CN113862275B - Partial sequence, cloning method and application of reference gene RPL14 stably expressed under different density treatments of wheat long tube aphid - Google Patents
Partial sequence, cloning method and application of reference gene RPL14 stably expressed under different density treatments of wheat long tube aphid Download PDFInfo
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Abstract
The invention discloses a partial sequence of an internal reference gene RPL14 stably expressed under different density treatments of a long tube aphid, a cloning method and application thereof, belongs to the field of molecular biology, and particularly relates to internal reference genes SA-HEL, SA-RPL14, SA-RPL11 and SA-28S of the long tube aphid, wherein the nucleotide sequences of the internal reference genes are respectively shown as SEQ ID No.1, SEQ ID No.6, SEQ ID No.11 and SEQ ID No. 16. The nucleotide sequence of the obtained wheat long tube aphid reference gene can be used as the reference gene for researching wheat long tube aphid functional gene or gene expression in different development periods, the reference gene for expressing the winged aphid and wingless aphid in vivo, or used as different density treatment, or used as pheromone E-beta-farnesene solution treatment, or used as a useful method for researching under pesticide or antibiotic treatment.
Description
The application is a divisional application of application number 201910608195.0 (the name of the invention: partial sequence, cloning method and application of the internal reference gene of the wheat long tube aphid).
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a SA-HEL gene, SA-RPL14 gene or SA-RPL11 gene or SA-28S gene partial sequence of a wheat long tube aphid internal reference gene, a cloning method and application of the wheat long tube aphid internal reference gene serving as the internal reference gene in RT-qPCR.
Background
The wheat long tube aphid (Sitobion avenae Fabricius) belongs to homoptera, and is one of main pests of wheat crops in China, has a large host species, and is also a grass weed such as rice, sorghum, corn, sugarcane and the like, a grass weed such as early-maturing grass, wheat-looking, crabgrass, club head grass, green bristlegrass, chinese green bristlegrass and the like besides the wheat crops such as harmful wheat, barley, oat and the like. The method has the characteristics of wide distribution, large quantity and strong fertility, and influences the normal growth of wheat plants, so that the wheat yield is seriously reduced, and huge losses are caused for agricultural production. The wheat yield can be reduced by 5.1% -16.5% in the general year, and the wheat yield is reduced by more than 40% in the most-occurring year. The aphids are generally wingless, the winged aphids and the winged aphids can be regulated and generated by external environment conditions and biological factors, the winged individuals can fly away for finding host plants in a long distance, and the winged individuals can directly pierce and suck wheat juice and are also an important medium for transmitting wheat virus diseases.
The wing-free parthenogenetic aphid body of the wheat long tube aphid is 3.1mm long, 1.4mm wide, oval long, grass green to orange red, the head is slightly grey, and the abdomen side has a grey green color spot. Feeler, coracoid end, tarsal, abdominal tube black, and caudal patch light. The sixth to eighth sections of the abdomen and the abdomen were textured with transverse lines, without edge tumors. Short handle of middle chest and abdomen bifurcation. The frontal tumor is significantly camber. The antenna is slender, the overall length is smaller than the body length, and the third section of base part is provided with 1-4 secondary sense loops. The beak is thick, exceeds the basal ganglia of the midfoot and the end node is in a cone shape, and is 1.8 times of the basal width. The abdominal tube is long and cylindrical, is 1/4 of the length of the body, and has more than ten rows of reticulation at the end. The tail piece is long and conical, and is 1/2 of the length of the abdominal tube, and has 6-8 pieces of bent hair. The winged parthenogenetic aphid body is 3.0mm long, elliptic, green and black in antenna, and the third section has 8-12 sense circles arranged in 1 row. The beak is short of the midfoot basal ganglia. The abdominal tube is long and cylindrical, black, and the end part is provided with 15-16 rows of transverse reticulation. The middle pulse of the anterior wing is trifurcated and the bifurcation is large.
In the research of insect gene transcription level, a real-time fluorescent quantitative PCR technology (qRT-PCR) is an effective means for detecting the mRNA expression level of specific genes and performing transcription analysis. The qRT-PCR technology has the advantages of real-time detection, strong specificity, rapidness, sensitivity and accuracy in quantification, and realizes the leap of the traditional PCR from qualitative to accurate quantification. When qRT-PCR assays are performed, the reference gene must be stably expressed under the given experimental conditions. In order to promote gene expression research and obtain more accurate expression quantity data, screening of relatively stable reference genes is a necessary basic work. The ideal reference gene is not influenced by any external environmental factors, and can be stably expressed under different experimental conditions. However, numerous studies have shown that there are no absolutely stably expressed genes (Xun, Z et al,. Selection and Evaluation of Reference Genes for Expression Analysis Using qRT-PCR in the Beet Armyworm Spo-doptera exigua (Lepidotera: noctaidae) PLoS ONE,2014,9 (1): e84730.Doi: 10.1371/journ.fine.0084730.; shakeel, M., et al, gene expression studies of reference genes for quantitative real-time PCR: an oversview in instruments.biotechnology Letters,2017.40 (2): p.227-236.). Currently, common insect reference genes are: 18S ribosomal RNA (18S ribosomal RNA,18S rRNA), elongation factor 1 (Elongationfactor-1 alpha, EF1-a), beta-actin gene (Beta-actin), 28S ribosomal RNA (28S ribosomal RNA,28S rRNA), beta-tubulin (Beta tubulin), the succinate dehydrogenase complex A subunit (Succinate dehydrogenase complex subunitA, SDHA) ribosomal protein S27 Ribosomal protein S (rsp 27), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and TATA box binding protein (TATA-Box binding protein) etc. (Xun, Z et al,. Selection and Evaluation of Reference Genes for Expression Analysis Using qRT-PCR in the Beet Armyworm Spo-doptera exigua (Lepidotera: noctaidae) PLoS ONE,2014,9 (1): e84730.Doi: 10.1371/joune. 0084730; shake. M. In insect samples, the same gene has differences in function and expression levels in different species of insects. Similarly, expression of candidate reference genes may vary from insect species to insect species or from experimental conditions to experimental conditions. Along with the wide application of qRT-PCR technology, researches show that the expression quantity of the housekeeping genes can be greatly changed under certain specific conditions due to different insect sample types and experimental conditions, and if the qRT-PCR technology is used for blind reference, the accuracy of the final quantitative result can be influenced. SA-HEL (HELicase), an enzyme that is widely found in many organisms and can break hydrogen bonds, generally acts to catalyze the unwinding of double-stranded DNA or RNA during the replication of the DNA or RNA. Helicases belong to the chaperone and are essential to ensure correct folding of DNA or RNA, preserving and modifying their specific secondary and tertiary structures. The SA-HEL gene is a very conserved housekeeping gene in organisms. Quantitative PCR detection shows that the gene can be stably expressed in the growth and development period of the myzus persicae and in the winged aphid and the winged aphid-free body, and provides an ideal internal reference gene for researching the function gene of the myzus persicae and the expression research of the winged aphid and the winged aphid-free body in different development periods. RPL14 (ribosomal protein, ribosomal protein L14) is a major component of ribosomes and plays an important role in intracellular protein biosynthesis. Ribosomal proteins are widely distributed in various tissues and together with ribonucleic acids they form ribosomes and play an important role in protein biosynthesis. There is increasing evidence that: many ribosomal proteins have a function independent of protein biosynthesis in addition to constituting ribosomes and being involved in protein biosynthesis. The RPL14 gene is a very conserved housekeeping gene in organisms. The quantitative PCR detection shows that the gene can be stably expressed in the body of the wheat long tube aphid, and a very ideal internal reference gene is provided for researching the wheat long tube aphid functional gene and the gene expression under different density treatments. SA-RPL11 (helicase) is an enzyme that is widely present in many organisms and can break hydrogen bonds and generally acts to catalyze the unwinding of double-stranded DNA or RNA during DNA or RNA replication. Helicases belong to the chaperone and are essential to ensure correct folding of DNA or RNA, preserving and modifying their specific secondary and tertiary structures. The SA-RPL11 gene is a very conserved housekeeping gene in organisms. The quantitative PCR detection shows that the gene can be stably expressed in the body of the wheat long tube aphid, and a very ideal internal reference gene is provided for researching the wheat long tube aphid functional gene and the gene expression under the treatment of the pheromone E-beta-farnesene solution. Ribosomes (ribosomal) are the only sites where cells synthesize proteins. Ribosomal RNA (rRNA) is a major component constituting a ribosome, and is a structural or functional component of the ribosome. Higher eukaryotes are often designated by their sedimentation coefficients, including 28S,18S, and 5S.28S ribosomal RNA (28S ribosomal RNA) is widely found in a variety of eukaryotic organisms. The SA-28S gene is a very conserved housekeeping gene in organisms. The quantitative PCR detection shows that the gene can be stably expressed in the body of the myzus persicae, and a very ideal internal reference gene is provided for researching the functional gene of the myzus persicae and researching the gene expression under the treatment of pesticides or antibiotics.
