CN109055507B - Primer and method for detecting EF gene expression characteristics of agasicles hygrophila - Google Patents
Primer and method for detecting EF gene expression characteristics of agasicles hygrophila Download PDFInfo
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Abstract
The invention provides a fluorescent quantitative PCR primer for detecting EF gene expression characteristics of agasicles hygrophila, which comprisesTwo pairs of the gene sequences are shown in SEQ ID NO.1-4, the invention establishes that the lotus grass thoracocentesis japonicas male and female worms take different CO2The fluorescence quantitative PCR method for detecting the EF gene expression level after the alligator alternanthera is cultivated at the concentration and carries out sequencing of a transcriptome, compares the EF gene fluorescence quantitative expression level trend with the transcriptome expression level trend, and lays a foundation for researching a defense mechanism and EF gene expression characteristics generated when the alligator alternanthera is stressed by the environment.
Description
Technical Field
The invention relates to a fluorescent quantitative PCR primer for detecting EF gene expression characteristics of agasicles hygrophila, belonging to the technical field of biology.
Background
Lotus herb, Thymus ananastus, belongs to Coleoptera, Leptospermum, is one of the main natural enemies of alternanthera philoxeroides in biocontrol. George Vogt found that the lotus seed thoracocentesis diver's beetle has a good control effect on alternanthera philoxeroides for the first time in 1962. Then, the lotus seed flea beetle is widely applied to the control of the alternanthera philoxeroides in the United states, Australia and the like, and achieves better control effect. Lily grass fleabane was introduced from the United states in 1986 in China, and the application of the Limonium sinense oliver to biological control of the alternanthera philoxeroides has succeeded to a certain extent in Sichuan, Zhejiang, Fujian, Guangxi and the like.
Elongation Factor (EF) is a protein factor involved in peptide chain elongation during protein synthesis, plays an important role in the metabolism of all multicellular organisms, and comprises EF-1 and EF-2. The elongation factor EF-1 is composed of 4 subunits of alpha, beta, gamma, delta and the like, is a multimeric ribosomal protein which is ubiquitous in cells and is expressed in a large amount, and plays an important role in the gene expression and translation processes. EF-2 is a protein factor essential for peptide chain extension in eukaryotic protein synthesis, and it catalyzes GTP hydrolysis to move peptidyl-tRNA from aminoacyl site to peptidyl site of ribosome, thereby moving ribosome along mRNA to complete transcription. Therefore, the normal expression of EF is related to the growth of body cells and the synthesis of proteins, the EF regulates the synthesis of total proteins by continuously changing the expression level of EF in the cells, and the activity and the expression level of EF are also changed under the action of different mechanisms.
Research shows that in the crustacean culture production, stress factors in the water environment can cause extensive cell damage including DNA damage, lipid peroxidation, protein oxidation and the like, and finally the expression level of EF is changed. Different insecticides acting on monochamus alternatus EF gene are measured by Luolulin et al (2014) to cause the monochamus alternatus EF gene to have up-regulated expression in different degrees, which indicates that the monochamus alternatus generates self-protection reaction. The ability of cells of biological organisms to adapt to environmental stresses is a key to their survival, and organisms have evolved to have a certain ability to respond to environmental and physiological stresses, a common feature of many responses being the selective regulation of gene transcription and translation, including allowing rapid expression of reversible genes. Current studies indicate that environmental stress can induce the expression of the EF gene, thereby regulating protein synthesis to account for changes in environmental conditions.
The research on the expression of EF gene of agasicles hygrophila is not reported at home and abroad, so that the real-time fluorescent quantitative PCR is adopted to research different CO2The expression characteristic of EF gene of agasicles hygrophila under concentration treatment shows that the result shows that the EF gene is expressed along with CO2The concentration is increased, the expression level of the EF genes of the male and female worms is increased, which is consistent with the change trend of the up-regulation of the expression level of the transcriptome, and the results show that the CO is increased2Elevated concentrations can affect regulation of the expression of the EF gene. The present study was aimed at understanding the presence of the EF gene in different CO2The reaction after concentration treatment provides a theoretical basis for the subsequent research of the action mechanism between the specific function of the Escholtzia zeylanica EF gene and the environmental stress.
