CN104988234A - Quantitative detection method of wheat fructan metabolizing enzyme gene - Google Patents
Quantitative detection method of wheat fructan metabolizing enzyme gene Download PDFInfo
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- CN104988234A CN104988234A CN201510415810.8A CN201510415810A CN104988234A CN 104988234 A CN104988234 A CN 104988234A CN 201510415810 A CN201510415810 A CN 201510415810A CN 104988234 A CN104988234 A CN 104988234A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- 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
Abstract
The invention discloses a quantitative detection method of wheat fructan metabolizing enzyme genes, relates to a real-time fluorescent quantitation PCR quantitative detection technique of wheat stem fructan metabolizing enzyme genes and belongs to the field of plant molecular biological techniques and gene engineering. The invention uses wheat stem fructan metabolism to synthesize key sucrose: fructan 6-fructosyltransferase (6-SFT) genes and degradation key enzyme genes fructan exocellular hydrolase (FEH) genes, and detects the expression quantity of 6-SFT genes and FEH genes to obtain a stem fructan metabolizing molecule physiological regulation mechanism at a wheat grain filling stage under drought regulation. The quantitative detection method of wheat fructan metabolizing enzyme genes has the beneficial effect that the method can quickly, accurately and quantitatively detect the differential expression of stem fructan metabolizing key enzyme genes 6-SFT and FEH at the wheat grain filling stage.
Description
Technical field
The present invention relates to the Real-Time Fluorescent Quantitative PCR Technique quantitative measurement technology of wheat culm Fructan metabolism enzyme gene, belong to plant molecular biotechnology and genetically engineered field, specifically a kind of method of triticin metabolic enzyme gene detection by quantitative.
Background technology
Polylevulosan is as bridge metabolic substd between wheat source (leaf) storehouse (stem and fringe portion), the injury of environment stress to wheat is not cushioned by means of only osmoregulation, it is also the required important carbon source of wheat grain grouting simultaneously, especially at Wheat in Grain Filling Stage, when the drought stress havoc wheat normal light cooperation used time, be stored in Polylevulosan in stem stalk before wheat flower to transport to seed, the wheat grain yield loss because arid causes can be compensated to a certain extent.
Research shows, sucrose: Polylevulosan-6-fructosyl transferase (6-SFT) and Fructan exohydrolase (FEH) are the key enzyme controlling Polylevulosan synthesis and degraded in grass body respectively.Wherein, 6-SFT is mainly substrate with sucrose, and the fructosyl on catalysing sucrose molecule is transferred on the C6 position of another sucrose molecules fructosyl, synthesis 6-kestose, with few Polylevulosan for substrate, can also generate branching type Polylevulosan simultaneously.FEH is mainly through the irreversible DeR of a kind of Polylevulosan, once separate next fructosyl from the fructosyl end of Polylevulosan, until surplus next sucrose molecules, the fructose molecule discharged can from new synthesis of sucrose as substrate, by phloem loading, long-distance transportation enters seed.Between Grains of Common Wheat during Grain-filling Stage, the environmental factors such as arid, high temperature all can induce the rising of FEH activity in cane, strengthen degraded and the output of Polylevulosan, thus facilitate the ability of mobilization of whole stem stalk Non-structure carbohydrate and Polylevulosan, promote that Polylevulosan degraded product sucrose is transported to seed, compensate grain milk.Therefore, be necessary that the differential expression for Wheat in Grain Filling Stage stem stalk Fructan metabolism key gene 6-SFT and FEH sets up quantitative detecting method fast and accurately, thus establish good technical foundation for further investigation arid regulates and controls Wheat in Grain Filling Stage stem stalk Fructan metabolism molecular physiology regulatory mechanism.
Summary of the invention
The object of this invention is to provide a kind of method of triticin metabolic enzyme gene detection by quantitative, detect Wheat in Grain Filling Stage stem stalk Fructan metabolism key gene 6-SFT and FEH, this detection method makes up the defect of Standard PCR technology, can Real-Time Monitoring amplified reaction, have fast, in real time, advantage reliably.
