geNorm).Consider, 23
In candidate's housekeeping gene, Tubulin_motif (Glyma.20G136000), Cons1 (Glyma.15G270900), Cons2
(Glyma.17G138500) and Cons9 (Glyma.10G152200) is the worst housekeeping gene of expression stability, and Cons6/
SKIP16(Glyma.12G051100)、Tubulin_motif(Glyma.15G132200)、Tubulin_motif
(Glyma.05G110200), TUB4 (Glyma.03G124400) and UKN2 (Glyma.06G038500) expression stability are preferable.
Under this experiment condition, carry out real-time quantitative PCR research when, can use Cons6/SKIP16 (Glyma.12G051100),
Tubulin_motif (Glyma.15G132200) or Tubulin_motif (Glyma.05G110200) is used as reference gene, or
Simultaneous selection Tubulin_motif (Glyma.15G132200) and Tubulin_motif (Glyma.05G110200) 2 run one's home
Gene combines as reference gene.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
For personnel it should be appreciated that the present invention is not limited to the above embodiments, that described in above-described embodiment and specification is only the present invention
Preference, be not intended to limit the present invention, without departing from the spirit and scope of the present invention, the present invention also have it is various
Changes and improvements, these changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by institute
Attached claims and its equivalent thereof.
Claims (1)
1. soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis, its
It is characterised by：Material to be tested is high anti-each 30 of the wild soybean seed with high sense soybean cyst nematode Heterodera glycines respectively；
Method and step is：
(1) sample treatment of material to be tested
By each 30 of the seed of two parts of materials to be tested with after 0.5%NaClO solution disinfections 3 minutes, 4 are rinsed with flowing running water
It is secondary；It is planted in after sterilized seed is carved into skin in the polypots for filling high-temperature sterilization perlite；At 27 DEG C, 9 hours illumination/15
After being cultivated 7 days under conditions of hour is dark, first trifoliolate leaf expansion；Seedling is carefully extracted, and rinsed well；
The plant of every part of material to be tested is divided into 6 groups, every group of 5 plant, wherein 3 groups are treatment group, 3 groups are control group, treatment group
Worm's ovum processing is inoculated with, control group inoculation liquid is running water；
Every group of plant is moved on in the polypots for filling high-temperature sterilization perlite, perlite accounts for the 2% of polypots volume, is handling
The root surface of group is inoculated with the fresh worm egg suspensions of 5ml, 23000 ovum/ml；Control group is inoculated with 5ml running water, then covers
The perlite of high-temperature sterilization thick 2cm；27 DEG C are positioned over, is cultivated under conditions of 9 hours illumination/15 hour dark；
9 days after inoculation, every part of material to be tested randomly selects 1 control group and 1 treatment group, by the plant of each polypots gently
Extract, and root system is rinsed well；Take the root system of 3 plant to be quickly positioned in liquid nitrogen at random from the root system of 5 plant, remain
Remaining 2 plants be used for soybean cyst nematode Heterodera glycines colonization status observations；The remaining 4 groups plant of material to be tested is gently extracted respectively, by root table
The worm's ovum in face and nematode are transplanted in the polypots for filling high-temperature sterilization perlite again after rinsing out, and are placed on 27 DEG C, 9 hours
Continue to cultivate under conditions of the dark of illumination/15 hour；
Difference 15 days and 20 days after inoculation, every part of material to be tested randomly selects 1 control group and 1 treatment group, will each mould
The plant of material alms bowl is gently extracted, and root system is rinsed well；Take the root system of 3 plant quick at random from the root system of 5 plant
It is positioned in liquid nitrogen, remaining 2 plants of observations for being used for soybean cyst nematode Heterodera glycines colonization status；
(2) RNA extraction and cDNA synthesis
The root system sample that will be preserved in liquid nitrogen, pulverizes last in liquid nitrogen, carries out RNA extraction, with 1.5% agarose
Detected through gel electrophoresis RNA integrality, detect RNA concentration and purity；CDNA synthesis is carried out, reverse transcription system is 40 μ L, its
In contain 14 μ L RNA solutions；
(3) design of primers
Select 24 housekeeping genes；QRT-PCR primer sequences are designed, synthesize cDNA；
(4)qRT-PCR
The cDNA solution synthesized is diluted into 6 times of initial concentrations as qRT-PCR；20 μ L qRT-PCR reaction systems include 10
μ L SYBR Premix Ex TaqTM solution, 8 μ L cDNA solution, 0.38 μM of sense primer and 0.38 μM of downstream are drawn
Thing；
(5) data analysis
Carry out data processing；Expression stability evaluation is carried out to candidate's housekeeping gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710775490.6A CN107338322A (en) | 2017-08-31 | 2017-08-31 | Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710775490.6A CN107338322A (en) | 2017-08-31 | 2017-08-31 | Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107338322A true CN107338322A (en) | 2017-11-10 |
Family
