CN108977514B - Primer for screening wax gourd real-time fluorescent quantitative PCR (polymerase chain reaction) reference gene EF-1 alpha - Google Patents
Primer for screening wax gourd real-time fluorescent quantitative PCR (polymerase chain reaction) reference gene EF-1 alpha Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and particularly relates to a primer for screening a wax gourd real-time fluorescence quantitative PCR EF-1 alpha reference gene. According to the invention, the Elongation Factor-1 alpha (EF-1 alpha) gene obtained by screening through the specific primer can be stably expressed under different tissues and low temperature stress of wax gourd, and is suitable for serving as an internal reference gene in the research of wax gourd gene expression. The invention uses the Elongation Factor-1 alpha (EF-1 alpha) gene as the internal reference gene for wax gourd gene expression analysis for the first time, which is beneficial to improving the stability, reliability and repeatability of the wax gourd gene expression analysis research; the detection primer provided by the invention has specificity, greatly improves the detection efficiency and improves the reliability of the detection result.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer for screening a real-time fluorescent quantitative PCR (polymerase chain reaction) reference gene EF-1 alpha of wax gourd different tissues and under low-temperature stress.
Background
White gourd (Benincasa hispida Cogn.) an annual vine herb of the genus Benincasa of the family Cucurbitaceae. The flower, leaf, peel, pulp and seed of wax gourd are good medicinesCan be used as food and beverage, and can be used for treating cough and asthma. Meanwhile, the melons are rich in active compounds such as amino acid, vitamin C, phenols and the like, the compounds have a certain effect on preventing chronic diseases, and the wax gourd is the only fat-free melon and vegetable in the melons and vegetables, is rich in tartronic acid components and can inhibit the conversion of saccharides into fat components. With the progress of research, the understanding of the functional components and the gene expression related to the regulation mechanism of wax gourd by using the molecular biology technology becomes an important research direction.
At present, no report is found about the research of reference genes of wax gourd at different parts under the condition of low temperature stress. The invention takes different tissues (roots, stems, leaves and flowers) of the white gourd with black skin and 2 true leaves under 0, 1, 6, 12 and 24h of low-temperature treatment at 4 ℃ as materials, analyzes and evaluates the expression stability of 7 candidate reference genes by utilizing GeNorm, NormFinder and BestKeeper, screens out an Elongation Factor-1 alpha (EF-1 alpha) gene as a proper reference gene, and provides reference for the subsequent related gene expression research of the white gourd.
Disclosure of Invention
The invention aims to provide primers for screening the real-time fluorescent quantitative PCR internal reference gene EF-1 alpha of different tissues of wax gourd, wherein the sequence specificity of the primer of the internal reference gene 1 is increased, the amplification efficiency is close, the linear correlation is good, and the primers can be used for screening the stable internal reference gene of different tissues of wax gourd and under low-temperature stress.
The invention solves the technical problems through the following technical scheme:
with the DNA of white gourd as a template and the sequencing of white gourd transcriptome as a basis, designing 7 pairs of fluorescent quantitative specific primers by using Primer Premier5.0 software and following the design principle of real-time fluorescent quantitative PCR primers, and finally determining the pair of fluorescent quantitative specific primers to be the real-time fluorescent quantitative PCR primers according to data analysis;
and the real-time fluorescent quantitative PCR primer is as follows:
the forward primer 5'-ACCGTCTATCAAGCACCC-3' is the primer that is used for the forward primer,
the reverse primer 5'-CCAAGTCCGCAGTCAAG-3'.
The invention has the beneficial effects that:
the invention provides a primer for screening wax gourd real-time fluorescent quantitative PCR reference genes, and when the designed real-time fluorescent quantitative PCR primer is used for wax gourd gene expression analysis, the stability, reliability and repeatability of the wax gourd gene expression analysis research can be improved; in addition, the designed real-time fluorescent quantitative PCR primer has strong specificity, so that the detection efficiency of the wax gourd by adopting real-time fluorescent quantitative detection can be greatly improved, and the reliability of the detection result is improved.
Drawings
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 shows the amplification results of 7 candidate reference genes of wax gourd.
FIG. 2A 28s candidate reference gene qRT-PCR melting curve.
FIG. 2B EF-1. alpha. candidate reference gene qRT-PCR melting curve.
FIG. 2C GAPDH candidate reference gene qRT-PCR melting curve.
FIG. 2D RP II candidate reference gene qRT-PCR melting curve.
FIG. 2E TUA candidate reference gene qRT-PCR melting curves.
FIG. 2F UBC21 candidate reference gene qRT-PCR melting curve.
FIG. 2G UBQ candidate reference gene qRT-PCR melting curve.
FIG. 3 shows the distribution of the mean CT values of 7 candidate reference genes.
FIG. 4 GeNorm software analyzes VN/VN +1 for different tissues and low temperature stress.