Disclosure of Invention
The invention provides a partial sequence, cloning method and application of a stable expression reference gene SA-HEL of wheat long tube aphid in different ages and different wing types, a stable expression reference gene SA-RPL14 under different density treatment, a stable expression reference gene SA-SA-RPL11 under pheromone E-beta-farnesene solution treatment, and a stable expression reference gene SA-28S under pesticide treatment or antibiotic treatment.
The invention also provides specific primers for cloning the SA-HEL, SA-RPL14, SA-SA-RPL11 and SA-28S of the wheat long tube aphids, and establishes an RT-PCR method based on SYBR Green1 dye technology, thereby providing a useful method for researching the SA-HEL, SA-RPL14, SA-RPL11 and SA-28S of the wheat long tube aphids as internal genes in the process of expressing the wheat long tube aphids functional genes or as genes in different development periods by utilizing qPCR, researching wheat aphid wing differentiation, treating the wheat long tube aphids as different densities, treating the wheat long tube aphids as an pheromone E-beta-farnesene solution, or treating the wheat long tube aphids as pesticides or antibiotics.
The technical scheme provided by the invention is as follows: the gene fragment of the wheat long tube aphid is SA-HEL gene fragment, SA-RPL14 gene fragment, SA-RPL11 gene fragment or SA-28S gene fragment, and the nucleotide sequences of the gene fragment are respectively shown as SEQ ID No.1, SEQ ID No.6, SEQ ID No.11 and SEQ ID No. 16. On the basis of fully analyzing and researching the gene sequences, the neck ring structure and non-specific amplification at a specific temperature are avoided, the amplification efficiency meeting the conditions is ensured, and the gene fragments are obtained through design and screening.
The invention also provides a cloning method of the partial sequence of the wheat long tube aphid gene, which comprises the following steps: extracting total RNA of a wheat long tube aphid genome, carrying out RT reaction by taking a mixed primer of a kit as a reaction primer, taking a first chain cDNA of a product as a template, respectively carrying out RT-PCR amplification by taking a primer pair SA-HEL-F/SA-HEL-R, SA-RPL14-F/SA-RPL14-R, SA-RPL11-F/SA-RPL11-R, SA-28S-F/SA-28S-R, and finally carrying out sequencing verification, wherein the primer pair has the following sequence:
SA-HEL-F：5’-ACTGGTTTGAACGAATATG-3’；
SA-HEL-R：5’-CATCCAAATAGTGTGTAAGA-3’；
the size of the obtained target fragment is 805bp.
SA-RPL14-F：5’-AATGTGAGGTTATGATTGTC-3’；
SA-RPL14-R：5’-TTAAACAGTGCTTGGTAG-3’；
The size of the obtained target fragment is 500bp.
SA-RPL11-F：5’-TCGTGTTGTTTCGCTTCGTC-3’；
SA-RPL11-R：5’-TGATACCCAAATCGATGTGTTCTTG-3’；
The size of the obtained target fragment is 418bp.
SA-28S-F：5’-CGAGTGAGCCAGAAACACAT-3’；
SA-28S-R：5’-ATCCCCAGTCTTTGGCCTTTT-3’；
The size of the obtained target fragment is 617bp.
The cloning method comprises the following steps: 10 XPCR Buffer 2.5. Mu.L, 2.5mmol/L dNTP 2.0. Mu.L, 25.0mmol/L MgCl 2 2.0. Mu.L, 5.0U/. Mu.L TaqDNA polymerase 0.5. Mu.L, 10.0. Mu.M upstream and downstream primers 1.0. Mu.L each, cDNA 2.5. Mu.L, ddH 2 O was made up to 25. Mu.L. The reaction conditions were as follows: pre-denaturing at 94 deg.c for 5 min; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, each cycle reduced by 0.5℃and extension at 72℃for 1 mm for a total of 30 cycles; denaturation at 94℃for 30sec, annealing at 45℃for 30sec and elongation at 72℃for 1 mm for 10 cycles; and finally, filling in at 72 ℃ for 7min, wherein the termination temperature is 8 ℃.
The invention also provides a method for qPCR amplification of the partial sequence of the wheat long tube aphid gene, which comprises the following steps: extracting total RNA of a wheat long tube aphid genome, carrying out RT reaction by taking a mixed primer of a kit as a reaction primer, carrying out fluorescent quantitative PCR amplification by taking a first-strand cDNA of a product as a template, and taking a fluorescent dye as SYBR Green 1, wherein the quantitative primer of the wheat long tube aphid in the quantitative PCR amplification has the following sequence:
QSA-HEL-F：5’-TGCTACCGGATGTGGGAAAA-3’；
QSA-HEL-R：5’-TCCAGCCACGTTCTCTGTTT-3’；
the size of the obtained target fragment is 152bp.
QSA-RPL14-F：5’-AAGCACCACAGTTCCAAGCG-3’；
QSA-RPL14-R：5’-ACTCGGCCAGTTTCCACAAA-3’；
The size of the obtained target fragment is 116bp.
QSA-RPL11-F：5’-CGTGTTGTTTCGCTTCGTCT-3’；
QSA-RPL11-R：5’-CTTTAAGTCGGCGAGGACCA-3’；
The size of the obtained target fragment is 91bp.
QSA-28S-F：5’-TCTATCAACCGGCAACCACA-3’；
QSA-28S-R：5’-TGGCGAATCTCGTTGCATTT-3’；
The size of the obtained target fragment is 109bp.
The reaction conditions for the fluorescent quantitative PCR amplification are as follows: pre-denaturing at 95deg.C for 15 min; 40 cycles of denaturation at 95℃for 10sec, annealing at 60℃for 32sec were run first, followed by 15sec at 95℃for the dissolution profile stage, 1min at 60℃for 30sec at 95℃for 15sec at 60 ℃.
The SA-HEL gene fragment of the invention can be used as an internal reference gene, and can be applied to the functional gene of the wheat long tube aphid or the gene expression in different development periods, and the quantitative PCR detection of the in-vivo gene expression of the winged aphid and the wingless aphid.
The SA-RPL14 gene fragment of the wheat long tube aphid can be used as an internal reference gene and applied to the functional gene of the wheat long tube aphid or the quantitative PCR detection of gene expression under different density treatments.
The SA-RPL11 gene fragment of the wheat long tube aphid can be used as an internal reference gene and applied to quantitative PCR detection of gene expression under the treatment of wheat long tube aphid functional genes or pheromone E-beta-farnesene solution.
The SA-28S gene fragment of the wheat long tube aphid can be used as an internal reference gene and applied to quantitative PCR detection of gene expression under the treatment of wheat long tube aphid functional genes or pesticides or antibiotics.
According to the invention, according to the transcriptome sequencing result of the wheat long tube aphid, the nucleotide sequence of the SA-HEL gene, the SA-RPL14 gene, the SA-RPL11 gene or the SA-28S gene is obtained by analysis and annotation, and the partial sequence of the SA-HEL, the SA-RPL14 gene, the SA-RPL11 gene or the SA-28S gene housekeeping gene of the wheat long tube aphid is cloned. The quantitative primer with the corresponding specificity is designed, and the neck ring structure and the nonspecific amplification are avoided and the amplification efficiency of the primer is ensured to meet the conditions on the basis of fully analyzing and researching the gene sequences in the primer design process. The RT-PCR method based on SYBR Green1 dye technology is established, so that a useful method is provided for taking SA-HEL, SA-RPL14, SA-RPL11 or SA-28S of the wheat long tube aphid as an internal reference gene, taking qPCR as a function gene for the wheat long tube aphid to express genes in different development periods, taking wheat aphid wing type differentiation research as different density treatment, taking the wheat aphid wing type differentiation research as pheromone E-beta-farnesene solution treatment or taking the wheat aphid wing type differentiation research as pesticide or antibiotic treatment.