For achieving the above object, the method for a kind of triticin metabolic enzyme gene detection by quantitative of the present invention, performing step is as follows:
(A) material sampling point and the time: save stem stalk under 5 ~ 6d clip stem stalk fringe after wheat flower, put into Liquid nitrogen storage immediately, total serum IgE in stem stalk is extracted;
The extracting method of described wheat culm total serum IgE is as follows:
1) wheat culm is immersed liquid nitrogen, THE ADIABATIC SHEAR IN wheat culm is the long fragment of 1 ~ 2cm, fully mixes fragment, takes Wheat Tissue 0.2g stem stalk fragment, add 2ml lysate RZ, carry out homogenized with Syrup-homogenizing instrument;
2) homogenised sample is placed 5min at 18 DEG C, make to adjust albumen composition and be separated completely, 4 DEG C of centrifugal 5min of 12000rpm, get supernatant, add 200 μ l chloroforms, build pipe lid, concuss 15sec, and room temperature places 3min; After 4 DEG C of centrifugal 10min of 12000rpm, aqueous phase is transferred in new pipe;
3) interior have in the new pipe of aqueous phase slowly add 0.5 times of volume dehydrated alcohol, mixing, to obtain solution proceeds in adsorption column together with precipitation, 4 DEG C of centrifugal 30s of 12000rpm, discard the waste liquid in collection tube, in adsorption column, add 500 μ l protein liquid removals, 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; In adsorption column, add 600 μ l rinsing liquids, room temperature leaves standstill 2min, and 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; This step of repetitive operation once; Adsorption column is put into 2ml collection tube, 4 DEG C of centrifugal 2min of 12000rpm, remove residual liquid, be placed on Bechtop and ventilate a moment, fully to dry;
4) adsorption column is proceeded in a new 1.5ml centrifuge tube, add 50 μ lRNase-Free ddH
2o, room temperature places 2min, 4 DEG C of centrifugal 2min of 12000rpm, and products therefrom is stand-by in-70 DEG C of preservations;
(C) wheat culm total serum IgE quality examination:
1) adopt the quality of spectrophotometry RNA, require that the ratio of OD260/OD280 is 2 ~ 2.1;
2) 1.0% sepharose is utilized to detect the quality of RNA;
(D) design of primers and synthesis: according to the cDNA sequence of the triticin key enzyme gene that NCBI registers, using Actin (GenBank ID:AB181991) as reference gene, sucrose: gene for the purpose of Polylevulosan-6-fructosyl transferase (6-SFT) and Fructan exohydrolase (FEH) (GenBank ID is respectively: AB029887 and AJ508387), utilize Primer Premier 5.0 software design Auele Specific Primer as follows, primer transfers to biotech firm to synthesize.
