CN108070673A - The fluorescent quantitation reference gene and its primer of two kinds of tree-like pears difference tree-crown location leaf textures and application - Google Patents
The fluorescent quantitation reference gene and its primer of two kinds of tree-like pears difference tree-crown location leaf textures and application Download PDFInfo
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- CN108070673A CN108070673A CN201810042855.9A CN201810042855A CN108070673A CN 108070673 A CN108070673 A CN 108070673A CN 201810042855 A CN201810042855 A CN 201810042855A CN 108070673 A CN108070673 A CN 108070673A
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- 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
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- 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
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- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q2600/13—Plant traits
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- 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
Abstract
The invention discloses the fluorescent quantitation reference gene and its primer of two kinds of tree-like pears difference tree-crown location leaf textures and applications.The present invention analyzes expression of 13 candidate's reference genes in the pears leaf tissue samples of two kinds of tree-like different tree-crown locations by using qRT PCR, the expression stationary value of candidate's reference gene is assessed with geNorm softwares, determines that ARM and Actin2 combines for optimal reference gene；Relative expression's normalized analysis is carried out to APX respectively with different reference genes, verifies that the reference gene combination ARM and Actin2 of the present invention is highly reliable as normalization factor.
Description
Technical field
The invention belongs to field of plant molecular biology, and in particular to two kinds of tree-like pears difference tree-crown location leaf textures it is glimmering
Light quantifies reference gene and its primer and application.
Background technology
Pears are one of fruit trees of the world and China most economic value.Different tree form structure affects photosynthetically active radiation and exists
Distribution and infiltration in canopy, and then there is adjustment effect to fruit quality.Traditional Pear trees shape tree body is tall and big, holds inside canopy
Easy closing；It is tree-like that recent year widelys popularize frame, and tree management is opened a business, and receives uniform illumination, is conducive to improve fruit
Commodity performance.It studies expression of the related gene at different each positions of pear tree shape to change, helps to parse tree-like structure regulating
The molecule mechanism of sink-source relation.
Real-time quantitative PCR (qRT-PCR) with the advantages such as its quick, sensitive, accurate, repeated high and practicability is wide,
It has been widely used in gene expression dose analysis.When using relative quantitation method, in order to eliminate different sample original templates
The deviations such as amount, RNA mass, the reference gene that expression need to be utilized stable are corrected experimental data.However currently without any
The expression of one gene is kept constant under all experiment conditions.Even same species, when experiment condition
Change, reference gene stability can also change, it is impossible to arbitrarily think to stablize in expression in some experiment condition
Ginseng gene can be suitably used for other all experiment conditions, otherwise likely results in experimental data and generates the deviation even conclusion of mistake.
Therefore, before gene expression is analyzed, the reference gene for stablizing expression under the specified conditions should be filtered out, this is accurate to obtaining
QRT-PCR experimental results are extremely important.It can be more accurately right in addition, multiple reference genes is selected to combine relatively single reference gene
Relative quantification data are corrected and standardize.
The content of the invention
The purpose of the present invention is being directed to using the not high deficiency of the single reference gene accuracy of tradition, exploitation one kind can be applied
Gene table in different tree form (frame is tree-like and evacuation is layered shape) and each tree-crown location (external, middle part, internal) leaf texture
Up to the double reference genes and its specific primer of analysis, reliable guarantee is provided for the accurate quantitative analysis of the gene under the experiment condition.
The present invention by using qRT-PCR analyze 13 candidate's reference genes two kinds it is tree-like (frame it is tree-like and evacuation be layered
Shape) different tree-crown locations (external, middle part, internal) pears leaf tissue samples expression；With geNorm softwares to candidate
The expression stationary value of reference gene is assessed, and determines that ARM and Actin2 combines for optimal reference gene；Again with different internal reference bases
Because carrying out relative expression's normalized analysis to APX respectively, verify that the assortment of genes has reliability as normalization factor.
First purpose of the present invention is to provide the fluorescent quantitation internal reference base of two kinds of tree-like pears difference tree-crown location leaf textures
Cause.The fluorescent quantitation reference gene of two kinds of tree-like pears difference tree-crown location leaf textures, be reference gene Actin2 and
ARM。
It is preferred that the reference gene Actin2, nucleotide sequence is as shown in SEQ ID NO.1；The internal reference base
Because of ARM, nucleotide sequence is as shown in SEQ ID NO.4.
