CN109136393A - Detect the primer and method of Agasicles hygrophila CCO gene expression characteristics - Google Patents
Detect the primer and method of Agasicles hygrophila CCO gene expression characteristics Download PDFInfo
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Abstract
The present invention provides the primers and method of detection Agasicles hygrophila CCO gene expression characteristics, and for primer sequence as shown in SEQ ID NO.1-2, the present invention establishes the female male worm feeding difference CO of Agasicles hygrophila2The fluorescence quantitative PCR method that CCO gene expression amount detects after the alternanthera philoxeroides that concentration is cultivated, and the sequencing of transcript profile is carried out, compared with having done to the expression quantity trend of CCO gene by fluorescence quantitative with the expression quantity trend of transcript profile, the defense mechanism and CCO gene expression characteristics generated when for research Agasicles hygrophila by environment-stress is laid a good foundation.
Description
Technical field
The present invention relates to the primers and method of detection Agasicles hygrophila CCO gene expression characteristics, belong to biotechnology neck
Domain.
Background technique
Agasicles hygrophila category coleoptera, Chrysomelidae are one of main biological and ecological methods to prevent plant disease, pests, and erosion natural enemies of alternanthera philoxeroides.George
Vogt had found that Agasicles hygrophila had good control action to alternanthera philoxeroides in 1962 for the first time.Thereafter, the straight chest of lotus grass
Flea beetle is widely used in the control of alternanthera philoxeroides on the U.S., Australia and other places, and achieves preferable control efficiency.I
State introduced Agasicles hygrophila from the U.S. in 1986, and was applied to the biological control of alternanthera philoxeroides, in Sichuan, Zhejiang, good fortune
It builds, the ground such as Guangxi obtain certain success.
Key enzyme of the cytochrome oxidase (CCO) as respiratory chain on mitochondrial inner membrane and oxidative phosphorylation system, load
In the presence of finally by electron transmission to the vital task of molecular oxygen, be electron transport chain the last one carrier n ".The generation of ATP must
Must be premised on electron transmission and basic, it is mutually auxiliary phase between the two that respiratory chain, which only has generation ATP that could push the transmitting of electronics,
At, mutual lotus root connection, COO as this lotus root connection process critical electronic carrier, content number and active height it is direct
The efficiency for affecting respiratory chain electron transfer process directly affects the energy production of body.Mitochondria is cellular respiration and generation
The position of ATP, the inner membrance of mitochondria contain the whole enzymes, including nadh dehydrogenase, CCO etc. of respiratory chain and oxidative phosphorylation system
A variety of enzymes, electronics finally pass to molecular oxygen by CCO by these enzymes in respiratory chain, and the various composition of electron transport chain is logical
Oxidization-reduction reaction successively transmits downwards the electronics that NADH is provided, and final electronics is received by oxygen molecule, is reduced to water, simultaneously
It gives off energy for body needs.In these enzymes, cytochrome oxidase (CCO) is its marker enzyme, it is in cytochromes
The end of system is the last one carrier of electron transport chain, including cytochromes a and a3, electronics of this enzyme substrate of respiration
It is directly passed to molecular oxygen by cytochrome system, electron transmission and being formed in normal tissue cell for ATP are phase couplings
Connection, the generation of ATP must be premised on electron transmission, and respiratory chain only generation ATP could push the transmitting of electronics.It is aerobic
Into the cell, the decomposition of sugar, fat and amino acid is all by respective decomposition approach, but finally all passes through one section of common oxidation
Process.In this process, sugar, fat and amino-acid oxidase take off hydrogen and the reduced coenzyme NADH and FADH2 that are formed, edge
Electron transport chain is eventually transferred into oxygen in the form of electronics and H+ and forms water, wherein the free energy discharged is i.e. for promoting
ADP and Pi forms ATP.Therefore the active height of CCO and content number directly affect the efficiency of respiratory chain transmitting electronics, determine
Body generate energy position and production capacity number, also just determine the maximum functional intensity that can reach when body movement and most
Long duration, it directly determines endurance exercise project achievement, this is most important during exercise, and entire movement instruction
Practice pursued target.
Guan Tao etc. (2007) research obtains hypoxic training, and rat heart muscle can be made to generate certain stress reaction, make myocardium group
CCO level in knitting obtains the raising of compensatory.The researchs such as the expression about Agasicles hygrophila CCO gene do not appear in the newspapers both at home and abroad
Road, therefore use real-time fluorescence quantitative PCR research difference CO2The expression characterization of the lower Agasicles hygrophila CCO gene of concentration processing,
The result shows that with CO2The expression quantity of the raising of concentration, female male worm CCO gene increases, the up-regulation of this and transcript profile expression quantity
Variation tendency is consistent, illustrates CO2Concentration increases the expression regulation that can influence CCO gene.