Compared with the prior art, the invention has the following advantages:
1. the invention clones the SA-HEL gene fragment, SA-RPL14 gene, SA-RPL11 gene or SA-28S gene of the wheat long tube aphid for the first time;
2. the invention firstly proposes that a general reference gene is established in quantitative PCR detection of the myzus persicae;
3. the invention provides quantitative PCR detection of SA-HEL gene of the wheat long tube aphid as an internal reference gene in different development periods of the wheat long tube aphid for the first time; firstly, putting forward quantitative PCR detection of SA-RPL14 genes of the wheat long tube aphids as internal reference genes in different density treatments of the wheat long tube aphids; firstly, putting forward quantitative PCR detection of SA-RPL11 gene of the wheat long tube aphid as an internal reference gene under the treatment of wheat long tube aphid pheromone E-beta-farnesene solution; firstly, putting forward quantitative PCR detection of SA-28S gene of the wheat long tube aphid as an internal reference gene under the treatment of wheat long tube aphid insecticide;
4. the invention provides that SA-HEL genes of the myzus persicae can be used as internal reference genes for relatively quantifying the relative expression level of genes in winged aphids and wingless aphids for the first time;
5. the invention provides a quantitative PCR detection of SA-28S gene of the wheat long tube aphid as an internal reference gene under the antibiotic treatment of the wheat long tube aphid for the first time;
6. the detection primer provided by the invention has specificity, optimizes the PCR amplification procedure, greatly improves the detection efficiency, shortens the detection time and improves the reliability of the detection result.
Drawings
FIG. 1 is a PCR electrophoresis diagram of SA-HEL reference genes of the myzus persicae; m is DNA molecular weight standard;
FIG. 2 is a melting curve of the SA-HEL reference gene of the Aphis glutinosa;
FIG. 3 is a standard curve of the SA-HEL reference gene of the Aphis glutinosa;
FIG. 4 shows the relative expression levels of SA-HEL reference genes in different periods of the wheat head aphid.
FIG. 5 is a PCR electrophoresis diagram of the reference gene SA-RPL14 of the wheat long tube aphid; m is DNA molecular weight standard;
FIG. 6 shows the melting curve of the reference gene of the SA-RPL14 of the Aphis glutinosa;
FIG. 7 is a standard curve of the reference gene SA-RPL14 of the Aphis glutinosa;
FIG. 8 shows the relative expression level of SA-RPL14 reference gene in the non-density treatment of Aphis glutinosa.
FIG. 9 is a PCR electrophoresis diagram of the SA-RPL11 reference gene of the wheat long tube aphid; m is DNA molecular weight standard;
FIG. 10 shows the melting curve of the reference gene SA-RPL11 of Aphis glutinosa;
FIG. 11 is a standard curve of the SA-RPL11 reference gene of Aphis glutinosa.
FIG. 12 is a PCR electrophoretogram of the SA-28S reference gene of the Aphis glutinosa; m is DNA molecular weight standard;
FIG. 13 shows the melting curve of the reference gene of A-28S of Aphis glutinosa;
FIG. 14 is a standard curve of the reference gene SA-28S of Aphis glutinosa;
FIG. 15 shows the relative expression levels of SA-28S reference genes under treatment with a wheat head aphid insecticide.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which is not intended to be limiting, but is made merely by way of example. The experimental procedure, in which specific conditions are not noted in the examples below, is generally followed by conventional conditions.
EXAMPLE 1 cloning of the SA-HEL Gene, SA-RPL14 Gene, SA-RPL11 Gene or SA-28S Gene from A.ophiopogon
1. Extracting total RNA of the myzus persicae:
total RNA of the wheat long tube aphid is extracted by TRizol (Amion, USA), and the specific method is as follows: taking 30mg of a wingless adult sample of the wheat long tube aphid, fully grinding the sample in liquid nitrogen, then rapidly adding 1ml of Trizol lysate, fully oscillating for 20s, and standing for 5-10 minutes; 200. Mu.L of chloroform was added thereto, the mixture was shaken for 20 seconds, and the mixture was allowed to stand still for 5 minutes, and centrifuged at 12,000rpm at 4℃for 15 minutes; adding the supernatant into a centrifuge tube containing 2.5 times of absolute ethyl alcohol, reversing and mixing, standing for 10min, and storing at-80deg.C for 2-4 hr; taking out the sample, and centrifuging at 7500rpm at 4 ℃ for 10min; the supernatant was discarded, 1ml of 75% ethanol was added, the pellet was washed twice and centrifuged at 12,000rpm for 10min at 4 ℃; 30. Mu.L of RNase-free water was added to dissolve the precipitate; the integrity of RNA is detected by 1% agarose gel electrophoresis, the concentration and quality of the total RNA is detected by an ultra-micro ultraviolet spectrophotometer Beckman DU 700, and the RNA sample with the quality parameter of 1.9 < A260/280 < 2.1 and 2.0 < A260/230 < 2.4 is taken as a qualified sample and stored in a refrigerator at-80 ℃ for standby.
2. Reverse transcription
Reverse transcription was performed using TAKARA PrimeScript st Strand cDNA synthesis kit, and the specific procedures were as follows: adding 1 mu g of total RNA of the wheat long tube aphid into a sterile PCR tube, adding 1 mu LOligo dT Primer and 1 mu LdNTP Mixture, and supplementing 10 mu L with sterilized deionized water without RNase; mixing, heating at 65deg.C for 5min, and rapidly cooling on ice; to 10. Mu.L of the reaction mixture were added 4. Mu.L of 5X PrimeScript Buffer, 0.5. Mu. L RNase Inhibitor, 1.0. Mu. L PrimeScript RTase, 4.5. Mu.L of RNase free H 2 O, carrying out reverse transcription reaction on a PCR instrument, wherein the reaction program is that the reaction is inactivated for 5min at 95 ℃ after the temperature bath is carried out for 60min at 42 ℃, and then the reaction is placed on ice after the reaction is finished. The synthesized first strand cDNA is stored at-20deg.C for a short period or at-80deg.C for a long period.
Design of PCR amplification primer sequences
Upstream primer of SA-HEL (SEQ ID NO. 2): 5'-ACTGGTTTGAACGAATATG-3';
downstream primer of SA-HEL (SEQ ID NO. 3): 5'-CATCCAAATAGTGTGTAAGA-3'.
The upstream primer of SA-RPL14 (SEQ ID NO. 7): 5'-AATGTGAGGTTATGATTGTC-3';
the SA-RPL14 downstream primer (SEQ ID NO. 8): 5'-TTAAACAGTGCTTGGTAG-3';
the SA-RPL11 upstream primer (SEQ ID NO. 12): 5'-TCGTGTTGTTTCGCTTCGTC-3';
The SA-RPL11 downstream primer (SEQ ID NO. 13): 5'-TGATACCCAAATCGATGTGTTCTTG-3';
the upstream primer of SA-28S (SEQ ID NO. 17): 5'-CGAGTGAGCCAGAAACACAT-3';
the SA-28S downstream primer (SEQ ID NO. 8): 5'-ATCCCCAGTCTTTGGCCTTTT-3'.
The nucleic acid partial sequences of candidate internal reference genes of the wheat long tube aphids HEL, SA-RPL14, SA-RPL11 and SA-28S are obtained from the transcription group of the wheat long tube aphids. The primers described above were used to analyze the secondary structure of the nucleic acid partial sequence of the gene using UNAFold on-line software (http:// mfold. Rna. Albany. Edu/: melting temperature,60 ℃; DNA sequence, linear; na+ concentration,50mM; mg2+ concentration,3mM; other options are initial settings. After obtaining the sites containing the stem-loop structure of the gene template sequence, primers were designed using NCBI-Primer-BLAST on-line software (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST/index. Cgilink_LOC=blastHome), the software set up as follows: primer melting temperature,57-63 ℃; primer GC content,40-60%; PCR product size,500-1500base bands; exposed regions, stem-loop structural sites; other options are initial settings.
4. Polymerase Chain Reaction (PCR)
PCR amplification was performed using the first strand cDNA obtained by RT as a template. The reaction system is as follows: 10 XPCR Buffer 2.5. Mu.L, 2.5mmol/L dNTP 2.0. Mu.L, 25.0mmol/L MgCl 2 2.0. Mu.L, 5.0U/. Mu.L TaqDNA polymerase 0.5. Mu.L, 10.0. Mu.M upstream and downstream primers 1.0. Mu.L each, cDNA 2.5. Mu.L, ddH 2 O was made up to 25. Mu.L. The reaction conditions were as follows: pre-denaturing at 94 deg.c for 5 min; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, each cycle reduced by 0.5℃and extension at 72℃for 1 mm for a total of 30 cycles; denaturation at 94℃for 30sec, annealing at 45℃for 30sec and elongation at 72℃for 1 mm for 10 cycles; and finally, filling in at 72 ℃ for 7min, wherein the termination temperature is 8 ℃. The PCR products were subjected to 1% agarose gel electrophoresis to detect the target fragment (as shown in FIG. 1, FIG. 5, FIG. 9, FIG. 12).