(E) synthesis of cDNA: adopt Reverse Transcription box to prepare cDNA, concrete operation method is:
1) in the centrifuge tube of the nuclease free of ice bath, following reaction mixture is added: 5 μ g total serum IgE, 2 μ l primers, 2 μ l Super Pure dNTPs (2.5mM), benefit RNase-Free ddH
2o is settled to 14.5 μ l;
2) rapid in cooled on ice 2min after 70 DEG C of heating 5min, brief centrifugation adds following component after collecting reaction solution: 4 μ l 5 × First-Strand Buffer (containing DTT), 0.5 μ l RNasin;
3) add 1 μ l (200U) TIANScript M-MLV, gently with pipettor mixing, centrifuge tube is put 25 DEG C of temperature bath 10min;
4) 42 DEG C of temperature bath 50min;
5) 95 DEG C of heating 5min termination reactions, put and carry out subsequent experimental on ice;
6) RNase-Free ddH is used
2reaction system is diluted to 50 μ l by O, gets 2-5 μ l and carries out pcr amplification reaction, detects product integrity;
(F) real-time fluorescence quantitative PCR testing goal gene differential expression: adopt fluorescence quantitative kit, in the enterprising performing PCR amplification of qRT-PCR instrument, 20 μ l reaction systems are as follows:
Reaction conditions is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations, gathers fluorescent signal, detect in real time at the end of the annealing temperature of each circulation.Each template concentrations does 3 repetitions, averages for interpretation of result.To increase the data (C obtained according to qRT-PCR
tvalue) adopt 2
-Δ Δ Ctmethod carries out the relative quantitative assay of destination gene expression, and specific analytical method is as follows:
C
tvalue relative method utilizes C
tthe mathematical relation that the logarithm of value and initiate dna concentration is inversely proportional to, calculate the relative percentage between different sample, its calculation formula is amount=2 of goal gene
-Δ Δ Ct, Δ Δ C
t=(the average C of experimental group goal gene
tthe average C of value-experimental group reference gene
tvalue)-(the average C of control group goal gene
tthe average C of value-control group reference gene
tvalue).Note: circulation threshold values (cycle threshold value, C
t) when namely the fluorescent signal of amplified production reaches the fluorescence threshold values of setting in pcr amplification process the cycle index of process.The mode that real time fluorescence quantifying PCR method adopts initial point quantitative, utilizes C
tconcept, detect in the initial stage of exponential amplification, now the tiny error of sample room is not yet amplified and amplification efficiency is also constant, therefore this C
tvalue has fabulous repeatability.
The method of a kind of triticin metabolic enzyme gene detection by quantitative of the present invention, it is characterized in that: the present invention utilizes wheat culm Fructan metabolism to synthesize key enzyme sucrose: Polylevulosan-6-fructosyl transferase gene (6-SFT) and degraded key gene Fructan exohydrolase gene, detection is carried out to the expression amount of 6-SFT and FEH gene and obtains arid regulation and control Wheat in Grain Filling Stage stem stalk Fructan metabolism molecular physiology regulatory mechanism, then apply qRT-PCR quantitative detecting method and verify; The present invention discloses a whole set of improve system of the expression checking of 6-SFT and FEH gene, there is important using value, and the method can the differential expression of detection by quantitative Wheat in Grain Filling Stage stem stalk Fructan metabolism key gene 6-SFT and FEH fast and accurately.
Accompanying drawing explanation
Fig. 1 is total serum IgE electrophoresis;
Fig. 2 is 6-SFT and FEH reverse transcription PCR amplification figure;
Fig. 3 is 6-SFT and FEH pcr amplification curve (A);
Fig. 4 is 6-SFT and FEH PCR solubility curve (B).
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
A method for triticin metabolic enzyme gene detection by quantitative, completes in the steps below:
1. the choosing of wheat experiment material: with Q9086 (strain that wheat institute of agricultural college of Xibei Univ. of Agricultural & Forest Science & Technology provides) for material, this kind is responsive to liquid manure reaction, especially the filling stage is responsive to reaction of moisture, Irrigation regime (contrast) is in the jointing stage, heading stage and flowering period Supplement irrigation, each irrigation quantity is 750m