Summary of the invention
The object of the present invention is to provide the primers and method of detection Agasicles hygrophila CCO gene expression characteristics, are comprehensive
The different disposal CO for understanding the expression characterization of Agasicles hygrophila CCO gene and designing2Concentration.
To achieve the above object, the technical solution adopted in the present invention is as follows:
By feeding difference CO2The female male worm of Agasicles hygrophila for the alternanthera philoxeroides that concentration is cultivated respectively adopts 9 and manages to Eppendorf
In, it is divided into 3 biology and repeats, totally 12 groups of sample.
The primer of a pair of of detection Agasicles hygrophila CCO gene expression characteristics of design, including meet quantitative fluorescent PCR reaction
The upstream and downstream primer of feature:
CCO-F:5`-GGTGGTGATTAGTCGGTTGTT-3`
CCO-R:5`-AATCTGTTCGCTTCATTCATTGC-3`
And the upstream and downstream primer as reference gene:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
The fluorescence quantitative PCR method of detection Agasicles hygrophila CCO gene expression characteristics of the present invention, carries out as follows:
(1) the first chain of cDNA synthesis: extracting the total serum IgE of sample and purifying, uses reverse transcription reagent box to obtain and is with the RNA of extraction
The cDNA that template reverse transcription obtains；
(2) Standard PCR detection is carried out to following primer
The fluorescent quantitation upstream and downstream primer of the gene:
CCO-F:5`-GGTGGTGATTAGTCGGTTGTT-3`
CCO-R:5`-AATCTGTTCGCTTCATTCATTGC-3`
And the upstream and downstream primer as reference gene:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
Standard PCR amplification system is 25 μ L:10 × buffer, 2.5 μ L, dNTP 1.0 μ L, 0.5 μ L of upstream primer, downstream primer
0.5 μ L, 100ng/ μ L cDNA template 1.0 μ L, Ex-TaqE 0.15 μ L, 19.35 μ L of water；
Routine PCR reaction program is as follows: 94 DEG C of 5 min of initial denaturation, then 94 DEG C of 30 s of denaturation, and 56 DEG C of 30 s of annealing, 72 DEG C are prolonged
1 min is stretched, under this condition 35 circulations, last 72 DEG C re-extend 10min；
(3) real-time fluorescence quantitative PCR reacts:
Primer described in step (2) is added as template in the cDNA obtained using step (1), carries out quantitative fluorescent PCR reaction, each
3 repetitions are arranged in sample, are averaged after amplification.
Real-time fluorescence quantitative PCR amplification system are as follows: SYBR® Premix Ex TaqTM 12.5 μ L, 0.5 μ L of upstream primer,
0.5 μ L, 100ng/ μ L cDNA of downstream primer, 1.0 μ L, supplies water to 25 μ L；Real-time fluorescence quantitative PCR response procedures are as follows:
95 DEG C of 30 s of initial denaturation, then 95 DEG C of 5 s of denaturation, 60 DEG C of annealing 30 s, 40 circulations.
The more detailed test method of the present invention is as follows:
The real-time fluorescence quantitative PCR detection method of Agasicles hygrophila CCO gene can be realized by following steps:
(1) design of primers: according to the sequence for the Agasicles hygrophila CCO gene that transcript profile sequencing result obtains, DNAMAN is used
Software design is suitble to the specific primer of fluorescence quantitative PCR detection, and primer sequence is as follows:
CCO-F:5`-GGTGGTGATTAGTCGGTTGTT-3`
CCO-R:5`-AATCTGTTCGCTTCATTCATTGC-3`
Meanwhile the sequence of the Agasicles hygrophila tubulin gene obtained according to transcript profile sequencing result, it is fixed designed for fluorescence
The primer of PCR internal reference object is measured, primer sequence is as follows:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
(2) Agasicles hygrophila processing test: in control CO2Concentration (420 μ LL- 1) and high CO2Concentration (750 μ LL- 1)
Alternanthera philoxeroides is cultivated in growth cabinet, for raising the female male worm of Agasicles hygrophila, different CO2The female male worm of concentration is respectively adopted
9, into Eppendorf pipe, are divided into 3 biology and repeat, totally 12 groups of samples.It is put into rapidly in liquid nitrogen and fixes after the processing of worm sample,
It is saved backup in -80 DEG C.