5. Obtaining and sequencing of the Gene fragment of interest
After agarose gel electrophoresis, the PCR products are subjected to gel recovery, the recovered and purified DNA fragments are respectively linked to a vector pMD 17-T Vevtor, transformed into E.coli DH5 alpha competent cells, screened by Amp antibiotics to obtain positive clone strains for confirming inserted target fragments, and subjected to nucleotide sequence determination to obtain nucleotide sequences of SA-HEL, SA-RPL14, SA-RPL11 and SA-28S of the wheat long pipe aphids, wherein the specific sequences are respectively shown in SEQ ID No.1, SEQ ID No.6, SEQ ID No.11 and SEQ ID No.16.
Example 2 design and Synthesis of fluorescent quantitative PCR amplification primers
Based on NCBI of the Long tube aphid and the sequence of each gene fragment cloned in example 1, primers for fluorescent quantitative PCR of the gene were designed. The secondary structure of the nucleic acid partial sequence of each gene was analyzed using UNAFold online software (http:// mfold. Rna. Albany. Edu/: melting temperature,60 ℃; DNA sequence, linear; na+ concentration,50mM; mg of 2+ concentration,3mM; other options are initial settings. After obtaining the sites containing the stem-loop structure for each gene template sequence, primers were designed using NCBI-Primer-BLAST on-line software (http:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST/index. Cgilink_LOC=blastHome), the software set as follows: primer melting temperature,57-63 ℃; primer GC content,40-60%; PCR product size,150-300base bands; exposed regions, stem-loop structural sites; other options are initial settings. Designing a plurality of pairs of primers for each gene sequence, firstly amplifying by using common PCR, screening the primers which can obtain single bands and should be quantified, and performing fluorescent quantitative PCR experiments. The primers for fluorescent quantitative PCR of each gene were as follows:
QSA-HEL-F(SEQ ID NO.4)：5’-TGCTACCGGATGTGGGAAAA-3’；
QSA-HEL-R(SEQ ID NO.5)：5’-TCCAGCCACGTTCTCTGTTT-3’。
QSA-RPL14-F(SEQ ID NO.9)：5’-AAGCACCACAGTTCCAAGCG-3’；
QSA-RPL14-R(SEQ ID NO.10)：5’-ACTCGGCCAGTTTCCACAAA-3’；
QSA-RPL11-F(SEQ ID NO.14)：5’-CGTGTTGTTTCGCTTCGTCT-3’；
QSA-RPL11-R(SEQ ID NO.15)：5’-CTTTAAGTCGGCGAGGACCA-3’；
QSA-28S-F(SEQ ID NO.19)：5’-TCTATCAACCGGCAACCACA-3’；
QSA-28S-R(SEQ ID NO.20)：5’-TGGCGAATCTCGTTGCATTT-3’。
Example 3 fluorescent quantitative PCR detection of SA-HEL Gene in Aphis glutinosa winged and wingless Aphis
1. Extracting total RNA of winged adult aphids and wingless adult aphids from myzus persicae:
total RNA of myzus persicae winged and wingless aphids is extracted by TRizol (Amion, USA) respectively, and the specific method is the same as that of the method for extracting the total RNA of myzus persicae in the example 1.
2. Reverse transcription
Reverse transcription was performed using TAKARA PrimeScript st Strand cDNA synthesis kit, and the specific procedures were as follows: adding 1 mu g of total RNA of the wheat long tube aphid into a sterile PCR tube, adding 1 mu LOligo dT Primer and 1 mu LdNTP Mixture, and supplementing 10 mu L with sterilized deionized water without RNase; mixing, heating at 65deg.C for 5min, and rapidly cooling on ice; to 10. Mu.L of the reaction mixture were added 4. Mu.L of 5X PrimeScript Buffer, 0.5. Mu. L RNase Inhibitor, 1.0. Mu. L PrimeScript RTase, 4.5. Mu.L of RNase free H 2 O, carrying out reverse transcription reaction on a PCR instrument, wherein the reaction program is that the reaction is inactivated for 5min at 95 ℃ after the temperature bath is carried out for 60min at 42 ℃, and then the reaction is placed on ice after the reaction is finished. The synthesized first strand cDNA is stored at-20deg.C for a short period or at-80deg.C for a long period.
3. Fluorescent quantitative PCR amplification primer synthesis
Fluorescent quantitative PCR upstream primer (SEQ ID NO. 4): 5'-TGCTACCGGATGTGGGAAAA-3';
Fluorescent quantitative PCR downstream primer (SEQ ID NO. 5): 5'-TCCAGCCACGTTCTCTGTTT-3'.
4. Fluorescent quantitative PCR
And (3) performing fluorescent quantitative PCR detection by taking the first-strand cDNA obtained by RT as a template. The reaction system of qPCR based on SYBR Green I dye is: ssoFast EvaGreen Supermix 10. Mu.L of each of the upstream and downstream primers (10.0. Mu.M), 1.0. Mu.L of cDNA, 1.0. Mu.L of ddH 2 O was made up to 20. Mu.L. The reaction conditions for the fluorescent quantitative PCR amplification are as follows: pre-denaturing at 95deg.C for 15 min; first run 40 cyclesIs denatured at 95 ℃ for 10sec, annealed at 60 ℃ for 32sec, and then run at 95 ℃ for 15sec,60 ℃ for 1min,95 ℃ for 30sec,60 ℃ for 15sec in the dissolution profile stage.
5. Amplification efficiency of primer
Serial gradient dilutions of the adult cDNA of the myzus persicae are used as templates for real-time fluorescent quantitative PCR, and an amplification efficiency standard curve of the primer is prepared. The primer amplification efficiency was calculated as follows: amplification efficiency (E) = (10) [ -1/slope] -1)×100。
6. Experimental results
According to the quantitative PCR reaction program, genome RNA samples of winged aphids and wingless aphids of the long tube aphids are subjected to quantitative PCR amplification to obtain a melting curve (figure 2), a quantitative cycle (quantification cycle, cq) and an amplification curve (amplification plot). The specificity of the amplified product is judged by combining the melting curve, and the ideal melting curve should be a unimodal curve. According to the analysis of the primer melting curve of the experiment, SA-HEL gene detection of winged aphid and wingless aphid samples of the wheat long pipe aphid shows a single peak curve, and no non-specific amplification such as primer dimer and the like is generated, which shows that the fluorescent quantitative PCR amplification primers (SEQ ID No.4 and 5) related to the invention have single amplified band and strong specificity, and no non-specific amplification is generated. The invention can be seen from the fluorescent quantitative PCR amplification curves of the winged aphids and the winged aphids of the wheat long pipe aphids, the pair of primers are amplified under different dilution concentrations of templates, and the product amplification amounts are subjected to typical amplification processes such as a baseline, an exponential amplification period, a linear period, a platform period and the like, so that the gene can be used for amplifying SA-HEL genes in the winged aphids and the winged aphids of the wheat long pipe aphids. Performing a series of 5-fold gradient dilutions on the wheat long tube aphid adult cDNA as a template, performing real-time fluorescence quantitative PCR detection to obtain a series of Cq values of the template, and drawing a fitting curve (shown in figure 3) of the wheat long tube aphid SA-HEL gene by taking the Cq values as ordinate and the dilution factors as abscissa, wherein the fitting curve is as follows: y= -3.2493x+31.709, correlation coefficient (R 2 ) The slope of the fitted curve was brought to 0.994 as the primer amplification efficiency calculation: amplification efficiency (E) = (10) [ -1/slope] -1). Times.100, the amplification efficiency was calculated to be 103%, the primerThe amplification efficiency of the primer is about 100%, which shows that the primer has high amplification efficiency and is in accordance with the fluorescent quantitative PCR detection reference genes of the winged aphids and the wingless aphids of the wheat long pipe aphids.