Second object of the present invention is to provide in the fluorescent quantitation of the tree-like pears difference tree-crown location leaf texture of above two
Join the specific primer of gene.
It is preferred that the specificity of the fluorescent quantitation reference gene of two kinds of tree-like pears difference tree-crown location leaf textures is drawn
Object, the forward primer and reverse primer of the specific primer of the reference gene Actin2 are respectively such as SEQ ID NO.2 and SEQ
Shown in ID NO.3；The forward primer and reverse primer of the specific primer of the reference gene ARM are respectively such as SEQ ID
Shown in NO.5 and SEQID NO.6.
Third object of the present invention is to provide the reference gene or the specific primer and determines preparing fluorescence
Measure the application in kit.
It is preferred that the kit further includes Standard PCR reagent.
The reference gene Actin2 and reference gene ARM is glimmering as two kinds of tree-like pears difference tree-crown location leaf textures
Light quantifies the application of reference gene.
Fluorescent quantitation specific primer of the specific primer as two kinds of tree-like pears difference tree-crown location leaf textures
Application.
It is preferred that the application is using quantitative fluorescent PCR, quantitative fluorescent PCR system is 10 μ L, comprising 5 μ L 2 ×
SYBRGreen PCR Master Mix, the forward primer and each 0.5 μ L of 0.5 μ L, cDNA template of reverse primer of 10 μm of ol/L add
ddH2O to 10 μ L；Quantitative fluorescent PCR response procedures be 50 DEG C of 5min, then 95 DEG C of 10min, 94 DEG C of 15sec, 60 DEG C of 1min into
40 Xun Huans of row.
It is that frame is tree-like and evacuation layering shape that described two kinds tree-like, and different tree-crown locations are the tree bodies done from center outside 1m
It is done in the middle part of tree body external, that 0.5-1.0m is done from center and from center inside the tree body of 0-0.5m.
The candidate's reference gene expression of the invention for analyzing two kinds of tree-like pears difference tree-crown locations comprehensively；Filter out use
The reference gene combination Actin2 and ARM of gene expression analysis in the leaf texture being distributed in different tree form pears in different spaces, and
It is highly reliable as normalization factor to demonstrate the assortment of genes.
It is an advantage of the invention that：
(1) the internal reference base of the gene expression analysis suitable for different tree form, different spaces position leaf texture is filtered out for the first time
Because combining Actin2 and ARM.
(2) reliability.The present invention takes into account different tree form and different tree-crown locations the two factors to gene table simultaneously
Up to the influence of stability, stability assessment is carried out to the expression Ct values of 13 candidate's reference genes by geNorm softwares,
Further verification shows that Actin2 and ARM reference genes combination application has reliability in target gene standardization.The present invention's
Actin2 and ARM stablizes expression in two kinds of tree-like different tree-crown location pears leaf tissues, is two kinds of tree-like pears difference tree body portions
The optimal double reference gene combination of the quantitative fluorescence analysis of position, application are substantially better than the standardization effect of common single reference gene
Fruit.New candidate's reference gene that wherein ARM is filtered out as inventor, at present standardization not yet for other purposes
Analysis present invention demonstrates that ARM stability is higher than traditional reference gene, is conducive to accurately detect gene under corresponding experiment condition
Expression analysis provides more structurally sound guarantee.
(3) present invention has wide applicability.It can be used for the pears leaf tissue sample of different tree form and different tree-crown locations
Reference gene during gene expression analysis in product, the gene expression analysis being organized in for pears leaf in spatial distribution provide one it is steady
Fixed standard of comparison.
Description of the drawings
Fig. 1 is the case line of Ct Distribution value of 13 candidate's reference genes in two kinds of tree-like pears difference tree-crown location samples
Scheme, the straight line, endpoint, the circle of target in chest up-and-down boundary, chest represent 25/75 quartile point, intermediate value, maximum/small respectively
Value, exceptional value.
Fig. 2 be geNorm softwares to 13 candidate's reference genes in the different tree-crown location leaf textures of two kinds of tree-like pears
Expression stability sorts.
Fig. 3 is that geNorm is determined for the optimal reference gene number of accurate quantitative analysis, in n-th normalization factor and
Analysis pairing difference, asterisk show most suitable reference gene number between (n+1)th normalization factor.