(3) the first chain of cDNA synthesizes: referring to Quan Shi King CompanyTransZol TMUp Plus RNA Kit kit explanation
Book extracts total serum IgE, according to the One-Step gDNA Removal and cDNA Synthesis of TransScript company
SuperMix kit specification synthesizes the first chain of cDNA.
(4) real-time fluorescence quantitative PCR reacts: real-time fluorescence quantitative PCR uses the SYBR of TCCOara company® Premix
Ex TaqTMKit carries out.Using the first chain of cDNA of above-mentioned synthesis as template, above-mentioned CCO-F, CCO-R and Tubulin-F,
Tubulin-R is specific primer, carries out quantitative fluorescent PCR program, and each sample sets 3 parallel repetitions, gained is taken after amplification
The average of parallel Ct value.
Real-time fluorescence quantitative PCR amplification system are as follows: SYBR® Premix Ex TaqTM 12.5 μ L, 0.5 μ L of upstream primer,
0.5 μ L, 100ng/ μ L cDNA of downstream primer, 1.0 μ L, supplies water to 25 μ L；
Real-time fluorescence quantitative PCR response procedures are as follows: 95 DEG C of 30 s of initial denaturation, then 95 DEG C of 5 s of denaturation, 60 DEG C of annealing 30
S, 40 circulations.
(5) calculation formula of the Agasicles hygrophila CCO gene relative to the expression quantity of tubulin gene are as follows: opposite mRNA
Expression=2-ΔΔCt× 100%, wherein Ct value=target gene Ct value-tubulin Ct value.
(6) Fig. 3 and Fig. 4 is respectively the female male worm feeding difference CO of Agasicles hygrophila2After the alternanthera philoxeroides that concentration is cultivated
The differential expression of CCO gene.The result shows that with CO2The expression quantity of the raising of concentration, female male worm CCO gene increases, and with
The expression quantity variation tendency of transcript profile is consistent, this provides weight for the research that we carry out Agasicles hygrophila CCO gene in a deep going way
The basic data wanted.
The present invention has the following advantages and effects with respect to the prior art:
(1) the different CO in the present invention2Concentration treatment measures are all suitable for insect overwhelming majority aibuhitensis cytochrome oxidase gene,
This has great importance to comprehensive further investigation gene expression characteristics.
(2) fluorescence quantitative PCR method of efficient quick of the present invention, including quantitative fluorescent PCR program etc., practical used time 50
Min substantially reduces the reaction time compared with the existing technology, the characteristic with efficient quick.
(3) fluorescence quantitative PCR method of the present invention provides Standard PCR electrophoresis result figure, intuitively can efficiently reflect
The specificity of primer out.
Detailed description of the invention
Attached drawing is the fluorescence quantitative PCR detection result of the female male worm CCO gene transcription level of Agasicles hygrophila.
Fig. 1: Agasicles hygrophila CCO gene by fluorescence quantitative primer Standard PCR product electrophoresis result: product length 126
Bp, Marker band are from top to bottom successively are as follows: 2000,1000,750,500,250,100bp.
Fig. 2: Agasicles hygrophila tubulin gene by fluorescence quantitative primer Standard PCR product electrophoresis result: product length is
199 bp, Marker band are from top to bottom successively are as follows: 2000,1000,750,500,250,100bp.
Fig. 3: Agasicles hygrophila female adult (F) feeding difference CO2The difference of CCO gene after the alternanthera philoxeroides that concentration is cultivated
Expression, cylindricality and left side reference axis are the expression quantity of fluorescent quantitation, and scatterplot and the right reference axis are the expression quantity of transcript profile.
Fig. 4: Agasicles hygrophila male worm (M) feeding difference CO2The difference of CCO gene after the alternanthera philoxeroides that concentration is cultivated
Expression, cylindricality and left side reference axis are the expression quantity of fluorescent quantitation, and scatterplot and the right reference axis are the expression quantity of transcript profile.
Specific embodiment
Illustrate that the present invention, the scheme of embodiment described here do not limit invention below in conjunction with specific embodiments and drawings,
His any changes, modifications, substitutions, combinations, simplifications done without departing from the spirit and principles of the present invention, should be equivalent
Substitute mode, be included within the scope of the present invention.
Embodiment 1
The processing of test material
In control CO2Concentration (420 μ LL- 1) and high CO2Concentration (750 μ LL- 1) growth cabinet in cultivate hollow lotus seeds
Grass, for raising the female male worm of Agasicles hygrophila, different CO2The female male worm of concentration respectively adopts 9 into Eppendorf pipe, is divided into 3
A biology repeats, totally 12 groups of samples.Worm sample is put into rapidly in liquid nitrogen after collecting and fixes, and saves backup in -80 DEG C.