ID=60215184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710775490.6A Pending CN107338322A (en) | 2017-08-31 | 2017-08-31 | Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107338322A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107904244A (en) * | 2017-11-28 | 2018-04-13 | 吉林省农业科学院 | Application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206651A (en) * | 2011-04-27 | 2011-10-05 | 东北农业大学 | Soybean cyst nematode resistance gene and application thereof |
| CN102246662A (en) * | 2011-05-16 | 2011-11-23 | 河南省农业科学院 | Method for identifying resistance against heterodera glycines |
| CN103866007A (en) * | 2014-02-19 | 2014-06-18 | 中国科学院新疆生态与地理研究所 | Method for screening real-time fluorescence quantification PCR internal reference molecules of syntrichia caninervis in desert |
| CN104293806A (en) * | 2014-08-28 | 2015-01-21 | 吉林省农业科学院 | Soybean SHMT novel allele and typing method thereof |
-
2017
- 2017-08-31 CN CN201710775490.6A patent/CN107338322A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206651A (en) * | 2011-04-27 | 2011-10-05 | 东北农业大学 | Soybean cyst nematode resistance gene and application thereof |
| CN102246662A (en) * | 2011-05-16 | 2011-11-23 | 河南省农业科学院 | Method for identifying resistance against heterodera glycines |
| CN103866007A (en) * | 2014-02-19 | 2014-06-18 | 中国科学院新疆生态与地理研究所 | Method for screening real-time fluorescence quantification PCR internal reference molecules of syntrichia caninervis in desert |
| CN104293806A (en) * | 2014-08-28 | 2015-01-21 | 吉林省农业科学院 | Soybean SHMT novel allele and typing method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 曾文韬 等: "适用于大豆实时荧光定量PCＲ分析的内参基因的筛选和验证", 《南京农业大学学报》 * |
| 王骁: "基于实时荧光定量PCR的大豆内参基因筛选", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 袁翠平 等: "大豆胞囊线虫侵染下野生大豆根组织实时定量PCR分析中内参基因的筛选", 《第十届全国大豆学术讨论会摘要集》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107904244A (en) * | 2017-11-28 | 2018-04-13 | 吉林省农业科学院 | Application of the wild soybean XLOC_023202 genes in soybean plants cyst nematode resistance is improved |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Root vacuolar Na+ sequestration but not exclusion from uptake correlates with barley salt tolerance | |
| Zhao et al. | An optimized protocol for stepwise optimization of real-time RT-PCR analysis | |
| Kuo et al. | Secret lifestyles of Neurospora crassa | |
| Thangavelu et al. | Genetic diversity of Fusarium oxysporum f. sp. cubense isolates (Foc) of India by inter simple sequence repeats (ISSR) analysis | |
| Hernandez Maldonado et al. | Spongospora subterranea root infection assessed in two potato cultivars differing in susceptibility to tuber powdery scab | |
| da Costa Ribeiro et al. | Physiological race characterization of Phytophthora capsici isolates from several host plant species in Brazil using New Mexico recombinant inbred lines of Capsicum annuum at two inoculum levels | |
| King et al. | A possible relationship between shoot N concentration and the sensitivity of N2 fixation to drought in soybean | |
| Zhang et al. | Measuring the damage of heavy metal cadmium in rice seedlings by SRAP analysis combined with physiological and biochemical parameters | |
| Tomkowiak et al. | Dependence of the heterosis effect on genetic distance, determined using various molecular markers | |
| Xi et al. | Species diversity and chemotypes of Fusarium species associated with maize stalk rot in Yunnan province of southwest China | |
| Zhao et al. | Differentially expressed gene transcripts related to the Cf-19-mediated resistance response to Cladosporium fulvum infection in tomato | |
| Sreedasyam et al. | JGI Plant Gene Atlas: an updateable transcriptome resource to improve structural annotations and functional gene descriptions across the plant kingdom | |
| Santiago et al. | Genetic structure of Ralstonia solanacearum and Ralstonia pseudosolanacearum in Brazil | |
| CN107338322A (en) | Soybean cyst nematode Heterodera glycines infect the screening technique of reference gene in lower wild soybean root tissue Real-time PCR Analysis | |
| Parma et al. | New insights into the evolution of C4 photosynthesis offered by the tarenaya cluster of Cleomaceae | |
| Chen et al. | Selection of potential reference genes for RT-qPCR in the plant pathogenic fungus Colletotrichum fructicola | |
| Li et al. | Selection of reference genes for quantitative real-time PCR normalization in Narcissus pseudonarcissus in different cultivars and different organs | |
| Wang et al. | Development of a real-time TaqMan PCR method for absolute quantification of the biocontrol agent Esteya vermicola | |
| Xue et al. | Phylogenetic analysis and temporal diversification of the tribe Alsineae (Caryophyllaceae) with the description of three new genera, Hesperostellaria, Reniostellaria and Torreyostellaria | |
| Mamba-Mbayi et al. | Characterization of Congolese Strains of Xanthomonas axonopodis pv. manihotis associated with Cassava Bacterial Blight | |
| Guo et al. | Ultraweak photon emission in strawberry fruit during ripening and aging is related to energy level | |
| CN107586879A (en) | A kind of primer pair group, kit, method and application for being used to detect flax SSR molecular marker | |
| Burra et al. | RNAseq and proteomics for analysing complex oomycete plant interactions | |
| Mekkerdchoo et al. | Genetic variation among Amorphophallus sp. from Northern Thailand and their glucomannan content | |
| Bennett et al. | Three inoculation methods for evaluating Sclerotinia blight resistance in peanut |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171110 |