Detailed Description
The present invention is illustrated by the following representative examples, which are intended to be illustrative only and not to limit the scope of the invention. And the instruments and reagents used in the examples were as follows: the American ABI7500 real-time quantitative PCR instrument, the American ABI Veriti 96-well thermal cycler, the American ABI Power SYBR Green PCR Master Mix, the cDNA first Strand Synthesis Kit (PrimeScript 1st Strand cDNA Synthesis Kit), the Marker DL 2000, the Taq DNA Polymerase and the dNTP are all products of Takara bioengineering (Dalian) company Limited, the primer Synthesis is finished by platane biotechnology (Shanghai) company Limited, and the other biochemical reagents are domestic analytical purity.
EXAMPLE 1 Total RNA extraction and cDNA Synthesis
The test material, black-skinned wax gourd, was uniformly sown in a vegetable research base of Town, east China, Fuqing, city, crop institute of agricultural sciences, Fujian province. And (5) plug seedling, and respectively placing the seedlings into an incubator for treatment when the seedlings grow to be three leaves and one heart. A first group: under normal cultivation conditions, taking four parts of roots, stems, leaves and flowers; second group: treating at 4 deg.C for 0, 1, 6, 12, 24h, and collecting 2 true leaves. RNA was extracted using a BioTeke's general plant Total RNA extraction kit, and total RNA integrity and concentration were checked by 1% agarose gel electrophoresis and UV1000 spectrophotometer. Sample was obtained using PrimeScript (Takara Co., Ltd.)™II 1st Strand cDNA Synthesis Kit method of first Strand cDNA, namely RNA reverse transcription cDNA; then, the obtained cDNA is taken as a template, and a real-time fluorescent quantitative PCR primer is taken as a primer pair for PCR amplification, and the reaction system and the reaction program of the PCR amplification are as follows:
and (3) PCR reaction system: the total volume of the reaction system was 25. mu.L, and contained 2. mu.L of cDNA of 100 ng/. mu.L, 1. mu.L of each of the forward and reverse primers of 10. mu.mol/L, 1. mu.L of dNTP of 10 mmol/L, 0.2. mu.L of Taq DNA polymerase of 5U/. mu.L, 2. mu.L of 10 XPCR buffer of 1.5 mmol/L MgCl2, and sterilized ultrapure water as the remaining component.
The PCR reaction program is: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension is carried out for 7 min at 72 ℃; stored at 4 ℃.
Example 2 primer design and PCR primer validation
Using the gene sequence obtained by sequencing wax gourd transcriptome in the early stage of the subject group as a template, 28s, UBQ, RP II, GAPDH, EF-1 alpha, UBC21, and TUA 7 candidate reference genes were selected, and primers were designed using Primer Premier5.0 according to the Primer design rule (Table 1). Carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, wherein the detection result is shown in figure 1; as can be seen from FIG. 1, the amplified products of the 7 reference genes have single bands and the same size as the target fragment, which indicates that the primers can specifically amplify, no primer dimer exists, and the primers are suitable for qRT-PCR research.
TABLE 1 primer sequences for candidate reference genes
Example 3 real-time fluorescent quantitative PCR primer validation
Extracting total RNA of wax gourd, and synthesizing a first Strand of cDNA according to the method of a PrimeScriptTM 1st Strand cDNA Synthesis Kit, namely, reversely transcribing the RNA into the cDNA; then, the obtained cDNA is taken as a template, and PCR reaction is carried out on an ABI7500 real-time quantitative PCR instrument according to the specification of Power SYBR Green PCR Master Mix, wherein the reaction system and the reaction program of the PCR reaction are as follows:
the reaction system is as follows: the total volume of the reaction system was 10. mu.L, 5. mu.L of Power SYBR Green PCR Master Mix, 2. mu.L template, 0.4. mu.L (10. mu. mol/L) of the forward primer of the real-time fluorescent quantitative PCR primer in example 2, 0.4. mu.L (10. mu. mol/L) of the reverse primer of the real-time fluorescent quantitative PCR primer in example 2, and distilled water was added to the total volume of 10. mu.L.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 30s, PCR reaction at 95 ℃ for 5s, PCR reaction at 60 ℃ for 34s, 40 cycles. Melting curve program: 95 deg.C, 60 deg.C for 1min, 95 deg.C for 15 s; storing at 4 deg.C; each reaction was repeated 3 times.
The reaction results are shown in fig. 2A-G, and the 7 candidate internal reference gene primers have good specificity, and the PCR melting curves have only one specific peak, which indicates that there is no primer dimer, the amplification band is single, the specificity is strong, and there is no non-specific amplification, thus indicating that the designed seven pairs of primers have strong specificity (i.e., the real-time fluorescent quantitative PCR primers in example 2), high amplification efficiency and strong specificity, and can be used for the internal reference primer experiments of wax gourd fluorescent quantitative PCR.
Example 4 analysis of expression stability of wax gourd reference Gene
Respectively extracting total RNA of the root, stem, leaf and flower of wax gourd, and then respectively synthesizing a first chain of cDNA according to the method of a PrimeScriptTM 1st Strand cDNA Synthesis Kit to obtain respective cDNA; the real-time fluorescent quantitative specific primers in example 2 were used as primer pairs, and the obtained cDNAs were used as templates for PCR amplification, respectively, wherein the PCR amplification system and the amplification procedure were as follows:
PCR amplification System: the total volume of the reaction system was 10. mu.L, 5. mu.L of Power SYBR Green PCR Master Mix, 2. mu.L template, 0.4. mu.L (10. mu. mol/L) of the forward primer of the real-time fluorescent quantitative PCR primer in example 2, 0.4. mu.L (10. mu. mol/L) of the reverse primer of the real-time fluorescent quantitative PCR primer in example 2, and distilled water was added to the total volume of 10. mu.L.