Embodiment 2
The design of primer
(1) design of primers: according to the sequence for the Agasicles hygrophila CCO gene that transcript profile is sequenced, DNAMAN software is used
The specific primer for being suitble to fluorescence quantitative PCR detection is designed, primer sequence is as follows:
CCO-F:5`-GGTGGTGATTAGTCGGTTGTT-3`
CCO-R:5`-AATCTGTTCGCTTCATTCATTGC-3`
Agasicles hygrophila CCO gene by fluorescence quantitative PCR product length is 126 bp, agarose gel electrophoresis results display amplification
Product length afterwards is consistent, and is single band, illustrates that designed primer has very strong specificity, suitable for glimmering in real time
Fluorescent Quantitative PCR detection, agarose gel electrophoresis results are as shown in Figure 1.
(2) simultaneously, the sequence for the Agasicles hygrophila tubulin gene being sequenced according to transcript profile, designed for glimmering
The primer of Fluorescent Quantitative PCR internal reference object, primer sequence are as follows:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
Agasicles hygrophila tubulin gene by fluorescence quantitative PCR product length is 199 bp, and agarose gel electrophoresis results are shown
Product length after amplification is consistent, and is single band, illustrates that designed primer has very strong specificity, suitable for real
When fluorescence quantitative PCR detection, agarose gel electrophoresis results are as shown in Figure 2.
Embodiment 3
The extraction of total serum IgE
Referring to Quan Shi King CompanyTransZol TMUp Plus RNA Kit kit specification extracts total serum IgE, will with liquid nitrogen
Polypide is pulverized, and 1ml is addedTransZol TMUp goes to 1.5 ml and turns in centrifuge tube, after being stored at room temperature 5 min, is added
200 μ L chloroforms/1 mlTransZol TMUp acutely vibrates 30 s, is incubated at room temperature 3 min.4 DEG C of 10000 g centrifugation 15
After min, upper strata aqueous phase (generally < 80%) is moved in new centrifuge tube, the dehydrated alcohol of 1/3 volume is added, is gently mixed by inversion.
RNA centrifugal column is placed in 2 ml collecting pipes, upper step mixture is all transferred in centrifugal column, 12000 g, 30 ~ 60 s centrifugation,
Mobile phase is abandoned, collecting pipe is reused.500 μ LCB9 are added, 12000 g of room temperature is centrifuged 30 s, abandons mobile phase, adds 500
μ LCB9,12000 g of room temperature are centrifuged 30 s, abandon mobile phase.Diluted 500 μ LWB9 is added, 12000 g are centrifuged 30 s, abandoned stream
Dynamic phase adds diluted 500 μ LWB9, and 12000 g are centrifuged 30 s, abandons mobile phase.12000 g are centrifuged 2 min, thoroughly go
Except remaining ethyl alcohol, centrifugal column is thoroughly dried being stored at room temperature several minutes.Centrifugal column is put into RNase-free Tube, is added
50 μ LRNase-free Water are stored at room temperature 1 min in the center of centrifugal column, and 12000 g are centrifuged 1 min, eluted rna.Institute
RNA be placed in -80 DEG C it is spare.
Embodiment 4
The first chain of cDNA synthesis: according to the One-Step gDNA Removal and cDNA of TransScript company
Synthesis SuperMix kit specification step, using the RNA in embodiment 3 as template, reverse transcription generates cDNA first
Chain, the specific steps are as follows:
(1) reverse transcription system
(2) 42 DEG C of 30 min of incubation.
(3) 85 DEG C of 5 min of heating, product can be placed on -20 DEG C, -80 DEG C of preservations.
Embodiment 5
Carry out quantitative fluorescent PCR reaction.Real-time fluorescence quantitative PCR uses the SYBR of TCCOara company® Premix Ex TaqTM
Kit carries out.Using the cDNA in embodiment 4 as template, it is anti-that real-time fluorescence quantitative PCR amplification is carried out with the primer in embodiment 2
It answers, each sample sets 3 parallel repetitions, and the average of the parallel Ct value of gained is taken after amplification.
(1) real-time fluorescence quantitative PCR amplification system is as follows:
SYBR® Premix Ex TaqTM 12.5 μ L, 0.5 μ L of upstream primer, 0.5 μ L, 100ng/ μ L cDNA 1.0 of downstream primer
μ L supplies water to 25 μ L；
(2) real-time fluorescence quantitative PCR response procedures are as follows: 95 DEG C of 30 s of initial denaturation, then 95 DEG C of 5 s of denaturation, 60 DEG C of annealing
30 s, 40 circulations.
