CN109161602A - Detect the primer and method of Agasicles hygrophila JHE gene transcription level - Google Patents

Detect the primer and method of Agasicles hygrophila JHE gene transcription level Download PDF

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CN109161602A
CN109161602A CN201811232458.4A CN201811232458A CN109161602A CN 109161602 A CN109161602 A CN 109161602A CN 201811232458 A CN201811232458 A CN 201811232458A CN 109161602 A CN109161602 A CN 109161602A
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primer
jhe
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郑丽祯
傅建炜
何肖云
李建宇
史梦竹
林凌鸿
王秋月
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Institute of Plant Protection of FAAS
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Abstract

The present invention provides the primers and method of detection Agasicles hygrophila JHE gene transcription level, 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 JHE 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 JHE gene by fluorescence quantitative with the expression quantity trend of transcript profile, the defense mechanism and JHE gene transcription level generated when for research Agasicles hygrophila by environment-stress is laid a good foundation.

Description

Detect the primer and method of Agasicles hygrophila JHE gene transcription level
Technical field
The present invention relates to the primers and method of detection Agasicles hygrophila JHE gene transcription level, 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.
JH esterase (juvenile hormone esterase, JHE), is most important one in most insects The specific esterase of kind degradation juvenile hormone (Ju-venile hormone, JH) plays JH titre in control insect bodies important Effect.In insectean metamorphosis growth course, JHE occurs in due course, extremely important to larva normal development pupa, in hemolymph When JHE activity is very high, JH titre can be made to decline.In development and reproduction, the degradation of JHE has the regular hour specific. JHE enzyme activity, content and distribution are variant because of the type of insect, histoorgan and developmental stage difference.
The decision of insectean metamorphosis is to be acted under conditions of shortage by moulting hormone, makes to synthesize larva type epidermis to synthesis Pupa type epidermis transformation etc., promotes larval metamorphosis to pupate, therefore, JHE is normally carried out with very important work insectean metamorphosis With, Abdel etc. studies have shown that when the activity for JHE occur when whole age is hindered, JH in blood and tissue is remained in not It can decompose completely, lead to the failure of larvae pupation, larva is prevented to form huge larva from timely nymphosis.JHE is by fat Body cell and epithelial cell generate, it can identify juvenile hormone in hemolymph-hemolymph JH binding protein (juvenile hormone-hemolymph juvenile hormone binding protein, JH-hJHBP) complex, JH is molten into hemolymph from separating in complex, then JH is removed by JHE.JHE has specificity to the decomposition of JH, The JH of free state can not only be decomposed, and the JH of JH-hJHBP bonding state also can be decomposed specifically.Touhara etc. Studies have shown that because JHE can identify JH-hJHBP complex, thus can decompose and hJHBP at bonding state JH, reach from JH effect is removed in blood and tissue rapidly.
The researchs such as the expression about Agasicles hygrophila JHE gene have not been reported both at home and abroad, therefore fixed using real-time fluorescence It measures PCR and studies different CO2The transcriptional level of the lower Agasicles hygrophila JHE gene of concentration processing, the results showed that, with CO2Concentration It increases, the expression quantity of female male worm JHE gene increases, this is consistent with the variation tendency of transcript profile expression quantity up-regulation, illustrates CO2It is dense Degree increases the expression regulation that can influence JHE gene.Corpus allatum hormone is a kind of insect growth regulator, IGR, can pass through interference The normal development of insect and make its death, have bioactivity high, selectivity is obvious, to people, animal safety and to natural enemy, beneficial insect It is harmless, it is free from environmental pollution the advantages that.The postembryonal development and breeding of vertebrate and invertebrate are all in endocrine regulation Under carry out, the research to Agasicles hygrophila JH esterase, it is intended to find efficiently, quick, green insect control it is new Direction.
Summary of the invention
The object of the present invention is to provide the primers and method of detection Agasicles hygrophila JHE gene transcription level, are comprehensive The different disposal CO for understanding the transcriptional level of Agasicles hygrophila JHE 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 JHE gene transcription level of design, including meet quantitative fluorescent PCR reaction The upstream and downstream primer of feature:
JHE-F:5`-GCCTATGGTAACTGGTCACAA-3`
JHE-R:5`-GCGTTGGATTTCTGTATTTAGCA-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 JHE gene transcription level 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:
JHE-F:5`-GCCTATGGTAACTGGTCACAA-3`
JHE-R:5`-GCGTTGGATTTCTGTATTTAGCA-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 is as follows:
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 JHE gene can be realized by following steps:
(1) design of primers: according to the sequence for the Agasicles hygrophila JHE 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:
JHE-F:5`-GCCTATGGTAACTGGTCACAA-3`
JHE-R:5`-GCGTTGGATTTCTGTATTTAGCA-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 Takara company® Premix Ex TaqTMKit carries out.Using the first chain of cDNA of above-mentioned synthesis as template, above-mentioned JHE-F, JHE-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 TaqTM12.5 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 annealing 30 S, 40 circulations.
(5) calculation formula of the Agasicles hygrophila JHE 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 JHE gene.The result shows that with CO2The expression quantity of the raising of concentration, female male worm JHE 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 JHE 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 JH esterase gene, this Have great importance to comprehensive further investigation gene transcription level.
(2) fluorescence quantitative PCR method of efficient quick of the present invention, including quantitative fluorescent PCR program etc., practical used time 55 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 JHE gene transcription level of Agasicles hygrophila.
Fig. 1: Agasicles hygrophila JHE gene by fluorescence quantitative primer Standard PCR product electrophoresis result: product length 109 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 JHE 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 JHE 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 JHE 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:
JHE-F:5`-GCCTATGGTAACTGGTCACAA-3`；
JHE-R:5`-GCGTTGGATTTCTGTATTTAGCA-3`.
Agasicles hygrophila JHE gene by fluorescence quantitative PCR product length is 109 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 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 Takara 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:
Real-time fluorescence quantitative PCR amplification system are as follows: SYBR® Premix Ex TaqTM12.5 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；
(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 JHE 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 JHE 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 JHE 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 hygrophila2JHE 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 JHE 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 JHE 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 JHE gene transcription level are detected
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<170> PatentIn version 3.3
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gcctatggta actggtcaca a 21
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gcgttggatt tctgtattta gca 23
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agatgtccgc caccttca 18
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gcctcttggt attgttggta ttca 24