3/ hm
2, drought stress (process) only to be poured water 750m in the jointing stage
3/ hm
2, save stem stalk under the wheat culm fringe of clip contrast and process respectively after wheat flower 5d, put into Liquid nitrogen storage immediately, for the extraction of total serum IgE in stem stalk;
2. the extraction of total serum IgE and detection:
1) total RNA extraction reagent box is adopted to extract RNA in wheat culm:
1. wheat culm is immersed liquid nitrogen, THE ADIABATIC SHEAR IN wheat culm is the long fragment of 1 ~ 2cm, fully mixes fragment, takes Wheat Tissue 0.2g stem stalk fragment, add 2ml lysate RZ, carry out homogenized with Syrup-homogenizing instrument;
2. homogenised sample is placed 5min at 18 DEG C, make to adjust albumen composition and be separated completely, 4 DEG C of centrifugal 5min of 12000rpm, get supernatant, add 200 μ l chloroforms, build pipe lid, concuss 15sec, and room temperature places 3min; After 4 DEG C of centrifugal 10min of 12000rpm, aqueous phase is transferred in new pipe;
3. interior have in the new pipe of aqueous phase slowly add 0.5 times of volume dehydrated alcohol, mixing, to obtain solution proceeds in adsorption column together with precipitation, 4 DEG C of centrifugal 30s of 12000rpm, discard the waste liquid in collection tube, in adsorption column, add 500 μ l protein liquid removals, 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; In adsorption column, add 600 μ l rinsing liquids, room temperature leaves standstill 2min, and 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; This step of repetitive operation once; Adsorption column is put into 2ml collection tube, 4 DEG C of centrifugal 2min of 12000rpm, remove residual liquid, be placed on Bechtop and ventilate a moment, fully to dry;
4. adsorption column is proceeded in a new 1.5ml centrifuge tube, add 50 μ lRNase-Free ddH
2o, room temperature places 2min, 4 DEG C of centrifugal 2min of 12000rpm, and products therefrom is stand-by in-70 DEG C of preservations;
2) adopt the quality of spectrophotometry RNA, require OD
260/ OD
280ratio 2 ~ 2.1;
3) 1.0% sepharose is utilized to detect the quality of RNA;
3. the Design and synthesis of Auele Specific Primer: according to the cDNA sequence of the triticin key enzyme gene of registration on NCBI, utilize Primer Premier 5.0 software design Auele Specific Primer, wherein the sequence of reference gene Actin (AB181991) is: F-CTATCCTTCGTTTGGACCTT, R-AGCGAGCTTCTCCTTTATGT; The sequence of 6-SFT (AB029887) is: F-CGCACCAGCGACAACTCAT, R-CACCTGTTTGCCGCAGAGT; The sequence of FEH (AJ508387) is: F-CAAGTGACATAGACGGTTG, R-GATCCAACCGGTTGTCGGAT; Primer transfers to biotech firm to synthesize;
The synthesis of 4.cDNA: adopt Reverse Transcription box to prepare cDNA, response procedures is: 37 DEG C, 15min, 85 DEG C, 5sec;
5. the qRT-PCR that key gene 6-SFT and FEH expresses detects and calculates: adopt fluorescence quantitative kit to carry out qRT-PCR detection, reaction system is: FastStart Universal SYBR GreenMaster (ROX) 10 μ l, cDNA 2 μ l, upstream primer (10 μMs) 0.6 μ l, downstream primer (10 μMs) 0.6 μ l, ddH
2o 6.8 μ l, response procedures is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations, gathers fluorescent signal, detect in real time at the end of the annealing temperature of each circulation.Each template concentrations does 3 repetitions, averages to calculate for result.Wherein, the average C of reference gene Actin Osmotic treatment
tvalue is 23.5073, contrasts average C
tvalue is the average C of 24.5031,6-SFT Osmotic treatment
tvalue is 36.7474, contrasts average C
tvalue is the average C of 37.5661, FEH Osmotic treatment
tvalue is 33.0168, contrasts average C
tvalue is 33.9875.
Wherein, the concrete calculation procedure of 6-SFT gene expression amount is as follows:
△ C
t Osmotic treatment=C
t Osmotic treatment-C
t Osmotic treatment reference gene=36.7474-23.5073=13.2401
△ C
t control treatment=C
t control treatment-C
t control treatment reference gene=37.5661-24.5031=13.0630
-△ △ C
t=-(△ C
osmotic treatment-△ C
t control treatment)=-(13.2401-13.0630)=0.1771
6-SFT gene relative fold expression (Osmotic treatment/control treatment)=2
-△ △ Ct=2
-0.1771=0.8845, be also that under Osmotic treatment, the expression multiple of 6-SFT gene improves 0.8845 times relative to contrast.