Example 4 fluorescent quantitative PCR detection of SA-HEL Gene at different developmental stages of Aphis glutinosa
1. Extracting total RNA of the myzus persicae:
extracting total RNA of 1-year-old nymphs, 2-year-old nymphs, 3-year-old nymphs, 4-year-old nymphs, adults and the like of the wheat long tube aphids respectively, and collecting samples of the wheat long tube aphids in different development periods: collecting about 40 1-year-old nymphs, 30 2-year-old nymphs, 20 3-year-old nymphs and 20-year-old 4-year-old nymphs and 20-year-old wingless adult aphids in each breeding period, collecting 4 tubes, numbering, quick-freezing in liquid nitrogen, and storing in a refrigerator at-80 ℃ for a short period. The RNA method is as follows: taking 30mg of sample, fully grinding the sample in liquid nitrogen, then quickly adding 1ml of Trizol lysate, fully oscillating for 20s, and standing for 5-10 minutes; 200. Mu.L of chloroform was added thereto, the mixture was shaken for 20 seconds, and the mixture was allowed to stand still for 5 minutes, and centrifuged at 12,000rpm at 4℃for 15 minutes; adding the supernatant into a centrifuge tube containing 2.5 times of absolute ethyl alcohol, reversing and mixing, standing for 10min, and storing at-80deg.C for 2-4 hr; taking out the sample, and centrifuging at 7500rpm at 4 ℃ for 10min; the supernatant was discarded, 1ml of 75% ethanol was added, the pellet was washed twice and centrifuged at 12,000rpm for 10min at 4 ℃; 30. Mu.L of RNase-free water was added to dissolve the precipitate; the integrity of RNA is detected by 1% agarose gel electrophoresis, the concentration and quality of the total RNA is detected by an ultra-micro ultraviolet spectrophotometer Beckman DU 700, and the RNA sample with the quality parameter of 1.9 < A260/280 < 2.1 and 2.0 < A260/230 < 2.4 is taken as a qualified sample and stored in a refrigerator at-80 ℃ for standby.
2. Reverse transcription
Reverse transcription was performed using TAKARA PrimeScript st Strand cDNA synthesis kit, and the procedure was the same as in example 3.
3. Fluorescent quantitative PCR amplification primer synthesis
Fluorescent quantitative PCR upstream primer (SEQ ID NO. 4): 5'-TGCTACCGGATGTGGGAAAA-3';
fluorescent quantitative PCR downstream primer (SEQ ID NO. 5): 5'-TCCAGCCACGTTCTCTGTTT-3'.
4. Fluorescent quantitative PCR
And (3) performing fluorescent quantitative PCR detection by taking the first-strand cDNA obtained by RT as a template. The reaction system of qPCR based on SYBR Green I dye is: ssoFast EvaGreen Supermix 10. Mu.L of each of the upstream and downstream primers (10.0. Mu.M), 1.0. Mu.L of cDNA, 1.0. Mu.L of ddH 2 O was made up to 20. Mu.L. The reaction conditions for the fluorescent quantitative PCR amplification are as follows: pre-denaturing at 95deg.C for 15 min; 40 cycles of denaturation at 95℃for 10sec, annealing at 60℃for 32sec were run first, followed by 15sec at 95℃for the dissolution profile stage, 1min at 60℃for 30sec at 95℃for 15sec at 60 ℃.
5. Experimental results
According to the quantitative PCR reaction program, genome RNA samples of 1-year-old nymphs, 2-year-old nymphs, 3-year-old nymphs, 4-year-old nymphs, adults and the like in different development periods are subjected to quantitative PCR amplification to obtain a melting curve (mering cut), a quantitative circulation (quantification cycle, cq) and an amplification curve (amplification plot). The specificity of the amplified product is judged by combining the melting curve, and the ideal melting curve should be a unimodal curve. The analysis of primer melting curve of the experiment shows that SA-HEL gene detection of samples of different development periods of 1-age nymphs, 2-age nymphs, 3-age nymphs, 4-age nymphs, adults and the like shows a unimodal curve, no non-specific amplification of primer dimers and the like is generated, and the fluorescent quantitative PCR amplification primers (SEQ ID No.4 and 5) related to the invention have single amplification band, strong specificity and no non-specific amplification. The expression condition of the SA-HEL gene in samples of different development periods of the wheat long tube aphid is analyzed (figure 4), cq values of the SA-HEL gene in samples of the wheat long tube aphid 1-age nymph, 2-age nymph, 3-age nymph, 4-age nymph and adults are statistically analyzed, the SA-HEL gene can be stably expressed in different development periods of the wheat long tube aphid, the difference is not obvious (P is more than 0.05), and the SA-HEL gene can be used as an internal reference gene for functional gene fluorescent quantitative PCR detection in samples of different development periods of the wheat long tube aphid 1-age nymph, 2-age nymph, 3-age nymph, 4-age nymph, adults and the like.
Example 5 fluorescent quantitative PCR detection of SA-RPL14 Gene under different Density treatments of Aphis glutinosa
1. Total RNA extraction of the myzus persicae:
the method is the same as in example 1, and aphid samples are collected after treatment for 24 hours according to different densities of 1 head/dish, 30 head/dish and 60 head/dish of 3-year-old myzus persicae and total RNA of the myzus persicae under each treatment is respectively extracted by TRizol (Amion, USA).
2. Reverse transcription
Reverse transcription was performed using TAKARA PrimeScript st Strand cDNA synthesis kit, and the procedure was the same as in example 3.
3. Fluorescent quantitative PCR amplification primer synthesis
Fluorescent quantitative PCR upstream primer (SEQ ID NO. 9): 5'-AAGCACCACAGTTCCAAGCG-3';
fluorescent quantitative PCR downstream primer (SEQ ID NO. 10): 5'-ACTCGGCCAGTTTCCACAAA-3'.
4. Fluorescent quantitative PCR
And (3) performing fluorescent quantitative PCR detection by taking the first-strand cDNA obtained by RT as a template. The reaction system of qPCR based on SYBR Green I dye is: ssoFast EvaGreen Supermix 10. Mu.L of each of the upstream and downstream primers (10.0. Mu.M), 1.0. Mu.L of cDNA, 1.0. Mu.L of ddH 2 O was made up to 20. Mu.L. The reaction conditions for the fluorescent quantitative PCR amplification are as follows: pre-denaturing at 95deg.C for 15 min; 40 cycles of denaturation at 95℃for 10sec, annealing at 60℃for 32sec were run first, followed by 15sec at 95℃for the dissolution profile stage, 1min at 60℃for 30sec at 95℃for 15sec at 60 ℃.
5. Amplification efficiency of primer
Serial gradient dilutions of the adult cDNA of the myzus persicae are used as templates for real-time fluorescent quantitative PCR, and an amplification efficiency standard curve of the primer is prepared. The primer amplification efficiency was calculated as follows: amplification efficiency (E) = (10) [ -1/slope] -1)×100。
6. Experimental results
According to the quantitative PCR reaction program, genome RNA samples of winged aphids and wingless aphids of the long tube aphids are subjected to quantitative PCR amplification, and a melting curve (shown in figure 6), a quantitative cycle (quantification cycle, cq) and an amplification curve (amplification plot) are obtained. Judging the specificity of the amplified product by combining with a melting curveThe desired melting curve should be a unimodal curve. According to the analysis of the primer melting curve of the experiment, SA-RPL14 gene detection of the winged aphid and wingless aphid samples of the wheat long pipe aphid shows a single peak curve, and no non-specific amplification such as primer dimer and the like is generated, which shows that the fluorescent quantitative PCR amplification primers (SEQ ID No.5 and 10) related to the invention have single amplification strip and strong specificity, and no non-specific amplification is generated. The invention can be seen from the fluorescent quantitative PCR amplification curves of the winged aphids and the winged aphids of the wheat long pipe aphids, the pair of primers are amplified under different dilution concentrations of templates, and the product amplification amounts are subjected to typical amplification processes such as a baseline, an exponential amplification period, a linear period, a platform period and the like, so that the gene can be used for amplifying SA-RPL14 genes in the winged aphids and the winged aphids of the wheat long pipe aphids. Performing a series of 5-fold gradient dilutions on the wheat long tube aphid adult cDNA as a template, performing real-time fluorescence quantitative PCR detection to obtain a series of Cq values of the template, and drawing a fitting curve (shown in figure 7) of the wheat long tube aphid SA-RPL14 gene by taking the Cq values as ordinate and the dilution times as abscissa, wherein the fitting curve is as follows: y= -3.2249x+29.113, correlation coefficient (R 2 ) The slope of the fitted curve was brought to 0.9945 as the primer amplification efficiency calculation: amplification efficiency (E) = (10) [ -1/slope] -1) x 100, the amplification efficiency of the primer is calculated to be 104%, and the amplification efficiency of the primer is about 100%, which shows that the primer has high amplification efficiency and is in line with the fluorescent quantitative PCR detection of the wheat long tube aphid under different density treatments as the wheat long tube aphid. Analysis of the expression of SA-RPL14 gene in different density treated wheat head aphid samples (as shown in FIG. 8), statistical analysis of Cq value of SA-RPL14 gene in each of wheat head aphid 1/dish, 30 head/dish and 60 head/dish, found that SA-RPL14 gene can be stably expressed under different density treatment of wheat head aphid, the difference is not obvious (P>0.05 The SA-RPL14 gene can be used as an internal reference gene for fluorescence quantitative PCR detection of functional genes in different density treatment samples of the wheat long tube aphid.