Fig. 4 be different reference genes respectively as normalization factor to APX genes in the different tree body portions of two kinds of tree-like pears
Position leaf texture in expression quantity variation analyzed, DP represent frame it is tree-like, SP represent evacuation layering shape, behind-EX ,-CE and-
IN is represented respectively outside tree body (being done from center outside 1m), middle part (doing 0.5-1.0m from center) and internal (doing 0-0.5m from center)
Leaf texture's sample, as DP-EX represent be the tree-like outside of frame pears leaf tissue samples, the rest may be inferred for other.
Specific embodiment
Following embodiment is the further explanation to the present invention rather than limitation of the present invention.
Embodiment 1：Filter out the optimal of the quantitative fluorescence analysis that Actin2 and ARM is two kinds of tree-like pears difference tree-crown locations
Double reference gene combinations
1. experiment material samples：With ' garden is yellow ' pears adult leaf tissue of two kinds tree-like (frame is tree-like and evacuation is layered shape)
For experiment material, the outside (being done from center outside 1m) of tree body is taken in mid-June, 2016 respectively, 0.5- (is done in middle part from center
1.0m) and internal (doing 0-0.5m from center) leaf texture.Each tree-like pear tree selects 3 trees respectively as 3 biology weights
It is multiple, it is quick-frozen in liquid nitrogen after leaf texture's sample is in vitro, then as -80 DEG C of preservations.
2. leaf material Total RNAs extraction：Using RNAprep Pure Plant Kit (Tiangeng) RNA extracts kits, according to
Specification step extraction leaf texture sample total serum IgE.
3. total serum IgE integrity detection：1.2% agarose gel electrophoresis, 1 × TAE buffer solutions, 100V, 20min；Quality and
Concentration monitor；Utilize NanoPhotometerTMSpectrophotometer (IMPLEN) micro-spectrophotometer detects
OD260/280 values and concentration, value are qualified samples between 1.9-2.1.
The synthesis of the first chain of 4.cDNA：According to RevertAidTM First Strand cDNA Synthesis Kit
(Fermentas) total serum IgE (detection qualified samples) that specification extracts step 2 is inverted to cDNA, for qRT-PCR templates.
1) reaction mixture is prepared on ice：1 μ g total serum IgEs, 1 μ l Oligo (dT) primer are sequentially added, DEPC water polishing is extremely
12μl。
2) 65 DEG C of warm bath 5min, cooled on ice 2min.
3) slightly centrifuge；Centrifuge tube is placed on ice, sequentially adds 4 μ l 5 × Reaction buffer, 1 μ l
Ribonuclease inhibitor ribalgilases repressor, 2 μ l 10mM dNTP mix and 1 μ l Revert AidTM M-
MuLV reverse transcriptase (200U/ μ l).
4) gently mixing, centrifugation.
5) 42 DEG C of warm bath 60min, 70 DEG C of warm bath 5min are put in cooled on ice afterwards, be stored in -20 DEG C it is spare.
5. using above-mentioned reverse transcription synthesis cDNA as template, select Actin2, ARM, EF1a, GAPC, Histone H3,
MYB10, SAND, SKD1, SR34A, TIP41-like, TUB7, UBQ5 and YLS8 are carried out as candidate's reference gene, design primer
QRT-PCR reacts.13 above-mentioned candidate's reference genes be according to inventor laboratory obtain transcriptome analysis result and its
What the pears reference gene sequence information screening that he has reported determined, for above-mentioned candidate gene, design corresponding amplimer.Candidate
The primer sequence and expansion of reference gene are shown in Table 1, and quantitative fluorescent PCR system is 10 μ L, includes 5 μ L2 × SYBR Green
PCR Master Mix, the forward primer and each 0.5 μ L of 0.5 μ L, cDNA template of reverse primer of 10 μm of ol/L add ddH2O to 10 μ
L；Quantitative fluorescent PCR response procedures are 50 DEG C of 5min；Then 95 DEG C of 10min, 94 DEG C of 15sec, 60 DEG C of 1min carry out 40 Xun Huans.
It is simple spike by observing the solubility curve generated after qRT-PCR reactions, judges that the primer is specifically to expand target fragment.
1 candidate's reference gene name of table, primer sequence and amplified production information
6. the fluorescence signal read in each reaction tube reaches the period undergone during the threshold value of setting, i.e. Ct values.Such as
Shown in Fig. 1, the transcriptional level of different reference genes shows different expression amplitudes of variation, the Ct values of 13 candidate's reference genes
Scope is in 15.93-26.86；GAPC and Histone H3 show relatively high transcriptional level, and Ct average values are respectively
18.90 with 16.75；MYB10 Average expression levels are minimum, and Ct average values are 26.21.
