CN110791580A - Detection method for CHS gene expression level of Indian wild peony - Google Patents
Detection method for CHS gene expression level of Indian wild peony Download PDFInfo
- Publication number
- CN110791580A CN110791580A CN201810867367.1A CN201810867367A CN110791580A CN 110791580 A CN110791580 A CN 110791580A CN 201810867367 A CN201810867367 A CN 201810867367A CN 110791580 A CN110791580 A CN 110791580A
- Authority
- CN
- China
- Prior art keywords
- detection
- primer
- detection method
- sequence
- wild peony
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 33
- 230000014509 gene expression Effects 0.000 title claims abstract description 24
- 241000736199 Paeonia Species 0.000 title claims abstract description 23
- 235000006484 Paeonia officinalis Nutrition 0.000 title claims abstract description 23
- 101150058917 CHS gene Proteins 0.000 title claims abstract description 14
- 238000003753 real-time PCR Methods 0.000 claims abstract description 12
- 239000002299 complementary DNA Substances 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims description 14
- 102000007469 Actins Human genes 0.000 claims description 6
- 108010085238 Actins Proteins 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 238000002844 melting Methods 0.000 claims description 6
- 230000008018 melting Effects 0.000 claims description 6
- 238000007781 pre-processing Methods 0.000 claims description 6
- 238000002123 RNA extraction Methods 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000011161 development Methods 0.000 abstract description 5
- 239000002773 nucleotide Substances 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 abstract description 2
- 102100020720 Calcium channel flower homolog Human genes 0.000 description 12
- 101000932468 Homo sapiens Calcium channel flower homolog Proteins 0.000 description 12
- 108010060641 flavanone synthetase Proteins 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 9
- 229920002477 rna polymer Polymers 0.000 description 8
- 229930002877 anthocyanin Natural products 0.000 description 5
- 235000010208 anthocyanin Nutrition 0.000 description 5
- 239000004410 anthocyanin Substances 0.000 description 5
- 150000004636 anthocyanins Chemical class 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000010802 RNA extraction kit Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000185686 Apocynum venetum Species 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000555675 Gentiana triflora Species 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 240000005338 Melastoma malabathricum Species 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Images
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
- C12Q2600/158—Expression markers
Abstract
The invention discloses a detection method of CHS gene expression quantity of Indian wild peony, and provides a quantitative primer designed according to a nucleotide sequence of Indian wild peony CHS, reverse-transcribed Indian wild peony cDNA is used as a template, and the CHS gene expression quantity of Indian wild peony at different development stages is effectively detected through a quantitative PCR reaction system.
Description
Technical Field
The invention belongs to the technical field of plant gene expression detection, and particularly relates to a detection method of CHS gene expression quantity of Indian wild peony.
Background
Chalcone synthase (CHS) is the first key enzyme in the metabolic pathway of anthocyanin, and deletion of CHS activity causes deletion of anthocyanin and other flavonoids, thereby generating white flower mutants (Hoshin et al, 2009; Hemlebe et al, 2004).
The spatio-temporal expression characteristics of genes involved in anthocyanin biosynthesis exhibit different patterns in different species, and the synergistic expression pattern of structural genes is also different in different plants (Gonget al., 1997; Lalusin et al., 2006; Lu and Yang, 2006). The structural gene shows different expression patterns in different stages of plant growth and development. The expression pattern of key genes in the anthocyanin biosynthesis pathway is also changed, usually with the opening of flowers, the stretching of leaves or the ripening of fruits. In asian lily, the expression level of CHS increases with the growth and development of flowers, reaching a maximum during flowering, suggesting that the expression of genes is coordinated with the production of anthocyanin (Nakatsuka et al, 2003). In Gentiana triflora, the structural gene is expressed only in the flower, and transcripts are barely detectable in the leaves and stems (Nakatsukaetael et al, 2005a, b; Nakatsuka et al, 2008). Ginger Cui et al (2016) para oil
The cloning and expression analysis of the CHS gene in the fruit shows that PsCHS is in
The expression level of the mature fruit is higher in the fruit early development stage and then begins to decrease, and the expression level is lower in the fruit mature stage. The plum seedling (2015) performs CHS expression analysis on different tissues of apocynum venetum, and the CHS expression level in a flower component is the highest and the CHS expression level in roots and leaves is the lowest.
