CN104789557B - A kind of musky gourd reference gene and its application - Google Patents
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Abstract
The invention discloses the 18S rRNA sequences that one is used for musky gourd reference gene, quantitative special primer is designed on this basis, musky gourd 18S rRNA genes can be stablized under each growth and development stage of musky gourd and various Abiotic stress conditions expresses, and is adapted in musky gourd gene expression research as reference gene.The present invention is used for musky gourd gene expression analysis using 18S rRNA genes as reference gene first, is conducive to improving stability, the reproducibility and reliability of the research of musky gourd gene expression analysis；Detection primer proposed by the present invention has specificity, greatly improves detection efficiency, improves the confidence level of testing result.
Description
【Technical field】
The invention belongs to technical field of molecular biology, and in particular to a kind of musky gourd reference gene and its application, tool
Body, a pair of fluorescent quantitation special primers are designed based on the nucleotide sequence using the musky gourd reference gene.
【Background technology】
Musky gourd (Cucurbita moschata Duch.) is Curcurbitaceae (Cucurbitaceae) Cucurbita
One of 3 main cultispecies in (Cucurbita spp.).Musky gourd strong adaptability, distributional region is wide, and species is various, product
It is nutritious, it is traditional vegetables that our people likes.Deepening continuously and develop, gene table with its molecular biology research
Just gradually it is being applied to up to analysis in the research of announcement musky gourd gene regulation mechanism.In gene expression analysis, in order to eliminate
The deviation of different tissues iuntercellular original template amount, RNA mass, enzymatic reaction efficiency etc., generally requires to introduce reference gene pair
It is corrected.Preferable reference gene should under histological types, growth phase and different experimental conditions expression
It is consistent, the house-keeping gene of the stable expression of often selection, such as actin gene (actin), glyceraldehyde -3- in practical study
Phosphate dehydrogenase gene (GAPDH), microtubule protein gene (tubulin), ubiquitin gene (ubiquitin), 18S rRNAs
(18S rRNA) etc. is used as reference gene.
18S rRNA are present in all eukaryotic cells, almost in a organized way in all high level expressions, same
The protein expression amount planted in cell or tissue is usually constant, and it is analyzing gene expression in plants as reference gene
And be widely used in the pathogen research in detection plant.But at present on musky gourd 18S rRNA genes clone and
Had not been reported as the research on musky gourd reference gene.
【The content of the invention】
The technical problems to be solved by the invention are to provide a kind of musky gourd reference gene, and disclose using in this
The real-time fluorescence quantitative PCR primer designed based on state's pumpkin reference gene.
The present invention is to solve above-mentioned technical problem by the following technical programs：A kind of musky gourd reference gene, it is described
The nucleotide sequence of reference gene such as SEQ ID NO：Shown in 3；And the reference gene be using musky gourd DNA as template and with
Following primer pair is carried out obtained by pcr amplification reaction；
Wherein primer pair is：
Forward primer 5'-CCAATCATACTCAAAAGAAGAGTT-3',
Reverse primer 5'-AGGATTCAATCCAGCCACAGGTT-3'.
Further, based on the nucleotide sequence of the reference gene, using Primer Premier5.0 softwares,
And follow the principle of real-time fluorescence quantitative PCR design of primers and design a pair of fluorescent quantitation special primers, this couple of fluorescent quantitation spy
Different primer is the real-time fluorescence quantitative PCR primer；
And the real-time fluorescence quantitative PCR primer is：
Forward primer 5'-AAACCTTACCAGCCCTTGAC-3',
Reverse primer 5'-CGCTCGTTATAGGACTTGACC-3'.
The beneficial effects of the present invention are：
The invention provides a kind of musky gourd reference gene, while it is base to disclose using the musky gourd reference gene
The real-time fluorescence quantitative PCR primer of plinth design, designed real-time fluorescence quantitative PCR primer is used for musky gourd gene expression analysis
During analysis, it is possible to increase the stability of musky gourd gene expression analysis research, reproducibility and reliability；In addition, designed reality
When fluorescence quantification PCR primer high specificity, so as to greatly improve using real time fluorescent quantitative detect musky gourd when
Detection efficiency, and improve the confidence level of testing result.
【Brief description of the drawings】
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is 1% agarose gel electrophoresis figure of embodiment 1 in the present invention.
Fig. 2 is 1% agarose gel electrophoresis figure of embodiment 2 in the present invention.
Fig. 3 is the PCR amplification curve diagrams of embodiment 3 in the present invention.
Fig. 4 is the solubility curve figure of embodiment 3 in the present invention.
Fig. 5 is 1% agarose gel electrophoresis figure of embodiment 4 in the present invention.