Disclosure of Invention
The invention aims to provide a real-time fluorescent quantitative PCR primer and a method for efficiently and quickly detecting EF gene expression quantity of a lotus grass flea beetle.
Another object of the present invention is toDifferent treatment CO designed for comprehensively understanding expression characteristics of EF gene of agasicles hygrophila2And (4) concentration.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
will eat different CO2The nelumbo nucifera gaertn gynandra of the nelumbo lutea cultured in concentration is respectively collected into 9 Eppendorf tubes and divided into 3 biological replicates, and 12 groups of samples are total.
Designing a pair of primers for detecting EF gene expression characteristics of the agasicles hygrophila, wherein the primers comprise an upstream primer and a downstream primer which accord with the characteristics of fluorescent quantitative PCR reaction:
EF-F：5`-TACCACGCTCACGCTCAG-3`
EF-R：5`-CGGCAAGTCCACCACAAC-3`
and upstream and downstream primers as reference genes:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
the invention relates to a fluorescence quantitative PCR method for detecting EF gene expression characteristics of agasicles hygrophila, which is characterized by comprising the following steps:
(1) first strand cDNA Synthesis: extracting and purifying total RNA of a sample, and obtaining cDNA obtained by reverse transcription by using the extracted RNA as a template by adopting a reverse transcription kit;
(2) the following primers were subjected to conventional PCR detection
Fluorescent quantitative upstream and downstream primers of the gene:
EF-F：5`-TACCACGCTCACGCTCAG-3`
EF-R：5`-CGGCAAGTCCACCACAAC-3`
and upstream and downstream primers as reference genes:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
the conventional PCR amplification system is as follows:
the conventional PCR reaction procedure is as follows: pre-denaturation at 94 deg.C for 5 min, denaturation at 94 deg.C for 30 s, annealing at 56 deg.C for 30 s, and extension at 72 deg.C for 1 min, under the condition for 35 cycles, and final extension at 72 deg.C for 10 min;
(3) real-time fluorescent quantitative PCR reaction:
and (3) taking the cDNA obtained in the step (1) as a template, adding the primer in the step (2) to perform fluorescent quantitative PCR reaction, setting 3 times of repetition for each sample, and taking an average value after amplification.
The real-time fluorescent quantitative PCR amplification system comprises the following components:
the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 30 s, followed by denaturation at 95 ℃ for 5 s and annealing at 60 ℃ for 30 s for 40 cycles.
The more detailed test method of the present invention is as follows:
the real-time fluorescence quantitative PCR detection method of the EF gene of the agasicles hygrophila can be realized by the following steps:
(1) designing a primer: according to the sequence of EF gene of the agasicles hygrophila obtained by the transcriptome sequencing result, DNAMAN software is used for designing a specific primer suitable for fluorescent quantitative PCR detection, and the primer sequence is as follows:
EF-F：5`-TACCACGCTCACGCTCAG-3`
EF-R：5`-CGGCAAGTCCACCACAAC-3`
meanwhile, according to the sequence of the lotus grass flea-beetle tubulin gene obtained by the sequencing result of the transcriptome, a primer for a fluorescent quantitative PCR internal control is designed, and the sequence of the primer is as follows:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
(2) lotus grass thoracocentesis scurf treatment test: in control CO2Concentration (420. mu.L. L)－1) And high CO2Concentration (750. mu.L.L)－1) Cultivating alternanthera philoxeroides in artificial climate boxIs used for feeding agasicles hygrophila, male and female insects with different CO2The female and male worms with the concentration are respectively taken into 9 Eppendorf tubes and divided into 3 biological replicates, and 12 groups of samples are obtained. After being treated, the insect sample is quickly put into liquid nitrogen for fixation and is stored at minus 80 ℃ for standby.
(3) First strand cDNA Synthesis: with reference to the whole body of goldTransZol TMTotal RNA was extracted using Up Plus RNA Kit instructions, and the first strand cDNA was synthesized according to One-Step gDNA Removal and cDNA Synthesis SuperMix Kit instructions from TransScript.