Indian wild peony is used as a garden ornamental plant and is mainly distributed in tropical and subtropical regions. The research on the ornamental plants of the melastoma family starts late in China, and the research on the plants of the family is mainly focused on the aspects of resource investigation, introduction and domestication, discussion and classification of cultivation measures and the like, and few reports are made on the aspects of flower color breeding, structural gene separation, functional analysis and the like.
Disclosure of Invention
The invention aims to provide a method for detecting CHS expression quantity of Indian wild peony by analyzing the expression level of CHS gene transcription activity in different development stages and different tissues of white flowers and pink flowers through real-time fluorescent quantitative PCR.
The technical scheme of the invention is as follows:
a detection method of CHS gene expression level of Indian wild peony comprises the following steps:
(1) total RNA extraction: extracting total RNA of Indian wild peony. Selecting flowers in the white flower and flower blooming period of the Indian wild peony, respectively taking 5 strains, respectively mixing the samples, and respectively extracting total RNA (ribonucleic acid) from 2 mixed samples;
(2) aiming at the 2 mixed samples obtained in the step (1), the following operations are respectively carried out: 2. mu.L of 5 Xg DNA Buffer, a total amount of the above total RNA not more than 2. mu.g, and RNase-Free ddH2Preparing a reaction system of 10 mu L by using O, uniformly mixing, incubating at 42 ℃ for 3min, and then placing on ice for later use;
(3) aiming at the 2 mixed samples obtained in the step (1), the following operations are respectively carried out: 10 XFast RT Buffer 2. mu.L, RTenzyme Mix 1. mu. L, FQ-RT Primer Mix 2. mu.L and RNase-Free ddH were added2Preparing a 10 mu L reaction system from O, adding the reaction system obtained in the step into the material obtained in the step (2), and incubating at 42 ℃ for 15min and then at 95 ℃ for 3min to obtain a cDNA template;
(4) aiming at the six mixed samples obtained in the step (1), the following operations are respectively carried out: mu.L of the cDNA template 2. mu. L, qPCRmasterMix 5. mu.L, 0.3. mu.L of forward detection primer at a concentration of 10. mu.M, 0.3. mu.L of reverse detection primer at a concentration of 10. mu.M, and ddH2After O2.4 mu L is uniformly mixed, carrying out fluorescent quantitative PCR detection, and making three multiple holes for each mixed sample, wherein the sequence of the forward detection primer is shown as SEQ ID NO 01, and the sequence of the reverse detection primer is shown as SEQ ID NO 02; simultaneously, actin is used as an internal reference to perform fluorescence quantitative PCR detection by the same system and conditions, wherein the sequence of a forward primer of the internal reference used by actin is shown as SEQ ID NO 03, and the sequence of a reverse primer of the internal reference is shown as SEQ ID NO 04;
(5) data processing: comprises the following steps
a. Data preprocessing: when Ct SD of three double holes of each sample is less than 0.2, no treatment is needed; when the Ct mean value of three complex holes of each sample is less than 30 and Ct SD is more than 0.2, after the Ct value of a certain complex hole deviating from the Ct values of 2 complex holes is removed, when the Ct SD is less than 0.3, the two remaining complex hole Ct values are considered to be reliable and effective; when the Ct SD of one of the multiple holes is still larger than 0.3 after the Ct value of the other hole is removed, the sample is considered to need to be subjected to repeated experiments to meet the requirement; when Ct mean is more than 30, the maximum limit of Ct SD is 0.5 due to the existence of Poisson distribution, and the data preprocessing mode is consistent with that before;
b. and (3) data calculation: experiment according to 2-ΔΔCtThe method processes the data pre-processed in step a.