【Embodiment】
The present invention lists following representative embodiment, and these embodiments are only exemplary, and are not used in limitation
The scope of the present invention, these embodiments are only used for that the present invention will be described.And instrument and reagent employed in each embodiment
It is as follows：American AB I7500 real-time PCRs, American AB I Veriti96-well thermal cyclers, American AB I PowerThe chain synthetic agent box of Green PCR Master Mix, pMD18-T-vector, cDNA first
(PrimeScriptTM 1st Strand cDNA Synthesis Kit)、Marker DL 2000、Taq DNA
Polymerase, dNTP are precious bioengineering (Dalian) Co., Ltd product, and glue reclaim kit, plasmid extraction kit are
Omega Products, primer synthesis and cloning and sequencing are completed by Bo Shang biotechnologys (Shanghai) Co., Ltd., remaining biochemical reagents
It is that domestic analysis is pure.
The acquisition of the reference gene of embodiment 1
Step (1), the watermelon announced according to Genbank, the 18S rRNA genes design pair of primers of muskmelon and cucurbita pepo,
And sequence alignment is carried out to the designed primer pair using clustalx softwares, and by platinum still biotechnology (Shanghai) limited public affairs
Department's this pair of primer of synthesis, specifically, this couple of primer (such as SEQ ID NO：1st, shown in 2)：Forward primer 5'-
CCAATCATACTCAAAAGAAGAGTT-3', reverse primer 5'-AGGATTCAATCCAGCCACAGGTT-3'.
Step (2), DNA are extracted：Musky gourd blade 0.5g is weighed in mortar, liquid nitrogen is added and 0.1 gram of PVP is quick
It is fully ground to powdered, powder is moved in 1.5mL centrifuge tubes；Often pipe adds the 2%CTAB extractions that 600 μ L are preheated to 65 DEG C
Buffer solution, while adding 7 μ L 2- coloured glaze base ethanol, is fully mixed, 65 DEG C of water-bath 30min, and reverse once every 10min；Take out
Centrifuge tube, is cooled to room temperature, is subsequently added into isometric chloroform/isoamyl alcohol (body of chloroform, both isoamyl alcohol in chloroform/isoamyl alcohol
Product is than being 24:1), light and slow reverse mixing, stands 10min, 12000rpm centrifuges 10min at room temperature；Supernatant is taken to another centrifugation
Pipe, plus 2/3 volume isopropanol, it is light and slow it is reverse mix, room temperature place 15min, 12000rpm is from 10min under the conditions of 4 DEG C；With
The ethanol washing DNA of volume fraction 70% is precipitated 2-3 times, is then air-dried；Gained DNA is dissolved in 500 μ L TE solution after air-drying,
Final concentration 50mg/mL RNase A 2uL are added, and 30min is incubated in 37 DEG C, electricity are carried out with 1% Ago-Gel afterwards
Swimming detection DNA mass；DNA is finally diluted to 50ng/ μ L, and saved backup in -20 DEG C of refrigerators.
Step (3), PCR amplifications：Using the DNA obtained by being handled through step (3) as template, and being drawn with acquisition in step (1)
Thing is that primer pair carries out pcr amplification reaction, obtains pcr amplification product.
PCR reaction systems：The cumulative volume of reaction system be the μ L of 25 μ L, μ containing 50ng/ L DNA 0.5,10 μm of ol/L it is positive and
Each 1 μ L of reverse primer, the μ L of 10mmol/L dNTP 0.5, the μ L of 5U/ μ L Taq archaeal dna polymerases 0.25,1.5mmol/L MgCl2's
The μ L of 10 × PCR buffer solutions 2.5, remaining composition are the ultra-pure water of sterilizing.
PCR response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 90s, 35
Circulation；Extend 10min at last 72 DEG C；In preservation at 4 DEG C.
Step (5), take after pcr amplification product obtained by step (4) detects through 1% agarose gel electrophoresis, obtain such as Fig. 1 institutes
Show the fragment of size, reclaim the fragment and be connected to conversion, picking positive clone molecule, PCR on pMD18-T carriers after purification
Sequencing is delivered to after detection, measured sequence is the nucleotide sequence of reference gene, it is such as SEQ ID NO：Shown in 3.
The real-time fluorescence quantitative PCR of embodiment 2 is designed and Standard PCR detection
Based on the nucleotide sequence of the reference gene obtained by embodiment 1, using Primer Premier5.0 softwares,
And follow the principle of real-time fluorescence quantitative PCR design of primers and design a pair of fluorescent quantitation special primers, its amplified fragments is
132bp, this pair of fluorescent quantitation special primer is the real-time fluorescence quantitative PCR primer (such as SEQ ID NO：4th, shown in 5)：Just
To primer 5'-AAACCTTACCAGCCCTTGAC-3', reverse primer 5'-CGCTCGTTATAGGACTTGACC-3'.