(4) Real-time fluorescent quantitative PCR reaction: SYBR available from Takara corporation was used for real-time fluorescent quantitative PCR® Premix Ex TaqTMAnd (4) carrying out kit. And (3) performing a fluorescent quantitative PCR program by using the synthesized first strand of the cDNA as a template and the EF-F, EF-R and Tubulin-F, Tubulin-R as specific primers, setting 3 parallel repeats for each sample, and taking the average of parallel Ct values obtained after amplification.
The real-time fluorescent quantitative PCR amplification system comprises the following components:
the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 30 s, followed by denaturation at 95 ℃ for 5 s and annealing at 60 ℃ for 30 s for 40 cycles.
(5) The expression of the EF gene of the agasicles hygrophila relative to the tubulin gene is calculated according to the following formula: relative mRNA expression = 2-ΔΔCt X 100%, where Ct value = target gene Ct value-tubulin Ct value.
(6) FIG. 3 and FIG. 4 show that the male and female insects of agasicles hygrophila feed different CO2Differential expression of EF gene after the alternanthera philoxeroides is cultivated in concentration. The results show that with CO2The concentration is increased, the expression quantity of the EF genes of the male and female worms is increased and is consistent with the change trend of the expression quantity of the transcriptome, so that important basic data are provided for deeply developing the research on the EF genes of the agasicles hygrophila.
Compared with the prior art, the invention has the following advantages and effects:
(1) different CO in the present invention2The concentration treatment measures are suitable for most of the elongation factor genes of the insects, and have important significance for comprehensively and deeply researching the gene expression characteristics.
(2) The efficient and fast fluorescence quantitative PCR method comprises a fluorescence quantitative PCR program and the like, is actually used for 50 min, greatly shortens the reaction time compared with the prior art, and has the characteristics of high efficiency and fast speed.
(3) The fluorescence quantitative PCR method provided by the invention provides a conventional PCR electrophoresis result picture, and can intuitively and quickly reflect the specificity of the primer.
Drawings
The attached figure is a fluorescence quantitative PCR detection result of EF gene transcription level of the female male sex weevil of the agasicles hygrophila.
FIG. 1: electrophoresis results of common PCR products of EF gene fluorescent quantitative primers of the agasicles hygrophila are as follows: the length of the product is 158 bp,
the Marker strip is as follows from top to bottom: 2000. 1000, 750, 500, 250 and 100 bp.
FIG. 2: electrophoresis results of conventional PCR products of fluorescence quantitative primers of agasicles hygrophila tubulin gene: the length of the product is 199 bp, and the Marker bands are sequentially from top to bottom: 2000. 1000, 750, 500, 250 and 100 bp.
FIG. 3: feeding by female Dactylicapnos lunulatus (F) with different CO2Differential expression of EF gene after the alligator alternanthera is cultivated at concentration, the column-shaped coordinate axis and the left coordinate axis are fluorescence quantitative expression quantity, and the scatter plot axis and the right coordinate axis are transcriptome expression quantity.
FIG. 4: feeding Nelumbo Nucifera Gaertn with different CO2Differential expression of EF gene after the alligator alternanthera is cultivated at concentration, the column-shaped coordinate axis and the left coordinate axis are fluorescence quantitative expression quantity, and the scatter plot axis and the right coordinate axis are transcriptome expression quantity.
Detailed description of the preferred embodiments
The present invention is described below with reference to the following embodiments and the accompanying drawings, wherein the embodiments are not limited to the invention, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be regarded as equivalent replacements within the scope of the present invention.
Example 1
Treatment of test materials
In control CO2Concentration (420. mu.L. L)－1) And high CO2Concentration (750. mu.L.L)－1) The artificial climate box is used for cultivating alternanthera philoxeroides which is used for cultivating alternanthera philoxeroides male and female flea beetles with different CO2The female and male worms with the concentration are respectively taken into 9 Eppendorf tubes and divided into 3 biological replicates, and 12 groups of samples are obtained. The insect sample is collected and then quickly placed into liquid nitrogen for fixation, and is stored at the temperature of minus 80 ℃ for standby.