In a preferred embodiment of the invention, the fluorescent quantitative PCR assay comprises a PCR procedure and a dissolution curve procedure, wherein
The PCR procedure was: 94-95 ℃ for 90 s; 94-95 ℃ for 5s, 59-61 ℃ for 15s, and 71-73 ℃ for 20s, for 38-42 cycles;
the melting curve program was: 94-95 ℃ for 15s and 64-66 ℃ for 1 min.
Further preferably, the PCR procedure is: 90s at 95 ℃; 5s at 95 ℃, 15s at 60 ℃ and 20s at 72 ℃ for 40 cycles.
More preferably, the melting curve program is: 95 ℃ for 15s, 65 ℃ for 1 min.
The invention has the beneficial effects that: the invention provides a quantitative primer designed according to a nucleotide sequence of the Indian wild peony CHS, and the CHS expression quantity of different developmental stages of the Indian wild peony is effectively detected by taking reverse-transcribed Indian wild peony cDNA as a template through a quantitative PCR reaction system.
Drawings
FIG. 1 is a CHS gene melting curve in example 1 of the present invention.
FIG. 2 is a CHS gene amplification curve in example 1 of the present invention.
FIG. 3 shows CHS gene expression levels of pollen (P2) and white flower (W2) of Indian wild peony in full bloom stage in example 1 of the present invention
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1
A detection method of CHS gene expression level of Indian wild peony comprises the following steps:
(1) total RNA extraction: total RNA of Indian wild peony petals is extracted according to the RNA extraction kit specification of Kangji century (CW 0559). Selecting flowers in the white flower and flower blooming period of the Indian wild peony, respectively taking 5 strains, respectively mixing the samples, and respectively extracting total RNA (ribonucleic acid) from 2 mixed samples; the RNA extraction kit used was purchased from Kong as a century (CW 0559).
(2) Aiming at the 2 mixed samples obtained in the step (1), the following operations are respectively carried out: 2. mu.L of 5 Xg DNA Buffer, a total amount of the above total RNA not more than 2. mu.g, and RNase-Free ddH2Preparing a reaction system of 10 mu L by using O, uniformly mixing, incubating at 42 ℃ for 3min, and then placing on ice for later use;
(3) aiming at the 2 mixed samples obtained in the step (1), the following operations are respectively carried out: 10 XFast RT Buffer 2. mu.L, RTenzyme Mix 1. mu. L, FQ-RT Primer Mix 2. mu.L and RNase-Free ddH were added2Preparing a 10 mu L reaction system from O, adding the reaction system obtained in the step into the material obtained in the step (2), and incubating at 42 ℃ for 15min and then at 95 ℃ for 3min to obtain a cDNA template; the reverse transcription Kit used in the steps (2) and (3) is FastQuantity RT Kit (KR106) from Tiangen.
(4) Aiming at the 2 mixed samples obtained in the step (1), the following operations are respectively carried out: mu.L of the cDNA template 2. mu. L, qPCRmasterMix 5. mu.L, 0.3. mu.L of forward detection primer at a concentration of 10. mu.M, 0.3. mu.L of reverse detection primer at a concentration of 10. mu.M, and ddH2After O2.4 mu L is uniformly mixed, carrying out fluorescent quantitative PCR detection, and making three multiple holes for each mixed sample, wherein the sequence of the forward detection primer is shown as SEQ ID NO 01, and the sequence of the reverse detection primer is shown as SEQ ID NO 02; simultaneously, actin is used as an internal reference to perform fluorescence quantitative PCR detection (the result is shown in figure 1 and figure 2) by using the same system and conditions, wherein the sequence of a forward primer of the internal reference used by actin is shown as SEQ ID NO 03, and the sequence of a reverse primer of the internal reference is shown as SEQ ID NO 04; the RT-QPCR reagent used in this step was purchased from TAKARA, SYBR Green fluorescent quantitation kit.