(3) after the completion of real-time fluorescence quantitative PCR, the ratio 2 of relative expression quantity under the conditions of different disposal is calculated according to Ct value-ΔΔCt.Calculation formula of the Agasicles hygrophila CCO gene relative to the expression quantity of tubulin gene are as follows: opposite mRNA expression=
2-ΔΔCt× 100%, wherein Ct value=target gene Ct value-tubulin Ct value.
Table 1: Agasicles hygrophila female adult (F) feeding difference CO2The C (T) of CCO gene after the alternanthera philoxeroides that concentration is cultivated
Value, average value, standard deviation, fluorescent quantitation expression quantity and transcript profile expression quantity
Table 2: Agasicles hygrophila male worm (M) feeding difference CO2Concentration cultivate alternanthera philoxeroides after CCO gene C (T) value,
Average value, standard deviation, fluorescent quantitation expression quantity and transcript profile expression quantity
(4) Fig. 3 and Fig. 4 is respectively the female male worm feeding difference CO of Agasicles hygrophila2CCO base after the alternanthera philoxeroides that concentration is cultivated
The differential expression of cause.The result shows that with CO2The expression quantity of the raising of concentration, female male worm CCO gene increases, with transcript profile
Expression quantity up-regulation variation tendency it is consistent.This provides weight for the research that we carry out Agasicles hygrophila CCO gene in a deep going way
The basic data wanted.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>primer and method of Agasicles hygrophila CCO gene expression characteristics are detected
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
ggtggtgatt agtcggttgt t 21
<210> 2
<211> 23
<212> DNA
<213>artificial sequence
<400> 2
aatctgttcg cttcattcat tgc 23
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
agatgtccgc caccttca 18
<210> 4
<211> 24
<212> DNA
<213>artificial sequence
<400> 4
gcctcttggt attgttggta ttca 24
Claims (2)
1. a kind of fluorescence quantification PCR primer for detecting Agasicles hygrophila CCO gene expression characteristics, it is characterised in that: including symbol
Close the upstream and downstream primer of quantitative fluorescent PCR reaction characteristics:
CCO-F:5`-GGTGGTGATTAGTCGGTTGTT-3`,
CCO-R:5`-AATCTGTTCGCTTCATTCATTGC-3`.
2. a kind of quantitative fluorescent PCR side using primer detection Agasicles hygrophila CCO gene expression characteristics described in claim 1
Method, it is characterised in that: as follows:
(1) the first chain of cDNA synthesis: extracting the total serum IgE of sample and purifying, uses reverse transcription reagent box to obtain and is with the RNA of extraction
The cDNA that template reverse transcription obtains；
(2) Standard PCR detection is carried out with following primer
The fluorescent quantitation upstream and downstream primer of the gene:
CCO-F:5`-GGTGGTGATTAGTCGGTTGTT-3`
CCO-R:5`-AATCTGTTCGCTTCATTCATTGC-3`
And the upstream and downstream primer as reference gene:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
Standard PCR amplification system is 25 μ L:10 × buffer, 2.5 μ L, dNTP 1.0 μ L, 0.5 μ L of upstream primer, downstream primer
0.5 μ L, 100ng/ μ L cDNA template 1.0 μ L, Ex-TaqE 0.15 μ L, 19.35 μ L of water；
Routine PCR reaction program are as follows: 94 DEG C of 5 min of initial denaturation, then 94 DEG C of 30 s of denaturation, 56 DEG C of 30 s of annealing, 72 DEG C are prolonged
1 min is stretched, under this condition 35 circulations, last 72 DEG C re-extend 10min；
(3) real-time fluorescence quantitative PCR reacts:
Primer described in step (2) is added as template in the cDNA obtained using step (1), carries out quantitative fluorescent PCR reaction, each
3 repetitions are arranged in sample, are averaged after amplification；
Real-time fluorescence quantitative PCR amplification system are as follows: SYBR® Premix Ex TaqTM 12.5 μ L, 0.5 μ L of upstream primer, downstream
0.5 μ L, 100ng/ μ L cDNA of primer, 1.0 μ L, supplies water to 25 μ L；
Real-time fluorescence quantitative PCR response procedures are as follows: 95 DEG C of 30 s of initial denaturation, then 95 DEG C of 5 s of denaturation, 60 DEG C of 30 s of annealing,
40 circulations.
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| CN111280165A (en) * | 2020-03-04 | 2020-06-16 | 山西农业大学 | Low-temperature preservation method for agasicles hygrophila eggs |
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