7. 13 reference gene stability are calculated and sorted using geNorm programs, as shown in Fig. 2, reference gene
Stability to weak by being ordered as Actin2/ARM by force>SR34A>SAND>GAPC>YLS8>MYB10>UBQ5>TIP41-like>
EF1a>SKD1>TUB7>Histone H3。
8. after the expression stability that candidate's reference gene is determined, it is poor that geNorm can be matched by the normalized factor
Different analysis (Vn/n+1) value judges the most suitable number of reference gene, if Vn/n+1Value is less than 0.15, then need not introduce (n+1)th
A reference gene.The results are shown in Figure 3, and left first pillar height of number is less than 0.15 in column diagram, has marked with an asterisk, corresponding
Abscissa be V2/V3, i.e. n=2.So stablize the reference gene group of expression in two kinds of tree-like different tree-crown location leaf textures
It is 2 to close minimal amount, i.e., the combination of optimal reference gene is Actin2 and ARM.The specificity of wherein reference gene Actin2 is drawn
The forward primer of object is 5'-CTCCCAGGGCTGTGTTTCCTA-3'(as shown in SEQ ID NO.2), reverse primer 5'-
CTCCATGTCATCCCAGTTGCT-3'(is as shown in SEQ ID NO.3), the expansion of the reference gene Actin2 of the primer pair amplifies
Increase production the nucleotide sequence of object as shown in SEQ ID NO.1；The forward primer of the specific primer of reference gene ARM is 5-
CAAGGGCATTCTTTCGG-3'(is as shown in SEQ ID NO.5), reverse primer 5-CAGGGACATCATTAGGAACAT-3'
(as shown in SEQ ID NO.6), the nucleotide sequence such as SEQ ID of the amplified production of the reference gene ARM of the primer pair amplifies
Shown in NO.4.
Example 2：Different reference genes respectively calculate the variation of pears APX gene expression amounts
Respectively with optimal reference gene combination (Actin2 and ARM), 2 unstable reference gene (Histone
H3 and TIP41-like) and 2 common reference genes (UBQ10 and TUB β) respectively as normalization factor to APX genes
It is standardized analysis (Fig. 4).The result shows that when selection 2 most stable of reference gene Actin2, ARM and combinations thereof are to gene
It is quantitative most accurate.And carry out the situation of quantitative analysis with the reference gene of unstable expression and 2 common reference genes
Under, APX gene expression doses generate relatively large deviation in some samples.
Sequence table
<110>Fruit Tree Tea Institute Of Hubei Academy of Agricultural Sciences
<120>The fluorescent quantitation reference gene and its primer of two kinds of tree-like pears difference tree-crown location leaf textures and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 173
<212> DNA
<213>Pears (Pyrus spp.)
<400> 1
ctcccagggc tgtgtttcct agtattgttg gtcgcccacg acacacaggt gtcatggttg 60
gtatgggtca gaaggatgcc tatgtaggtg atgaagcaca gtcgaaaaga ggtatcctta 120
ccttgaagta tcccattgag cacggtatag tgagcaactg ggatgacatg gag 173
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
ctcccagggc tgtgtttcct a 21
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 3
ctccatgtca tcccagttgc t 21
<210> 4
<211> 101
<212> DNA
<213>Pears (Pyrus spp.)
<400> 4
caagggcatt ctttcggagc aactggtttc tgtgaaagaa gaaagcatga gaatattgaa 60
ggacttcatc accagacaca atgttcctaa tgatgtccct g 101
<210> 5
<211> 17
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 5
caagggcatt ctttcgg 17
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 6
cagggacatc attaggaaca t 21
Claims (10)
1. the fluorescent quantitation reference gene of two kinds of tree-like pears difference tree-crown location leaf textures, which is characterized in that including reference gene
Actin2 and ARM.
2. reference gene according to claim 1, which is characterized in that the reference gene Actin2, nucleotides sequence
Row are as shown in SEQ ID NO.1；The reference gene ARM, nucleotide sequence is as shown in SEQ ID NO.4.