Example 6 fluorescence quantitative PCR detection of SA-RPL11 Gene under treatment with Mettube aphid pheromone E-beta-farnesene solution 1. Total RNA extraction of Mettube aphid:
the method is the same as in example 1, and the adult myzus persicae is treated with pheromone E-beta-farnesene solution, and after 24 hours of treatment, aphid samples are collected, and total RNA of the myzus persicae under each treatment is extracted by TRizol (Amion, USA) respectively.
2. Reverse transcription
Reverse transcription was performed using TAKARA PrimeScript st Strand cDNA synthesis kit, and the procedure was the same as in example 3.
3. Fluorescent quantitative PCR amplification primer synthesis
Fluorescent quantitative PCR upstream primer (SEQ ID NO. 14): 5'-CGTGTTGTTTCGCTTCGTCT-3';
fluorescent quantitative PCR downstream primer (SEQ ID NO. 15): 5'-CTTTAAGTCGGCGAGGACCA-3'.
4. Fluorescent quantitative PCR
And (3) performing fluorescent quantitative PCR detection by taking the first-strand cDNA obtained by RT as a template. The reaction system of qPCR based on SYBR Green I dye is: ssoFast EvaGreen Supermix 10. Mu.L of each of the upstream and downstream primers (10.0. Mu.M), 1.0. Mu.L of cDNA, 1.0. Mu.L of ddH 2 O was made up to 20. Mu.L. The reaction conditions for the fluorescent quantitative PCR amplification are as follows: pre-denaturing at 95deg.C for 15 min; 40 cycles of denaturation at 95℃for 10sec, annealing at 60℃for 32sec were run first, followed by 15sec at 95℃for the dissolution profile stage, 1min at 60℃for 30sec at 95℃for 15sec at 60 ℃.
5. Amplification efficiency of primer
Serial gradient dilutions of the adult cDNA of the myzus persicae are used as templates for real-time fluorescent quantitative PCR, and an amplification efficiency standard curve of the primer is prepared. The primer amplification efficiency was calculated as follows: amplification efficiency (E) = (10) [ -1/slope] -1)×100。
6. Experimental results
According to the quantitative PCR reaction program, genome RNA samples of winged aphids and wingless aphids of the long tube aphids are subjected to quantitative PCR amplification, and a melting curve (shown in figure 10), a quantitative cycle (quantification cycle, cq) and an amplification curve (amplification plot) are obtained. The specificity of the amplified product is judged by combining the melting curve, and the ideal melting curve should be a unimodal curve. From the analysis of the primer melting curve of this experiment,the SA-RPL11 gene detection of the winged aphid and wingless aphid samples shows a unimodal curve, and no primer dimer and other nonspecific amplification is generated, which indicates that the fluorescent quantitative PCR amplification primers (SEQ ID No.14 and 15) related to the invention have single amplified band and strong specificity, and no nonspecific amplification is generated. The invention can be seen from the fluorescent quantitative PCR amplification curves of the winged aphids and the winged aphids of the wheat long pipe aphids, the pair of primers are amplified under different dilution concentrations of templates, and the product amplification amounts are subjected to typical amplification processes such as a baseline, an exponential amplification period, a linear period, a platform period and the like, so that the gene can be used for amplifying SA-RPL11 genes in the winged aphids and the winged aphids of the wheat long pipe aphids. Performing a series of 5-fold gradient dilutions on the wheat long tube aphid adult cDNA as a template, performing real-time fluorescence quantitative PCR detection to obtain a series of Cq values of the template, and drawing a fitting curve (shown in figure 11) of the wheat long tube aphid SA-RPL11 gene by taking the Cq values as ordinate and the dilution times as abscissa, wherein the fitting curve is as follows: y= -3.558x+37.779, correlation coefficient (R 2 ) The slope of the fitted curve was brought to 0.9997 as a primer amplification efficiency calculation: amplification efficiency (E) = (10) [ -1/slope] -1) x 100, the amplification efficiency of the primer obtained by calculation is 91%, and the amplification efficiency of the primer is about 100%, which shows that the primer has high amplification efficiency and accords with the internal reference gene used as the fluorescent quantitative PCR detection of the wheat long tube aphid. Analysis of the expression of the SA-SA-RPL11 gene in the wheat long tube aphid pheromone E-beta-farnesene solution treatment sample, statistical analysis of Cq values of the SA-SA-RPL11 gene in the wheat long tube aphid pheromone E-beta-farnesene solution treatment sample and the control group, and found that the SA-SA-RPL11 gene can be stably expressed in the wheat long tube aphid pheromone E-beta-farnesene solution treatment sample, and the difference is not obvious (P>0.05 The SA-SA-RPL11 gene can be used as an internal reference gene for fluorescence quantitative PCR detection of functional genes in a sample body treated by a long tube semiochemical E-beta-farnesene solution.
Example 7 fluorescent quantitative PCR detection of SA-28S Gene under treatment with Aphis glutinosa antibiotic
1. Total RNA extraction of the myzus persicae:
the method is the same as in example 1, except that the adult aphid of the myzus persicae is treated with 50. Mu.g/ml rifampicin for 24 hours, and then the aphid sample is collected, and total RNA of the myzus persicae under each treatment is extracted by TRizol (Amion, USA).
2. Reverse transcription
Reverse transcription was performed using TAKARA PrimeScript st Strand cDNA synthesis kit, and the procedure was the same as in example 3.
3. Fluorescent quantitative PCR amplification primer synthesis
Fluorescent quantitative PCR upstream primer (SEQ ID NO. 19): 5'-TCTATCAACCGGCAACCACA-3';
fluorescent quantitative PCR downstream primer (SEQ ID NO. 20): 5'-TGGCGAATCTCGTTGCATTT-3'.
4. Fluorescent quantitative PCR
And (3) performing fluorescent quantitative PCR detection by taking the first-strand cDNA obtained by RT as a template. The reaction system of qPCR based on SYBR Green I dye is: ssoFast EvaGreen Supermix 10. Mu.L of each of the upstream and downstream primers (10.0. Mu.M), 1.0. Mu.L of cDNA, 1.0. Mu.L of ddH 2 O was made up to 20. Mu.L. The reaction conditions for the fluorescent quantitative PCR amplification are as follows: pre-denaturing at 95deg.C for 15 min; 40 cycles of denaturation at 95℃for 10sec, annealing at 60℃for 32sec were run first, followed by 15sec at 95℃for the dissolution profile stage, 1min at 60℃for 30sec at 95℃for 15sec at 60 ℃.