The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 34s, and 40 cycles; finally, extending for 10 min at 72 ℃; stored at 4 ℃.
The expression stability of 7 candidate reference genes in different tissues is analyzed and evaluated by utilizing GeNorm, NormFinder and BestKeeper3 software, the GeNorm software determines the optimal gene data according to the figure 4, and finally the analysis result of the GeNorm software (table 2) shows that the M values of the 7 reference genes in different tissues of white gourd are sequenced to be UBQ = EF-1 alpha < RP II < UBC21<28s < TUA < GAPDH; the NormFinder software analyzes (Table 3), and the expression stability values S of the candidate reference genes are ranked as EF-1 alpha < RP II < UBQ < UBC21< TUA <28S < GAPDH; the results of BestKeeper software (Table 4) were ranked top with EF-1. alpha. and RP II, and the SD values of TUA and GAPDH were small but the TUA correlation was poor, while GAPDH was the least stable and negative and was not suitable as an internal reference gene. The analysis results of the three software are combined to obtain the following results: EF-1 alpha has the best expression stability in different tissues.
The expression stability of 7 candidate reference genes in different tissues is analyzed and evaluated by GeNorm, NormFinder and BestKeeper3 software, the expression stability values of the 7 candidate reference genes are calculated by GeNorm software (Table 4), the M values of the wax gourd under the condition of low temperature treatment in different periods are ranked as RP II = EF-1 alpha < UBC21< UBQ < TUA <28S < GAPDH, the analysis result of the NormFinder software is known (Table 5), the expression stability values S of the 7 candidate reference genes under the condition of low temperature treatment at 4 ℃ are ranked as EF-1 alpha < RP II < TUA <28S < UBC21< UBQ < GAPDH, and the BestKeepKeer software directly utilizes the CT value (figure 3) to carry out data processing analysis. The results show (Table 6), that EF-1. alpha. is ranked at position 1 under the low-temperature stress condition, indicating that EF-1. alpha. is suitable as an internal reference gene under the low-temperature treatment. The analysis results of the three software are combined to obtain the following results: EF-1 alpha has the best expression stability under low-temperature stress.
TABLE 2 GeNorm software analysis of expression stability of 7 candidate reference genes in different tissues
TABLE 3NormFinder software analysis of expression stability of 7 candidate reference genes in different tissues
TABLE 4 BestKeeper software analysis of expression stability of 7 candidate reference genes in different tissues
TABLE 5 GeNorm software analysis of expression stability of 7 candidate reference genes under Low temperature stress test
TABLE 6 NormFinder software analysis of expression stability of 7 candidate reference genes under Low temperature stress
Table 7 BestKeeper software analysis of expression stability of 7 candidate reference genes under low temperature stress
In conclusion, the invention provides the real-time fluorescent quantitative PCR primer designed on the basis of the wax gourd internal reference gene, and when the designed real-time fluorescent quantitative PCR primer is used for wax gourd gene expression analysis, the stability, reliability and repeatability of wax gourd gene expression analysis research can be improved; in addition, the designed real-time fluorescent quantitative PCR primer has strong specificity, so that the detection efficiency of the wax gourd adopting the real-time fluorescent quantitative detection can be greatly improved, and the reliability of the detection result is improved.
SEQUENCE LISTING
<110> institute of agricultural sciences college of Fujian province
<120> primer for screening wax gourd real-time fluorescence quantitative PCR reference gene EF-1 alpha
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<170> PatentIn version 3.3
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Claims (1)
1. The primer for screening the real-time fluorescent quantitative PCR internal reference gene EF-1 alpha under the low-temperature stress of the wax gourd is characterized by comprising the following steps of: using DNA of wax gourd as a template, and based on the result of transcriptome, designing a pair of fluorescent quantitative specific primers by using Primer Premier5.0 software and following the principle of real-time fluorescent quantitative PCR Primer design, wherein the primers are primers for screening the real-time fluorescent quantitative PCR EF-1 alpha reference gene under the low-temperature stress of wax gourd; the sequence is as follows:
the forward primer 5'-ACCGTCTATCAAGCACCC-3' is the primer that is used for the forward primer,
the reverse primer 5'-CCAAGTCCGCAGTCAAG-3'.
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| CN110016518A (en) * | 2019-04-28 | 2019-07-16 | 福建省农业科学院作物研究所 | A kind of okra reference gene EF-1 α and its application |
| CN111575401A (en) * | 2020-07-07 | 2020-08-25 | 福建省农业科学院作物研究所 | Primer of towel gourd reference gene UBQ and application |
| CN111676230B (en) * | 2020-07-07 | 2022-04-08 | 福建省农业科学院作物研究所 | Towel gourd reference gene EF-1 alpha, and primer and application thereof |
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