3. the specificity of the fluorescent quantitation reference gene of two kinds of tree-like pears difference tree-crown location leaf textures described in claim 2 is drawn
Object, which is characterized in that the forward primer and reverse primer of the specific primer of the reference gene Actin2 are respectively such as SEQ
Shown in ID NO.2 and SEQ ID NO.3；The forward primer and reverse primer of the specific primer of the reference gene ARM point
Not as shown in SEQ ID NO.5 and SEQ ID NO.6.
4. the specific primer described in reference gene described in claim 1 or claim 3 is preparing fluorescence quantitative kit
In application.
5. application according to claim 4, which is characterized in that the kit further includes Standard PCR reagent.
6. reference gene Actin2 and ARM described in claim 1 is glimmering as two kinds of tree-like pears difference tree-crown location leaf textures
Light quantifies the application of reference gene.
7. the specific primer described in claim 3 is special as the fluorescent quantitation of two kinds of tree-like pears difference tree-crown location leaf textures
The application of property primer.
8. application according to claim 7, which is characterized in that the application is using quantitative fluorescent PCR, fluorescent quantitation
PCR system is 10 μ L, comprising 5 μ L 2 × SYBR Green PCR Master Mix, the forward primer of 10 μm of ol/L and reversely
Each 0.5 μ L of 0.5 μ L, cDNA template of primer, add ddH2O to 10 μ L；Quantitative fluorescent PCR response procedures are 50 DEG C of 5min, then 95
DEG C 10min, 94 DEG C of 15sec, 60 DEG C of 1min carry out 40 cycles.
9. according to the application described in claim 6 or claim 7, which is characterized in that it is that frame is tree-like that described two kinds tree-like
Shape is layered with evacuation.
10. according to the application described in claim 6 or claim 7, which is characterized in that the different tree-crown locations are from
The heart, which is done, to be done in the middle part of the tree body of 0.5-1.0m outside tree body outside 1m, from center and is done from center inside the tree body of 0-0.5m.
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| CN109706260A (en) * | 2019-01-10 | 2019-05-03 | 贵州大学 | The screening and application of flue fruits different development stage reference gene |
| CN111118198A (en) * | 2020-01-20 | 2020-05-08 | 福建农林大学 | Pear peel qRT-PCR (quantitative reverse transcription-polymerase chain reaction) internal reference genes and primers and application thereof |
| CN111118197A (en) * | 2020-01-20 | 2020-05-08 | 福建农林大学 | Pear pulp qRT-PCR (quantitative reverse transcription-polymerase chain reaction) reference genes as well as primers and application thereof |
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| CN107190062A (en) * | 2017-06-05 | 2017-09-22 | 南京农业大学 | The screening and application of pear fruit different development stage fluorescent quantitation reference gene |
| CN107988407A (en) * | 2017-11-29 | 2018-05-04 | 湖北省农业科学院果树茶叶研究所 | The fluorescent quantitation reference gene of two kinds of tree-like pears different development stage leaf textures and its primer and application |
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| CN107190062A (en) * | 2017-06-05 | 2017-09-22 | 南京农业大学 | The screening and application of pear fruit different development stage fluorescent quantitation reference gene |
| CN107988407A (en) * | 2017-11-29 | 2018-05-04 | 湖北省农业科学院果树茶叶研究所 | The fluorescent quantitation reference gene of two kinds of tree-like pears different development stage leaf textures and its primer and application |
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| CN109706260A (en) * | 2019-01-10 | 2019-05-03 | 贵州大学 | The screening and application of flue fruits different development stage reference gene |
| CN109706260B (en) * | 2019-01-10 | 2022-07-26 | 贵州大学 | Screening and application of reference genes of pitaya fruits in different development periods |
| CN111118198A (en) * | 2020-01-20 | 2020-05-08 | 福建农林大学 | Pear peel qRT-PCR (quantitative reverse transcription-polymerase chain reaction) internal reference genes and primers and application thereof |
| CN111118197A (en) * | 2020-01-20 | 2020-05-08 | 福建农林大学 | Pear pulp qRT-PCR (quantitative reverse transcription-polymerase chain reaction) reference genes as well as primers and application thereof |
| CN111118197B (en) * | 2020-01-20 | 2022-07-15 | 福建农林大学 | Pear pulp qRT-PCR reference gene as well as primer and application thereof |
| CN111118198B (en) * | 2020-01-20 | 2022-07-15 | 福建农林大学 | Pear peel qRT-PCR (quantitative reverse transcription-polymerase chain reaction) reference genes as well as primers and application thereof |
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