The fluorescent quantitative PCR detection comprises a PCR program and a dissolution curve program, wherein
The PCR procedure was: 90s at 95 ℃; 5s at 95 ℃, 15s at 60 ℃ and 20s at 72 ℃ for 40 cycles.
The melting curve program was: 95 ℃ for 15s, 65 ℃ for 1 min.
(5) Data processing: comprises the following steps
a. Data preprocessing: when Ct SD of three double holes of each sample is less than 0.2, no treatment is needed; when the Ct mean value of three complex holes of each sample is less than 30 and Ct SD is more than 0.2, after the Ct value of a certain complex hole deviating from the Ct values of 2 complex holes is removed, when the Ct SD is less than 0.3, the two remaining complex hole Ct values are considered to be reliable and effective; when the Ct SD of one of the multiple holes is still larger than 0.3 after the Ct value of the other hole is removed, the sample is considered to need to be subjected to repeated experiments to meet the requirement; when Ct mean is more than 30, the maximum limit of Ct SD is 0.5 due to the existence of Poisson distribution, and the data preprocessing mode is consistent with that before;
b. and (3) data calculation: experiment according to 2-ΔΔCtThe method processes data, and the detection results of the pollen and white flower in the full bloom stage are shown in fig. 3, wherein,
Δ Ct ═ i (mean Ct of target gene-mean Ct of internal reference)
Δ Δ Ct ═ target gene Δ Ct in the mixed sample-reference target gene Δ Ct in the mixed sample)
Δ Ct mean ═ AVERAGE (Ct value for all replicate wells)
Ct SD ═ STDEV (all samples replicate Ct values)
Error＝SE Ct sample
The quantitative data and analysis results of 2 mixed samples (white flower for W2-QPCR, pink flower for P2-QPCR) are shown in Table 1, wherein the W2 treatment of CHS has one complex pore value greatly deviating from other complex pore values, so the complex pore data are removed:
TABLE 1
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Sequence listing
<110> institute for science of tropical crops in Fujian province
<120> detection method for CHS gene expression level of Indian wild peony
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
<210>2
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
tcaccaacag cgagcacaa 19
<210>3
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
agttgttgtt ccgctttcct c 21
<210>4
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ttttatcctc ctcaccctcc c 21
Claims (4)
1. A detection method of CHS gene expression level of Indian wild peony is characterized in that: the method comprises the following steps:
(1) total RNA extraction: extracting total RNA of Indian wild peony, selecting flowers in the white flower and pink flower petal-spitting period of Indian wild peony, respectively taking 5 strains, mixing the samples, and respectively extracting the total RNA from 2 mixed samples;
(2) aiming at the 2 mixed samples obtained in the step (1), the following operations are respectively carried out: 2. mu.L of 5 Xg DNA Buffer, a total amount of the above total RNA not more than 2. mu.g, and RNase-Free ddH2Preparing a reaction system of 10 mu L by using O, uniformly mixing, incubating at 42 ℃ for 3min, and then placing on ice for later use;
(3) aiming at the 2 mixed samples obtained in the step (1), the following operations are respectively carried out: 10 XFast RT Buffer 2. mu.L, RTenzyme Mix 1. mu. L, FQ-RT Primer Mix 2. mu.L and RNase-Free ddH were added2Preparing a 10 mu L reaction system from O, adding the reaction system obtained in the step into the material obtained in the step (2), and incubating at 42 ℃ for 15min and then at 95 ℃ for 3min to obtain a cDNA template;