The concrete calculation procedure of FEH gene expression amount is as follows:
△ C
t Osmotic treatment=C
t Osmotic treatment-C
t Osmotic treatment reference gene=33.0168-23.5073=9.5095
△ C
t control treatment=C
t control treatment-C
t control treatment reference gene=33.9875-24.5031=9.4844
-△ △ C
t=-(△ C
osmotic treatment-△ C
t control treatment)=-(9.5095-9.4844)=0.0251
FEH gene relative fold expression (Osmotic treatment/control treatment)=2
-△ △ Ct=2
-0.0251=0.9828, be also that under Osmotic treatment, the expression multiple of FEH gene improves 0.9828 times relative to contrast.
From embodiment 1: under Osmotic treatment, the expression multiple of FEH gene improves 0.9828 times relative to contrast.
Embodiment 2
1. wheat culm sampling: save stem stalk under 5-6d clip stem stalk fringe after wheat flower, put into Liquid nitrogen storage immediately, stem stalk total serum IgE to be extracted;
2. the extraction of wheat culm total serum IgE:
1. wheat culm is immersed liquid nitrogen, THE ADIABATIC SHEAR IN wheat culm is the long fragment of 1 ~ 2cm, fully mixes fragment, takes Wheat Tissue 0.2g stem stalk fragment, add 2ml lysate RZ, carry out homogenized with Syrup-homogenizing instrument;
2. homogenised sample is placed 5min at 18 DEG C, make to adjust albumen composition and be separated completely, 4 DEG C of centrifugal 5min of 12000rpm, get supernatant, add 200 μ l chloroforms, build pipe lid, concuss 15sec, and room temperature places 3min; After 4 DEG C of centrifugal 10min of 12000rpm, aqueous phase is transferred in new pipe;
3. interior have in the new pipe of aqueous phase slowly add 0.5 times of volume dehydrated alcohol, mixing, to obtain solution proceeds in adsorption column together with precipitation, 4 DEG C of centrifugal 30s of 12000rpm, discard the waste liquid in collection tube, in adsorption column, add 500 μ l protein liquid removals, 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; In adsorption column, add 600 μ l rinsing liquids, room temperature leaves standstill 2min, and 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; This step of repetitive operation once; Adsorption column is put into 2ml collection tube, 4 DEG C of centrifugal 2min of 12000rpm, remove residual liquid, be placed on Bechtop and ventilate a moment, fully to dry;
4. adsorption column is proceeded in a new 1.5ml centrifuge tube, add 50 μ lRNase-Free ddH
2o, room temperature places 2min, 4 DEG C of centrifugal 2min of 12000rpm, and products therefrom is stand-by in-70 DEG C of preservations;
3. wheat culm total serum IgE quality examination: with sepharose and the ultramicrospectrophotometer of 1%, detects the quality of the wheat culm total serum IgE extracted, concentration and relative purity;
4. design of primers and synthesis: according to the cDNA sequence of the upper triticin key enzyme gene registered of NCBI (www.ncbi.nlm.nih.gov), using Actin (GenBank ID:AB181991) as reference gene, sucrose: gene for the purpose of Polylevulosan-6-fructosyl transferase (6-SFT) and Fructan exohydrolase (FEH) (GenBank ID is respectively: AB029887 and AJ508387), utilize Primer Premier5.0 software design Auele Specific Primer (table 1), primer transfers to biotech firm to synthesize;
Table 1PCR amplimer sequence
The synthesis of 5.cDNA: adopt TIAN script RT Kit test kit (Beijing Tian Gen biochemical technology company limited), the Article 1 chain of reverse transcription synthesis cDNA, and detect product integrity, concrete operation method is as follows:
(1) in the centrifuge tube of the nuclease free of ice bath, following reaction mixture is added:
5 μ g total serum IgE;
2 μ l primers;
2μl Super Pure dNTPs(2.5mM)；
Mend RNase-Free ddH
2o is settled to 14.5 μ l;