Claims (2)

1. a kind of fluorescence quantification PCR primer for detecting Agasicles hygrophila JHE gene transcription level, it is characterised in that: including symbol Close the upstream and downstream primer of quantitative fluorescent PCR reaction characteristics:
JHE-F:5`-GCCTATGGTAACTGGTCACAA-3`,
JHE-R:5`-GCGTTGGATTTCTGTATTTAGCA-3`.
2. a kind of quantitative fluorescent PCR side using primer detection Agasicles hygrophila JHE gene transcription level 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:
JHE-F:5`-GCCTATGGTAACTGGTCACAA-3`,
JHE-R:5`-GCGTTGGATTTCTGTATTTAGCA-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 TaqTM12.5 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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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002060940A1 (en) * 2001-02-01 2002-08-08 Commonwealth Scientific And Industrial Research Organisation Juvenile hormone esterase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002060940A1 (en) * 2001-02-01 2002-08-08 Commonwealth Scientific And Industrial Research Organisation Juvenile hormone esterase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FU JW等: "登录号：KF792065.1", 《GENBANK》 *
赵梅婷: "莲草直胸跳甲不同地理种群响应高温的生态学表现及转录组分析", 《中国博士学位论文全文数据库 农业科技辑》 *
郭艳琼等: "莲草直胸跳甲羧酸酯酶基因AhCesB1的克隆、序列分析及表达特征", 《植物保护学报》 *
马兰: "保幼激素酯酶的生化及结构特性研究进展", 《陕西农业科学》 *

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