(4) aiming at the 2 mixed samples obtained in the step (1), the following operations are respectively carried out: mu.L of the cDNA template 2. mu. L, qPCRmasterMix 5. mu.L, 0.3. mu.L of forward detection primer at a concentration of 10. mu.M, 0.3. mu.L of reverse detection primer at a concentration of 10. mu.M, and ddH2After O2.4 mu L is uniformly mixed, carrying out fluorescent quantitative PCR detection, and making three multiple holes for each mixed sample, wherein the sequence of the forward detection primer is shown as SEQ ID NO 01, and the sequence of the reverse detection primer is shown as SEQ ID NO 02; simultaneously, actin is used as an internal reference to perform fluorescence quantitative PCR detection by the same system and conditions, wherein the sequence of a forward primer of the internal reference used by actin is shown as SEQ ID NO 03, and the sequence of a reverse primer of the internal reference is shown as SEQ ID NO 04;
(5) data processing: comprises the following steps
a. Data preprocessing: when Ct SD of three double holes of each sample is less than 0.2, no treatment is needed; when the Ct mean value of three complex holes of each sample is less than 30 and Ct SD is more than 0.2, after the Ct value of a certain complex hole deviating from the Ct values of 2 complex holes is removed, when the Ct SD is less than 0.3, the two remaining complex hole Ct values are considered to be reliable and effective; when the Ct SD of one of the multiple holes is still larger than 0.3 after the Ct value of the other hole is removed, the sample is considered to need to be subjected to repeated experiments to meet the requirement; when Ct mean is more than 30, the maximum limit of Ct SD is 0.5 due to the existence of Poisson distribution, and the data preprocessing mode is consistent with that before;
b. and (3) data calculation: experiment according to 2-ΔΔCtThe method processes the data pre-processed in step a.
2. The detection method according to claim 1, characterized in that: the fluorescent quantitative PCR detection comprises a PCR program and a dissolution curve program, wherein
The PCR procedure was: 94-95 ℃ for 90 s; 94-95 ℃ for 5s, 59-61 ℃ for 15s, and 71-73 ℃ for 20s, for 38-42 cycles;
the melting curve program was: 94-95 ℃ for 15s and 64-66 ℃ for 1 min.
3. The detection method according to claim 2, characterized in that: the PCR program is as follows: 90s at 95 ℃; 5s at 95 ℃, 15s at 60 ℃ and 20s at 72 ℃ for 40 cycles.
4. The detection method according to claim 2, characterized in that: the melting curve program is: 95 ℃ for 15s, 65 ℃ for 1 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810867367.1A CN110791580A (en) | 2018-08-01 | 2018-08-01 | Detection method for CHS gene expression level of Indian wild peony |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810867367.1A CN110791580A (en) | 2018-08-01 | 2018-08-01 | Detection method for CHS gene expression level of Indian wild peony |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110791580A true CN110791580A (en) | 2020-02-14 |
Family
ID=69425545
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810867367.1A Pending CN110791580A (en) | 2018-08-01 | 2018-08-01 | Detection method for CHS gene expression level of Indian wild peony |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110791580A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080286766A1 (en) * | 2004-07-09 | 2008-11-20 | Guohua Zhou | Comparing Method for Expression Amount of the Same Gene from Different Sources by Base Sequence Measurement |
| CN104073557A (en) * | 2014-06-11 | 2014-10-01 | 中国科学院海洋研究所 | Fluorescence quantitative reference genes for crassostrea gigas Poly (I:C) stress experiment as well as primers and applications of fluorescence quantitative reference genes |
| CN107936104A (en) * | 2017-12-29 | 2018-04-20 | 中国科学院植物研究所 | Tree peony PsMYB12 transcription factors and its encoding gene and application |
| WO2018086263A1 (en) * | 2016-11-10 | 2018-05-17 | 三生国健药业（上海）股份有限公司 | Real-time fluorescent quantitative pcr detection method, and standard sample and detection kit thereof |
-
2018
- 2018-08-01 CN CN201810867367.1A patent/CN110791580A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080286766A1 (en) * | 2004-07-09 | 2008-11-20 | Guohua Zhou | Comparing Method for Expression Amount of the Same Gene from Different Sources by Base Sequence Measurement |