5. Amplification efficiency of primer
Serial gradient dilutions of the adult cDNA of the myzus persicae are used as templates for real-time fluorescent quantitative PCR, and an amplification efficiency standard curve of the primer is prepared. The primer amplification efficiency was calculated as follows: amplification efficiency (E) = (10) [ -1/slope] -1)×100。
6. Experimental results
According to the invention, a quantitative PCR (polymerase chain reaction) amplification is carried out on a genomic RNA sample of an antibiotic treatment and a control of the myzus persicae according to a quantitative PCR reaction program, and a melting curve (figure 13), a quantitative cycle (quantification cycle, cq) and an amplification curve (amplification plot) are obtained. The specificity of the amplified product is judged by combining the melting curve, and the ideal melting curve should be a unimodal curve. From the analysis of the primer melting curve of the experiment, SA-28S gene detection of the aphid-forming sample treated by the wheat long tube aphid antibiotic shows a unimodal curve without primerThe non-specific amplification of dimer and the like is generated, which shows that the fluorescent quantitative PCR amplification primers (SEQ ID No.19 and 20) related to the invention have single amplified band and strong specificity, and no non-specific amplification occurs. The invention can be seen from the fluorescent quantitative PCR amplification curve of the adult aphids treated by the wheat long tube aphid antibiotics, the pair of primers are amplified under different dilution concentrations of the templates, and the product amplification amount is subjected to typical amplification processes such as a baseline, an index amplification period, a linear period, a platform period and the like, so that the gene can be used for amplifying SA-28S genes in the adult aphids treated by the wheat long tube aphids antibiotics. Performing a series of 5-fold gradient dilutions on the wheat long tube aphid adult cDNA as a template, performing real-time fluorescence quantitative PCR detection to obtain a series of Cq values of the template, and drawing a fitting curve (shown in figure 14) of the wheat long tube aphid SA-28S gene by taking the Cq values as ordinate and the dilution times as abscissa, wherein the fitting curve is as follows: y= -3.0672x+30.912, correlation coefficient (R 2 ) For 0.9886, the slope of the fitted curve was taken into the primer amplification efficiency calculation: amplification efficiency (E) = (10) [ -1/slope] -1) x 100, the amplification efficiency of which is calculated to be 112%, the amplification efficiency of the primer is about 100%, which shows that the amplification efficiency of the primer is very high, and the primer accords with the internal reference gene of the fluorescent quantitative PCR detection of the adult aphid treated by the antibiotics of the myzus persicae.
Example 8 fluorescent quantitative PCR detection of SA-28S Gene under treatment with A. Ophiopogonis insecticide
1. Total RNA extraction of the myzus persicae:
adults of the aphid Aphis gracilis were treated with different concentrations of insecticide (LC 20 Mortality of 20% and LC 50 Insecticide concentration with mortality of 50%) after 24h treatment, aphid samples were collected. The selected pesticide comprises imidacloprid, thiamethoxam and chlorpyrifos, biological activity measurement is carried out on the wheat aphid adults, wheat seedlings with wheat aphids are all soaked in a test agent, and the test agent is dried for 5-10min at room temperature after staying for 10s and then placed in a 9cm culture dish. With 0.1% Triton X-100 as control, 4 tubes were collected for each treatment, about 50 test insects per tube, quick frozen in liquid nitrogen and stored in a refrigerator at-80℃for a short period. Total RNA from each of the treated Aphis glutinosa was extracted by TRizol (Amion, USA) in the same manner as in example 1.
2. Reverse transcription
Reverse transcription was performed using TAKARA PrimeScript st Strand cDNA synthesis kit, and the procedure was the same as in example 3.
3. Fluorescent quantitative PCR amplification primer synthesis
Fluorescent quantitative PCR upstream primer (SEQ ID NO 4): 5'-TCTATCAACCGGCAACCACA-3';
fluorescent quantitative PCR downstream primer (SEQ ID NO 5): 5'-TGGCGAATCTCGTTGCATTT-3'.
4. Fluorescent quantitative PCR
And (3) performing fluorescent quantitative PCR detection by taking the first-strand cDNA obtained by RT as a template. The reaction system of qPCR based on SYBR Green I dye is: ssoFast EvaGreen Supermix 10. Mu.L of each of the upstream and downstream primers (10.0. Mu.M), 1.0. Mu.L of cDNA, 1.0. Mu.L of ddH 2 O was made up to 20. Mu.L. The reaction conditions for the fluorescent quantitative PCR amplification are as follows: pre-denaturing at 95deg.C for 15 min; 40 cycles of denaturation at 95℃for 10sec, annealing at 60℃for 32sec were run first, followed by 15sec at 95℃for the dissolution profile stage, 1min at 60℃for 30sec at 95℃for 15sec at 60 ℃.
5. Amplification efficiency of primer
Serial gradient dilutions of the adult cDNA of the myzus persicae are used as templates for real-time fluorescent quantitative PCR, and an amplification efficiency standard curve of the primer is prepared. The primer amplification efficiency was calculated as follows: amplification efficiency (E) = (10) [ -1/slope] -1)×100。
6. Experimental results
According to the quantitative PCR reaction program, genome RNA samples of winged adult aphids and wingless adult aphids of the long tube aphids are subjected to quantitative PCR amplification to obtain a melting curve (mering cut), a quantitative circulation (quantification cycle, cq) and an amplification curve (amplification plot). The specificity of the amplified product is judged by combining the melting curve, and the ideal melting curve should be a unimodal curve. From the analysis of the primer melting curve of the experiment, SA-28S gene detection of the aphid-forming sample treated by the wheat long tube aphid insecticide shows a single peak curve, and No primer dimer and other nonspecific amplification is generated, which shows that the fluorescent quantitative PCR amplification primers (SEQ ID No. 4 and 5) related by the invention amplify a single bandFirst, the specificity is strong, and no nonspecific amplification occurs. The invention can be seen from the fluorescent quantitative PCR amplification curve of the adult aphids treated by the wheat long tube aphid insecticide, the pair of primers are amplified under different dilution concentrations of templates, and the product amplification amount is subjected to typical amplification processes such as a baseline, an exponential amplification period, a linear period, a platform period and the like, so that the gene can be used for amplifying SA-28S genes in the winged adult aphids and the wingless adult aphids of the wheat long tube aphids. Carrying out a series of 5-fold gradient dilutions on the wheat long tube aphid adult cDNA as a template, carrying out real-time fluorescence quantitative PCR detection to obtain Cq values of a series of templates, and drawing a fitting curve of the wheat long tube aphid SA-28S gene by taking the Cq values as an ordinate and the dilution factors as an abscissa, wherein the fitting curve is as follows: y= -3.0672x+30.912, correlation coefficient (R 2 ) For 0.9886, the slope of the fitted curve was taken into the primer amplification efficiency calculation: amplification efficiency (E) = (10) [ -1/slope] -1) x 100, the amplification efficiency of the primer is calculated to be 112%, and the amplification efficiency of the primer is about 100%, which shows that the primer has high amplification efficiency and accords with the fluorescent quantitative PCR detection reference gene treated by the pesticide of the wheat long pipe aphid. Analysis of the expression of the SA-28S Gene in samples treated with different pesticide concentrations from the wheat head aphid (as shown in FIG. 14), statistical analysis of the SA-28S Gene in wheat head aphid in LC 20 、LC 50 And Cq value in CK, the SA-28S gene can be stably expressed under the treatment of different pesticide concentrations of the wheat long tube aphid, the difference is not obvious (P>0.05 The SA-28S gene can be used as an internal reference gene for functional gene fluorescence quantitative PCR detection in a sample body treated by the wheat long tube aphid insecticide.