Rapidly at cooled on ice 2min after (2) 70 DEG C of heating 5min, brief centrifugation adds following component after collecting reaction solution:
4 μ l 5 × First-Strand Buffer (containing DTT); 0.5 μ l RNasin;
(3) add 1 μ l (200U) TIANScript M-MLV, gently with pipettor mixing, centrifuge tube is put 25 DEG C of temperature bath 10min;
(4) 42 DEG C of temperature bath 50min;
(5) 95 DEG C of heating 5min termination reactions, put and carry out subsequent experimental on ice;
(6) RNase-Free ddH is used
2reaction system is diluted to 50 μ l by O, gets 2-5 μ l and carries out pcr amplification reaction, detects product integrity;
6. real-time fluorescence quantitative PCR testing goal gene differential expression: adopt Fast Start UniversalSYBR Green Master Rox test kit (Shanghai Roche Diagnistics Products Co., Ltd), FTC-3000qRT-PCR instrument is tested through the amplification of performing PCR, and 20 μ l reaction systems are as follows:
| FastStart Universal SYBR Green Master(ROX) | 10μl |
| cDNA | 2μl |
| Upstream primer (10 μMs) | 0.6μl |
| Downstream primer (10 μMs) | 0.6μl |
| ddH 2O | 6.8μl |
Reaction conditions is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations, gather fluorescent signal, detect in real time at the end of the annealing temperature of each circulation, each template concentrations does 3 repetitions, averages for interpretation of result; Adopt 2-Δ Δ Ct method to carry out the relative quantitative assay of destination gene expression according to the increase data (Ct value) that obtain of qRT-PCR, specific analytical method is as follows:
Ct value relative method is the mathematical relation utilizing the logarithm of Ct value and initiate dna concentration to be inversely proportional to, calculate the relative percentage between different sample, its calculation formula is amount=2-Δ Δ Ct, Δ Δ Ct=(experimental group goal gene average Ct Zhi – experimental group reference gene Average Ct values)-(the control group goal gene average Ct Zhi – control group reference gene Average Ct values) of goal gene.
Adopting said method respectively organizes Fructan metabolism key gene 6-SFT and FEH expression to carry out Real-Time Monitoring to wheat, is conducive to the collection of data, ensures the reliability of data, contribute to the research expressed complex condition wheat cdna simultaneously.
Embodiment 3
1. wheat culm sampling: save stem stalk under 5-6d clip stem stalk fringe after wheat flower, put into Liquid nitrogen storage immediately, stem stalk total serum IgE to be extracted;
2. the extraction of wheat culm total serum IgE:
1. wheat culm is immersed liquid nitrogen, THE ADIABATIC SHEAR IN wheat culm is the long fragment of 1 ~ 2cm, fully mixes fragment, takes Wheat Tissue 0.2g stem stalk fragment, add 2ml lysate RZ, carry out homogenized with Syrup-homogenizing instrument;
2. homogenised sample is placed 5min at 18 DEG C, make to adjust albumen composition and be separated completely, 4 DEG C of centrifugal 5min of 12000rpm, get supernatant, add 200 μ l chloroforms, build pipe lid, concuss 15sec, and room temperature places 3min; After 4 DEG C of centrifugal 10min of 12000rpm, aqueous phase is transferred in new pipe;
3. interior have in the new pipe of aqueous phase slowly add 0.5 times of volume dehydrated alcohol, mixing, to obtain solution proceeds in adsorption column together with precipitation, 4 DEG C of centrifugal 30s of 12000rpm, discard the waste liquid in collection tube, in adsorption column, add 500 μ l protein liquid removals, 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; In adsorption column, add 600 μ l rinsing liquids, room temperature leaves standstill 2min, and 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; This step of repetitive operation once; Adsorption column is put into 2ml collection tube, 4 DEG C of centrifugal 2min of 12000rpm, remove residual liquid, be placed on Bechtop and ventilate a moment, fully to dry;