| CN104073557A (en) * | 2014-06-11 | 2014-10-01 | 中国科学院海洋研究所 | Fluorescence quantitative reference genes for crassostrea gigas Poly (I:C) stress experiment as well as primers and applications of fluorescence quantitative reference genes |
| WO2018086263A1 (en) * | 2016-11-10 | 2018-05-17 | 三生国健药业（上海）股份有限公司 | Real-time fluorescent quantitative pcr detection method, and standard sample and detection kit thereof |
| CN107936104A (en) * | 2017-12-29 | 2018-04-20 | 中国科学院植物研究所 | Tree peony PsMYB12 transcription factors and its encoding gene and application |
Non-Patent Citations (2)
| Title |
|---|
| HIDAYAH JAMALNASIR等: "Molecular cloning and characterization of a cDNA encoding a polyketide synthase from Melastoma decemfidum", SECTION CELLULAR AND MOLECULAR BIOLOGY, pages 1482 - 1491 * |
| 周琳;王雁;彭镇华;: "牡丹查耳酮合酶基因Ps-CHS1的克隆及其组织特异性表达", 园艺学报, no. 08, pages 97 - 104 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107557369B (en) | Characteristic sequence, labeled primer and identification method of apocarya variety Nacono | |
| CN108165653B (en) | InDel molecular marker for identifying pepper maturity and application thereof | |
| CN108330163B (en) | Characteristic sequence, primer and identification method of apocarya variety Nacono and Sumner | |
| CN113637794B (en) | SSR molecular marker of new variety of mulberry, namely Guangdong mulberry 201, and core primer group, kit and application thereof | |
| Maroufi | Selection of reference genes for real-time quantitative PCR analysis of gene expression in Glycyrrhiza glabra under drought stress | |
| CN108424913A (en) | Muskmelon U6 genes and its application | |
| CN107586866B (en) | Characteristic sequence, labeled primer and identification method of apocarya variety Moore | |
| CN106350591B (en) | Construct SSR primer, constructed finger-print and its application of coloured silk Ye Luanshu Variety fingerprinting | |
| CN110791580A (en) | Detection method for CHS gene expression level of Indian wild peony | |
| CN106834534B (en) | Method for detecting iron chelate reductase family gene of citrus | |
| CN106755526B (en) | Functional molecular marker related to lipped and petal sepal character of cymbidium and identification method thereof | |
| CN108728569A (en) | A kind of okra reference gene and its application | |
| CN108588091A (en) | A kind of okra reference gene and its application | |
| CN105274219B (en) | Purposes of the CL5547.Contig2 gene in the analysis of pumpkin gene expression real-time fluorescence quantitative PCR as reference gene | |
| CN112695125B (en) | Katelia SSR molecular marker primer composition and application thereof | |
| CN104404156A (en) | Rapid identification molecular marker of self-compatible variety of loquat, marker primer and identification method | |
| CN108949780B (en) | The Ravenna grass genoid EfNAC44 that Ravenna grass wild species are expressed by low temperature stress | |
| CN108085405A (en) | A kind of molecular labeling, primer pair and its application for being used to differentiate candleberry male and female | |
| CN109207625B (en) | SSR-PCR identification method for distinguishing 4 main cultivars of agaricus bisporus | |
| CN109055599B (en) | Molecular marker primer for identifying Jinmei variety of kiwi fruit and application thereof | |
| CN108330164B (en) | Characteristic sequence, primer and identification method of apocarya variety Moore | |
| CN112980989A (en) | MiRNA fluorescent quantitative reference gene of different tissues of cryptomeria fortunei as well as primer and application thereof | |
| CN105950729B (en) | One kind SNP marker relevant to rubber tree stem girth and its application | |
| Maroufi et al. | Differential expression of fructan 1-exohydrolase genes involved in inulin biodegradation in chicory ('Cichorium intybus') cultivars | |
| CN108841842B (en) | Festuca arundinacea gene EfWRKY62 expressed by wild species of the saccharum arundinacea under low temperature stress |
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 |