<110> institute of plant protection of national academy of agricultural sciences
<120> partial sequence of internal reference gene RPL14 stably expressed under different density treatment of wheat long tube aphid, cloning method and application
<160> 20
<210> 1
<211> 805
<212> DNA
<213> nucleotide sequence of SA-HEL Gene fragment
<400> 1
ACTGGTTTGA ACGAATATGA AAAGGAAAAT GAAGAGTATA ATGAACTATC AAAAAAAGCT 60
TTACATGATT CAAATATGAG TTCTGTTGAT AGTATTTCTA ATGTAAATTC AGTGGCAGAT 120
CTGAAAGATT TGGATATAGA TGAAATTAAG CTTTTTGAAA ATTATAAATT CAAGGATCCA 180
TCGAATAAGT CTAATGATTT AGAAATTGCT GCATATAAAG ATAAGATAAT TTCAATTGTC 240
AATAGTAATA ATACAGTCGT GATATCTGGT GCTACCGGAT GTGGGAAAAG TACACAAGTT 300
CCTCAGTTTA TTTTAGATGA CTGTATGAGT AAAAAGAAAT ATTGTAATAT TATTGTTACT 360
CAACCTAGAC GTATAGCTGC AATTTCAGTA TCTAAACAAG TAAACAGAGA ACGTGGCTGG 420
AAAGATGGGC TTTTAGTCGG TTATCAAGTT GGGCGTAAAA AAGATTTTGA TCCTACCACA 480
ACAAAAATTT TATATTGCAC TACAGGAATT TTGTTACTAA GAATTATTAA AGCAAAGAGC 540
CTTGCTGAAT TTTCTCACAT TATCCTAGAC GAAGTTCATG AAAGAACACT TGAAATGGAT 600
TTTTTATTAT TAATTATAAA GAAGTTAAAA AAGAAAAACT ATGAGTCAAC TCGTGTAATA 660
CTAATGTCTG CCACAGCAGA CGCATTGAAA TTACAAGATT ATTTTGGTGA TTATTATGGA 720
CATCCTTATT ATAGACACGT AACAGCACCA TTAATTAAGA TCGAAAAGCC ATCTAACTAT 780
ACTATTCTTA CACACTATTT GGATG 805
<210> 2
<211> 19
<212> DNA
<213> PCR amplification upstream primer for SA-HEL
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 2
ACTGGTTTGAACGAATATG 19
<210> 3
<211> 20
<212> DNA
<213> PCR amplification downstream primer for SA-HEL
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 3
CATCCAAATAGTGTGTAAGA 20
<210> 4
<211> 20
<212> DNA
<213> qPCR amplification upstream primer of SA-HEL
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 4
TGCTACCGGATGTGGGAAAA 20
<210> 5
<211> 20
<212> DNA
<213> qPCR amplification downstream primer of SA-HEL
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 5
TCCAGCCACGTTCTCTGTTT 20
<210> 6
<211> 500
<212> DNA
<213> nucleotide sequence of SA-RPL14 Gene fragment
<400> 6
AATGTGAGGT TATGATTGTC TTCTAAGCAC CACAGTTCCA AGCGTTTGGT TTCTCATTCT 60
TTTAGGTTCA ACAATCGACG TGTGAGAGAG CATTATTTCG GAAAAATGCC TTTCAAGAGG 120
TTTGTGGAAA CTGGCCGAGT GGTCTACGTT GTAGATGGTC CTTACAAAGG CAAAATTGTT 180
TCAATAGTCG ACTGCATCGA TCAAAAAACT GTTTTGGTAG ATGGACCAGA AACTGGAGTA 240
CCCAGGTCAA AGATGCGTAT TAGCCAAATC CACTTGACCA AGTTTAAGAT TAATTTCCCT 300
TACAATGGAT CAACCAGGAC TGTTCGCCAA GCATGGAAAA AAGCCGATTT AAACAAATTG 360
TGGGTTCAAA GCAGGTGGGC AGAAAAAGCC GCCAACAGGG AGAAGCGTGC TTCTTTGGGC 420
GATTTTGAAA GATTTAAGCT TAAGAGAGCA AGAAAAATCA GAAACAAGAT CAGGACTAAC 480
GTCTACCAAG CACTGTTTAA 500
<210> 7
<211> 20
<212> DNA
<213> PCR amplification upstream primer for SA-RPL14
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 7
AATGTGAGGTTATGATTGTC 20
<210> 8
<211> 18
<212> DNA
<213> PCR amplification downstream primer for SA-RPL14
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 8
TTAAACAGTGCTTGGTAG 18
<210> 9
<211> 20
<212> DNA
<213> qPCR amplification upstream primer of SA-RPL14
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 9
AAGCACCACAGTTCCAAGCG 20
<210> 10
<211> 20
<212> DNA
<213> qPCR amplification downstream primer of SA-RPL14
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 10
ACTCGGCCAGTTTCCACAAA 20
<210> 11
<211> 418
<212> DNA
<213> nucleotide sequence of SA-RPL11 Gene fragment
<400> 11
TTTCCTCGTT GGACGTGAGA TAGCCGCTAC GAATCCATCA TGGTCCTCGC CGACTTAAAG 60
AAAGGAGGCA ACAAGTCTTC TGAGAAAACG AAAAATCGTA TGCGCGAATT GAGGATTCGC 120
AAATTATGTC TGAACATTTG TGTCGGTGAA TCTGGTGACA AGCTCACCCG TGCCGCTAAG 180
GTGTTGGAAC AATTGACTGG CCAACAACCA GTGTTCTCTA AAGCTCGTTA TACAGTCAGA 240
TCTTTCAGTA TCAGAAGAAA TGAAAAAATT GCAGTGCACT GCACGGTTCG AGGTGCTAAG 300
GCTGAAGAAA TCTTGGAACG TGGATTAAAG GTTCGTGAAT ACGAATTGAG GCGAGAAAAC 360
TTCTCGGACA CCGGTAACTT CGGTTTTGGT ATTCAAGAAC ACATCGATTT GGGTATCA 418
<210> 12
<211> 20
<212> DNA
<213> PCR amplification upstream primer for SA-RPL11
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 12
TCGTGTTGTTTCGCTTCGTC 20
<210> 13
<211> 25
<212> DNA
<213> PCR amplification downstream primer for SA-RPL11
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 13
TGATACCCAAATCGATGTGTTCTTG 25
<210> 14
<211> 20
<212> DNA
<213> qPCR amplification upstream primer of SA-RPL11
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 14
CGTGTTGTTTCGCTTCGTCT 20
<210> 15
<211> 20
<212> DNA
<213> qPCR amplification downstream primer of SA-RPL11
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400>15
CTTTAAGTCGGCGAGGACCA 20
<210> 16
<211> 617
<212> DNA
<213> nucleotide sequence of SA-28S Gene fragment
<400> 16
CGAGTGAGCC AGAAACACAT TTTACAATAA TTTTTGTTAC ATATTTTCGA TATAATTTAT 60
TATTTGAGTA ACTAGGTTTT AAGAAAACTA TAACAATGTT TTTGAACACT ATTCGCACAT 120
TGTCGTCTAC ACTTTTCAAA GCATCAAGTG GTCTATCAAC CGGCAACCAC ATTGTGCCTA 180
AAAACGGAAG AAATGTCTAC ACAAACGTGG TCAAATATAA TGAATTATCA ACTAAAGATG 240
AAATGCAACG AGATTCGCCA AAAGATAGAT CGACTGTAAT TCCTGCCGAA ACAAGTATTG 300
AATATCTAAA AAGTGATGCA TACAAATCCA CTTATGGAGA TAATCCTGTT TGGAAAGAAT 360
ATCGCCGTAA TCATAAAGGA AGTATTCCAC CAAGAAAAAC TAGGAAAACG TGTATTAGAT 420
CTGATATGAT TGCAACTGGT AATCCATGTC CAGTATGTCG TGACGAATAC CTCATACTCG 480
ACTACAGAAA TGTGGATTTG CTCAAGCAGT TCATATCTCC TTATTCTGGA AAACTGTTAT 540
CCTATAAATT AACTGGAATT TGCCAAAAAC AGCATGAAAA TTTGATAGTT GCCGTAAAAA 600
AGGCCAAAGA CTGGGGA 617
<210> 17
<211> 20
<212> DNA
<213> PCR amplification upstream primer for SA-28S
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 17
CGAGTGAGCCAGAAACACAT 20
<210> 18
<211> 21
<212> DNA
<213> PCR amplification downstream primer for SA-28S
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 18
ATCCCCAGTCTTTGGCCTTTT 21
<210> 19
<211> 20
<212> DNA
<213> qPCR amplification upstream primer of SA-28S
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 19
TCTATCAACCGGCAACCACA 20
<210> 20
<211> 20
<212> DNA
<213> qPCR amplification downstream primer of SA-28S
<220>
<223> description of artificial sequence: artificially synthesized sequences
<400> 20
TGGCGAATCTCGTTGCATTT 20
Claims (4)
1. The application of the wheat long tube aphid gene fragment as an internal reference gene is characterized in that the wheat long tube aphid gene fragment is an SA-RPL14 gene fragment, and the nucleotide sequence of the wheat long tube aphid gene fragment is shown as SEQ ID No. 6; the SA-RPL14 gene fragment is used for quantitative PCR detection of the functional gene of the wheat long tube aphid or in-vivo gene expression under different density treatments.
2. The use according to claim 1, wherein the primer pair sequences for detecting the SA-RPL14 gene fragment are as follows:
SA-RPL14-F：5’ -AATGTGAGGTTATGATTGTC-3’ ；
SA-RPL14-R：5’ -TTAAACAGTGCTTGGTAG-3’。
3. the use according to claim 2, wherein the SA-RPL14 gene fragment is amplified by fluorescent quantitative PCR, comprising the steps of extracting total genome RNA of the myzus persicae, carrying out RT reaction with a kit-of-parts mixed primer as a reaction primer, and carrying out fluorescent quantitative PCR amplification with the first-strand cDNA of the product as a template, wherein the fluorescent dye of the primer pair is SYBR Green 1.
4. The use according to claim 3, wherein the reaction conditions for the fluorescent quantitative PCR amplification are as follows:
pre-denaturing at 95deg.C for 15 min; 40 cycles of denaturation at 95℃for 10sec, annealing at 60℃for 32sec were run first, followed by 15sec at 95℃for the dissolution profile stage, 1min at 60℃for 30sec at 95℃for 15sec at 60 ℃.
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