Example 2
Design of primers
(1) Designing a primer: according to the sequence of EF gene of the agasicles hygrophila obtained by transcriptome sequencing, DNAMAN software is used for designing a specific primer suitable for fluorescent quantitative PCR detection, and the primer sequence is as follows:
EF-F：5`-TACCACGCTCACGCTCAG-3`
EF-R：5`-CGGCAAGTCCACCACAAC-3`
the length of the fluorescence quantitative PCR product of the EF gene of the agasicles hygrophila is 158 bp, and the agarose gel electrophoresis result shows that the amplified products have the same length and are single strips, which indicates that the designed primer has strong specificity and is suitable for real-time fluorescence quantitative PCR detection, and the agarose gel electrophoresis result is shown in figure 1.
(2) Meanwhile, according to the sequence of the lotus grass fleabane tubulin gene obtained by sequencing the transcriptome, a primer for a fluorescent quantitative PCR internal control is designed, and the sequence of the primer is as follows:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
the length of the fluorescence quantitative PCR product of the agasicles hygrophila tubulin gene is 199 bp, and the agarose gel electrophoresis result shows that the amplified products have the same length and are single bands, which indicates that the designed primer has strong specificity and is suitable for real-time fluorescence quantitative PCR detection, and the agarose gel electrophoresis result is shown in figure 2.
Example 3
Extraction of Total RNA
With reference to the whole body of goldTransZol TMUp Plus RNA Kit instructions for total RNA extraction, liquid nitrogen grinding the worm body into powder, adding 1mlTransZol TMUp, transferred to a 1.5 ml centrifuge tube, left to stand at room temperature for 5 min, and added with 200. mu.L chloroform/1 mlTransZol TMUp, vigorously shaken for 30 s and incubated at room temperature for 3 min. Centrifuging at 4 deg.C for 15 min at 10000 g, and transferring the upper aqueous phase (generally<80%) into a new centrifuge tube, 1/3 volumes of absolute ethanol were added and mixed by gentle inversion. Sheathing the RNA centrifugal column in a 2 ml collecting pipe, transferring the mixture in the previous step into the centrifugal column, centrifuging at 12000 g for 30-60 s, discarding the mobile phase, and reusing the collecting pipe. Add 500 μ LCB9, centrifuge at 12000 g at room temperature for 30 s, discard mobile phase, add 500 μ LCB9, centrifuge at 12000 g at room temperature for 30 s, discard mobile phase. Diluted 500. mu. LWB9 was added, 12000 g was centrifuged for 30 s, the mobile phase was discarded, and diluted 500. mu. LWB9, 12000 g was added, centrifuged for 30 s, and the mobile phase was discarded. Centrifuging at 12000 g for 2 min to completely remove residual ethanol, standing at room temperature for several minutes, and completely air drying the centrifugal column. The column was placed in RNase-free Tube, 50. mu.L Nase-free Water was added to the center of the column, and the column was left standing at room temperature for 1 min, centrifuged at 12000 g for 1 min, and RNA was eluted. The RNA obtained was stored at-80 ℃ until use.
Example 4
First strand cDNA Synthesis: the first strand of cDNA was generated by reverse transcription using RNA as template according to the procedure of One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransScript Co., Ltd.) as follows:
(1) reverse transcription system
(2) Incubate at 42 ℃ for 30 min.
(3) Heating at 85 deg.C for 5 min, and storing at-20 deg.C and-80 deg.C.
Example 5
And carrying out fluorescent quantitative PCR reaction. SYBR available from Takara corporation was used for real-time fluorescent quantitative PCR® Premix Ex TaqTMAnd (4) carrying out kit. Real-time fluorescent quantitative PCR amplification reaction was performed using the cDNA of example 4 as a template and the primers of example 2, 3 replicates were taken for each sample, and the average of parallel Ct values obtained after amplification was taken.
(1) The real-time fluorescent quantitative PCR amplification system comprises the following components:
(2) the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 30 s, followed by denaturation at 95 ℃ for 5 s and annealing at 60 ℃ for 30 s for 40 cycles.