4. adsorption column is proceeded in a new 1.5ml centrifuge tube, add 50 μ lRNase-Free ddH
2o, room temperature places 2min, 4 DEG C of centrifugal 2min of 12000rpm, and products therefrom is stand-by in-70 DEG C of preservations;
3. wheat culm total serum IgE quality examination: with sepharose and the ultramicrospectrophotometer of 1%, detects the quality of the wheat culm total serum IgE extracted, concentration and relative purity;
4. design of primers and synthesis: according to the cDNA sequence of the upper triticin key enzyme gene registered of NCBI (www.ncbi.nlm.nih.gov), using Actin (GenBank ID:AB181991) as reference gene, sucrose: gene for the purpose of Polylevulosan-6-fructosyl transferase (6-SFT) and Fructan exohydrolase (FEH) (GenBank ID is respectively: AB029887 and AJ508387), utilize Primer Premier5.0 software design Auele Specific Primer (table 1), primer transfers to biotech firm to synthesize;
Table 1PCR amplimer sequence
The synthesis of 5.cDNA: adopt TIAN script RT Kit test kit (Beijing Tian Gen biochemical technology company limited), the Article 1 chain of reverse transcription synthesis cDNA, and detect product integrity, concrete operation method is as follows:
(1) in the centrifuge tube of the nuclease free of ice bath, following reaction mixture is added:
5 μ g total serum IgE;
2 μ l primers;
2μl Super Pure dNTPs(2.5mM)；
Mend RNase-Free ddH2O and be settled to 14.5 μ l;
Rapidly at cooled on ice 2min after (2) 70 DEG C of heating 5min, brief centrifugation adds following component after collecting reaction solution:
4 μ l 5 × First-Strand Buffer (containing DTT); 0.5 μ l RNasin;
(3) add 1 μ l (200U) TIANScript M-MLV, gently with pipettor mixing, centrifuge tube is put 25 DEG C of temperature bath 10min;
(4) 42 DEG C of temperature bath 50min;
(5) 95 DEG C of heating 5min termination reactions, put and carry out subsequent experimental on ice;
(6) with RNase-Free ddH2O, reaction system is diluted to 50 μ l, gets 2-5 μ l and carry out pcr amplification reaction, detect product integrity;
6. real-time fluorescence quantitative PCR testing goal gene differential expression: adopt Fast Start UniversalSYBR Green Master Rox test kit (Shanghai Roche Diagnistics Products Co., Ltd), FTC-3000qRT-PCR instrument is tested through the amplification of performing PCR, and 20 μ l reaction systems are as follows:
| FastStart Universal SYBR Green Master(ROX) | 10μl |
| cDNA | 2μl |
| Upstream primer (10 μMs) | 0.6μl |
| Downstream primer (10 μMs) | 0.6μl |
| ddH 2O | 6.8μl |
Reaction conditions is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations, gathers fluorescent signal, detect in real time at the end of the annealing temperature of each circulation; Each template concentrations does 3 repetitions, averages for interpretation of result; Adopt 2-Δ Δ Ct method to carry out the relative quantitative assay of destination gene expression according to the increase data (Ct value) that obtain of qRT-PCR, specific analytical method is as follows:
Ct value relative method is the mathematical relation utilizing the logarithm of Ct value and initiate dna concentration to be inversely proportional to, calculate the relative percentage between different sample, its calculation formula is amount=2-Δ Δ Ct, Δ Δ Ct=(experimental group goal gene average Ct Zhi – experimental group reference gene Average Ct values)-(the control group goal gene average Ct Zhi – control group reference gene Average Ct values) of goal gene.