Musky gourd total serum IgE is extracted, and is tried according to PrimeScriptTM 1st Strand cDNA Synthesis Kit
The method synthesis chains of cDNA first of agent box, i.e., be cDNA by RNA reverse transcriptions；Afterwards using gained cDNA as template, with real-time fluorescence
Quantification PCR primer is that primer pair enters performing PCR amplification, and the reaction system and response procedures of PCR amplifications are as follows：
PCR reaction systems：The cumulative volume of reaction system is the μ L of 25 μ L, μ containing 50ng/ L cDNA 0.5,10 μm of ol/L forward directions
With each 1 μ L of reverse primer, the μ L of 10mmol/L dNTP 0.5, the μ L of 5U/ μ L Taq archaeal dna polymerases 0.25,1.5mmol/L MgCl2
The μ L of 10 × PCR buffer solutions 2.5, remaining composition for sterilizing ultra-pure water.
PCR response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, 35
Circulation；Extend 10min at last 72 DEG C；In preservation at 4 DEG C.
Pcr amplification product is subjected to 1% agarose gel electrophoresis detection, testing result is as shown in Figure 2；As shown in Figure 2,
PCR amplifications obtain a single band, non-specific amplification band (Fig. 2) do not occur, are 132bp through size is sequenced, meet
It is expected that size；The real-time fluorescence quantitative PCR primer checking in downstream can then be continued.
The real-time fluorescence quantitative PCR primer of embodiment 3 is verified
Musky gourd total serum IgE is extracted, and is tried according to PrimeScriptTM 1st Strand cDNA Synthesis Kit
The method synthesis chains of cDNA first of agent box, i.e., be cDNA by RNA reverse transcriptions；Afterwards using gained cDNA as template, according to PowerGreen PCR Master Mix specifications are in the enterprising performing PCR reaction of ABI7500 real-time PCRs, PCR reactions
Reaction system and response procedures it is as follows：
Reaction system is：The cumulative volume of reaction system is 25 μ L, 12.5 μ L PowerGreen PCR
(concentration is 10 μm of ol/ to the μ L of forward primer 0.5 of real-time fluorescence quantitative PCR primer in Master Mix, 1 μ L templates, embodiment 2
L), in embodiment 2 real-time fluorescence quantitative PCR primer the μ L of reverse primer 0.5 (concentration be 10 μm ol/L), mend distilled water to totality
25 μ L of product.
Response procedures are：95 DEG C of pre-degeneration 10min；95 DEG C of denaturation 15s, 60 DEG C of annealing 1min, totally 40 circulations；4 DEG C of guarantors
Deposit；3 repetitions are done in each reaction.
Shown in the result of reaction such as Fig. 3 (wherein, Δ Rn refers to fluorescence intensity, cycle and refers to period), from figure 3, it can be seen that
The fluorescent quantitative PCR curve of 3 repetitions is fine.And to examine the specificity of reaction, applicant is melted after PCR
Tracing analysis, analysis result as shown in figure 4, can be shown from Fig. 4, the Tm (solution temperature) of 3 repetitions is respectively 82.89 DEG C,
82.89 DEG C, 82.7 DEG C, and only one of which specific peak, show primer free dimer, amplified band is single, high specificity does not have
Non-specific amplification occurs, so as to show the designed pair of primers high specificity (real time fluorescent quantitative i.e. in embodiment 2
PCR primer), amplification efficiency is high, high specificity, can be used in the internal control primer experiment of musky gourd quantitative fluorescent PCR.
The musky gourd 18S rRNA gene expression stability analysis of experimental example 4
Musky gourd root, stem, leaf, flower, young melon, old melon, high-temperature process (38 DEG C) blade, low-temperature treatment (8 are extracted respectively
DEG C) blade, strong light processing (2000umolm-2·s-1) blade and dim light processing (200umolm-2·s-1) blade it is total
RNA, is synthesized according to the method for PrimeScriptTM 1st Strand cDNA Synthesis Kit kits respectively afterwards
The chains of cDNA first, to obtain respective cDNA；It is primer pair using the real time fluorescent quantitative special primer in embodiment 2, to obtain
10 kinds of the cDNA obtained is template, and performing PCR amplification is entered respectively, and PCR amplification system is as follows with amplification program：
PCR amplification system：The cumulative volume of reaction system is the μ L of 25 μ L, μ containing 50ng/ L cDNA 0.5,10 μm of ol/L forward directions
With each 1 μ L of reverse primer, the μ L of 10mmol/L dNTP 0.5, the μ L of 5U/ μ L Taq archaeal dna polymerases 0.25,1.5mmol/L MgCl2
The μ L of 10 × PCR buffer solutions 2.5, remaining composition for sterilizing ultra-pure water.