(3) After the real-time fluorescent quantitative PCR is finished, calculating the ratio 2 of relative expression amounts under different treatment conditions according to the Ct value-ΔΔCt. The expression of the EF gene of the agasicles hygrophila relative to the tubulin gene is calculated according to the following formula: relative mRNA expression = 2-ΔΔCtX 100%, where Ct value = target gene Ct value-tubulin Ct value.
Table 1: feeding by female Dactylicapnos lunulatus (F) with different CO2C (T) value, average value, standard deviation, fluorescent quantitative expression level and transcriptome expression level of EF gene after cultivation of alligator alternanthera at concentration
Table 2: feeding Nelumbo Nucifera Gaertn with different CO2C (T) value, average value, standard deviation, fluorescent quantitative expression level and transcriptome expression level of EF gene after cultivation of alligator alternanthera at concentration
(4) FIG. 3 and FIG. 4 are each a schematic view of a display deviceFeeding different CO for pistacia stratiotes and pistacia nutans2Differential expression of EF gene after the alternanthera philoxeroides is cultivated in concentration. The results show that with CO2The increase of the concentration and the expression quantity of the EF genes of the male and female worms are increased, and the change trend is consistent with the change trend of the up-regulation of the expression quantity of the transcriptome. The method provides important basic data for deeply developing the research on EF genes of the agasicles hygrophila.
SEQUENCE LISTING
<110> institute of plant protection of academy of agricultural sciences of Fujian province
<120> primer and method for detecting EF gene expression characteristics of agasicles hygrophila
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence
<400> 1
taccacgctc acgctcag 18
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence
<400> 2
cggcaagtcc accacaac 18
<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 fluorescence quantitative PCR primer for detecting EF gene expression characteristics of agasicles hygrophila is characterized in that: comprises upstream and downstream primers which accord with the characteristics of fluorescent quantitative PCR reaction:
EF-F：5`-TACCACGCTCACGCTCAG-3`，
EF-R：5`-CGGCAAGTCCACCACAAC-3`；
and upstream and downstream primers as reference genes:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3` ，
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`。
2. a fluorescence quantitative PCR method for detecting EF gene expression characteristics of agasicles hygrophila by using the primer of claim 1 is characterized in that: comprises the following steps:
(1) first strand cDNA Synthesis: extracting and purifying total RNA of a sample, and obtaining cDNA obtained by reverse transcription by using the extracted RNA as a template by adopting a reverse transcription kit;
(2) the following primers were subjected to conventional PCR detection
Fluorescent quantitative upstream and downstream primers of the gene:
EF-F：5`-TACCACGCTCACGCTCAG-3`
EF-R：5`-CGGCAAGTCCACCACAAC-3`
and upstream and downstream primers as reference genes:
Tubulin-F: 5`-AGATGTCCGCCACCTTCA-3`
Tubulin-R: 5`-GCCTCTTGGTATTGTTGGTATTCA-3`
the conventional PCR amplification system is 25 muL: 10 × buffer 2.5 muL, dNTP 1.0 muL, an upstream primer 0.5 muL, a downstream primer 0.5 muL, a 100 ng/muL cDNA template 1.0 muL, Ex-TaqE 0.15 muL and water 19.35 muL;
the conventional PCR reaction procedure was: pre-denaturation at 94 deg.C for 5 min, denaturation at 94 deg.C for 30 s, annealing at 56 deg.C for 30 s, and extension at 72 deg.C for 1 min, under the condition for 35 cycles, and final extension at 72 deg.C for 10 min;
(3) real-time fluorescent quantitative PCR reaction:
adding the primers obtained in the step (2) into the cDNA obtained in the step (1) as a template to perform fluorescent quantitative PCR reaction, setting 3 times of repetition for each sample, and averaging after amplification;
the real-time fluorescent quantitative PCR amplification system is as follows: SYBR® Premix Ex TaqTM12.5 muL, 0.5 muL of an upstream primer, 0.5 muL of a downstream primer, 1.0 muL of 100 ng/muL cDNA, and supplementing water to 25 muL;
the real-time fluorescent quantitative PCR reaction program is as follows: pre-denaturation at 95 ℃ for 30 s, followed by denaturation at 95 ℃ for 5 s and annealing at 60 ℃ for 30 s for 40 cycles.
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