Adopting said method respectively can organize Fructan metabolism key gene 6-SFT and FEH expression to carry out Real-Time Monitoring to wheat, is conducive to the collection of data, ensures the reliability of data, contribute to the research expressed complex condition wheat cdna simultaneously.
Claims (5)
1. a method for triticin metabolic enzyme gene detection by quantitative, is characterized in that: Wheat in Grain Filling Stage stem stalk Total RNAs extraction, 6-SFT and FEH gene-specific primer Design and synthesis, prepare reverse transcription product cDNA, qRT-PCR and detect and the calculating of gene relative expression quantity;
Described Wheat in Grain Filling Stage stem stalk Total RNAs extraction, method is as follows:
1) wheat culm is immersed liquid nitrogen, THE ADIABATIC SHEAR IN wheat culm is the long fragment of 1 ~ 2cm, fully mixes fragment, takes Wheat Tissue 0.2g stem stalk fragment, add 2ml lysate RZ, carry out homogenized with Syrup-homogenizing instrument;
2) homogenised sample is placed 5min at 18 DEG C, make to adjust albumen composition and be separated completely, 4 DEG C of centrifugal 5min of 12000rpm, get supernatant, add 200 μ l chloroforms, build pipe lid, concuss 15sec, and room temperature places 3min; After 4 DEG C of centrifugal 10min of 12000rpm, aqueous phase is transferred in new pipe;
3) interior have in the new pipe of aqueous phase slowly add 0.5 times of volume dehydrated alcohol, mixing, to obtain solution proceeds in adsorption column together with precipitation, 4 DEG C of centrifugal 30s of 12000rpm, discard the waste liquid in collection tube, in adsorption column, add 500 μ l protein liquid removals, 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; In adsorption column, add 600 μ l rinsing liquids, room temperature leaves standstill 2min, and 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; This step of repetitive operation once; Adsorption column is put into 2ml collection tube, 4 DEG C of centrifugal 2min of 12000rpm, remove residual liquid, be placed on Bechtop and ventilate a moment, fully to dry;
4) adsorption column is proceeded in a new 1.5ml centrifuge tube, add 50 μ lRNase-Free ddH
2o, room temperature places 2min, 4 DEG C of centrifugal 2min of 12000rpm, and products therefrom is stand-by in-70 DEG C of preservations.
2. the method for a kind of triticin metabolic enzyme gene detection by quantitative as claimed in claim 1, is characterized in that: described wheat saves stem stalk under Post flowering 5 ~ 6d clip stem stalk fringe, puts into Liquid nitrogen storage stand-by.
3. the method for a kind of triticin metabolic enzyme gene detection by quantitative as claimed in claim 1, it is characterized in that: described 6-SFT and FEH gene-specific primer Design and synthesis, the sequence of gene is: F-CTATCCTTCGTTTGGACCTT, R-AGCGAGCTTCTCCTTTATGT; The sequence of 6-SFT is: F-CGCACCAGCGACAACTCAT, R-CACCTGTTTGCCGCAGAGT; The sequence of FEH is: F-CAAGTGACATAGACGGTTG, R-GATCCAACCGGTTGTCGGAT.
4. the method for a kind of triticin metabolic enzyme gene detection by quantitative as claimed in claim 1, it is characterized in that: described cDNA sequence, 6-SFT is gene for the purpose of AB029887; FEH is gene for the purpose of AJ508387; The reaction conditions of the synthesis of cDNA is: 37 DEG C, 15min, 85 DEG C, 5sec.
5. the method for a kind of triticin metabolic enzyme gene detection by quantitative as claimed in claim 1, it is characterized in that: the qRT-PCR that described 6-SFT and FEH expresses detects and calculates, adopt qRT-PCR detection method, response procedures is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations, at the end of the annealing temperature of each circulation, gather fluorescent signal, detect in real time; Each template concentrations does 3 repetitions, averages to calculate for result; Adopt 2
-Δ Δ Ctmethod of calculation carry out the relative quantification of destination gene expression.
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