PCR amplification programs are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s, 28
Circulation；Extend 10min at last 72 DEG C；In preservation at 4 DEG C.
Pcr amplification product is subjected to 1% agarose gel electrophoresis detection, testing result as shown in figure 5, and identifying 1 in Fig. 5
To 10 be respectively musky gourd root, stem, leaf, flower, young melon, old melon, high-temperature process (38 DEG C) blade, low-temperature treatment (8 DEG C) blade,
The PCR amplifications of strong light processing (2000umolm-2s-1) blade and dim light processing (200umolm-2s-1) blade are anti-
Answer result；Shown by Fig. 5, when using the real time fluorescent quantitative special primer in embodiment 2 for primer pair, 10 obtained by amplification
Band brightness is basically identical, so as to show in musky gourd different tissues, each growth and development stage and various abiotic stress bars
It can stablize under part and express, that is, illustrate stability of the real time fluorescent quantitative special primer in musky gourd gene expression, from
And suitable for the research of musky gourd gene expression.
To sum up, the invention provides a kind of musky gourd reference gene, and disclose and utilize the musky gourd reference gene
Based on the real-time fluorescence quantitative PCR primer that designs, designed real-time fluorescence quantitative PCR primer is used for musky gourd gene table
Up to during analysis, it is possible to increase the stability of musky gourd gene expression analysis research, reproducibility and reliability；In addition, designed
Real-time fluorescence quantitative PCR primer specificity it is strong, South China is detected using real time fluorescent quantitative so as to greatly improve
Detection efficiency during melon, and improve the confidence level of testing result.
Claims (2)
1. a kind of musky gourd reference gene, it is characterised in that：The nucleotide sequence of the reference gene such as SEQ ID NO：3 institutes
Show；And the reference gene is using musky gourd DNA as template and carried out with following primer pair obtained by pcr amplification reaction；
Wherein primer pair is：
Forward primer 5'-CCAATCATACTCAAAAGAAGAGTT-3',
Reverse primer 5'-AGGATTCAATCCAGCCACAGGTT-3'.
2. a kind of real-time fluorescence quantitative PCR primer, it is characterised in that：With the nucleotides sequence of reference gene described in claim 1
Basis is classified as, is designed using Primer Premier5.0 softwares and the principle that follows real-time fluorescence quantitative PCR design of primers
A pair of fluorescent quantitation special primers, this pair of fluorescent quantitation special primer is the real-time fluorescence quantitative PCR primer；
And the real-time fluorescence quantitative PCR primer is：
Forward primer 5'-AAACCTTACCAGCCCTTGAC-3',
Reverse primer 5'-CGCTCGTTATAGGACTTGACC-3'.
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| CN105274219B (en) * | 2015-10-13 | 2018-12-07 | 华中农业大学 | Purposes of the CL5547.Contig2 gene in the analysis of pumpkin gene expression real-time fluorescence quantitative PCR as reference gene |
| CN107287306B (en) * | 2017-07-03 | 2021-04-06 | 中国农业科学院农产品加工研究所 | Purple cabbage reference gene, primer pair and application |
| CN107653250B (en) * | 2017-10-31 | 2020-10-27 | 福建省农业科学院作物研究所 | Acer palmatum reference gene and application thereof |
| CN108486131A (en) * | 2018-05-25 | 2018-09-04 | 福建省农业科学院作物研究所 | A kind of american pumpkin TUA genes and application |
| CN108728569A (en) * | 2018-05-25 | 2018-11-02 | 福建省农业科学院作物研究所 | A kind of okra reference gene and its application |
| CN108588091A (en) * | 2018-06-22 | 2018-09-28 | 福建省农业科学院作物研究所 | A kind of okra reference gene and its application |
| CN108754012A (en) * | 2018-06-22 | 2018-11-06 | 福建省农业科学院作物研究所 | A kind of cucurbita pepo Actin reference genes and its application |
| CN112080577B (en) * | 2020-09-15 | 2022-03-15 | 华南农业大学 | Reference gene for phytophthora litchi growth, development and infection stages and primer and application thereof |
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| AY357215.1;Bartoszewski,G.et al.,;《GenBank》;20040809;全文 * |
| Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles;Shaogui Guo et al.;《BMC Genomics》;20100921;第12卷;摘要以及第11页左栏第2段 * |
| Reference genes in real-time PCR;Bartłomiej Kozera et al.;《J Appl Genetics》;20130928;第54卷;391–406页 * |
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