CN117004760A - Phyllostachys pubescens real-time fluorescence quantitative PCR reference gene, primer pair and application thereof - Google Patents

Phyllostachys pubescens real-time fluorescence quantitative PCR reference gene, primer pair and application thereof Download PDF

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CN117004760A
CN117004760A CN202311194684.9A CN202311194684A CN117004760A CN 117004760 A CN117004760 A CN 117004760A CN 202311194684 A CN202311194684 A CN 202311194684A CN 117004760 A CN117004760 A CN 117004760A
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高志民
朱成磊
刘燕
林泽铭
李慧
狄小琳
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International Center for Bamboo and Rattan
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Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to a moso bamboo real-time fluorescence quantitative PCR internal reference gene, a primer pair and application thereof. The reference gene comprises ADPRE gene with nucleic acid sequence shown in SEQ ID NO. 1, GAPDH gene with nucleic acid sequence shown in SEQ ID NO. 2, NRP gene with nucleic acid sequence shown in SEQ ID NO. 3, MBF gene with nucleic acid sequence shown in SEQ ID NO. 4, TRX gene with nucleic acid sequence shown in SEQ ID NO. 5 or CAM1 gene with nucleic acid sequence shown in SEQ ID NO. 6. The internal reference gene can be conserved in different tissues, different treatments or different moso bamboo varieties, and the expression quantity is stable; when the gene is used as an internal reference gene for the real-time fluorescence quantitative PCR analysis of moso bamboos, the analysis accuracy is high.

Description

Phyllostachys pubescens real-time fluorescence quantitative PCR reference gene, primer pair and application thereof
Technical Field
The invention belongs to the technical field of plant molecular biology, and particularly relates to a moso bamboo real-time fluorescence quantitative PCR internal reference gene, a primer pair and application thereof.
Background
Moso bamboo (Phyllostachys edulis) is an important non-wood forest resource in China, and is widely concerned due to the characteristics of good wood performance, high wood forming speed and the like, and plays an important role in human production and life. In addition, phyllostachys pubescens is an important forest carbon fixation resource and plays an important role in improving global climate deterioration. In recent years, extensive researches on breeding and cultivation of phyllostachys pubescens and wood characteristics thereof have been carried out, and great contributions are made to alleviating wood shortage. Specifically, the research on the breeding, cultivation and wood characteristics of phyllostachys pubescens is usually carried out by analyzing the gene expression condition of phyllostachys pubescens so as to achieve the research purpose.
As a commonly used method for detecting gene expression, the real-time fluorescence quantitative PCR (RealtimefluorescencequantitativePCR, RT-qPCR) technology is widely applied to the scientific research fields of animal and plant breeding and the like, and has the advantages of high accuracy, high sensitivity, strong specificity, wide detection range, simplicity in operation and the like. However, when quantitative PCR is performed to calculate the relative expression level of a target gene, the result is affected by factors such as total RNA quality, primer specificity, amplification efficiency, etc., and therefore, it is necessary to combine a relatively stable reference gene to perform uniformity correction and to improve the accuracy of the quantitative result. Commonly used internal genes are housekeeping genes whose expression is relatively stable in cells, genes encoding proteins necessary for maintaining basic vital activities of cells, including gene EF1 alpha encoding a transcriptional elongation factor 1-alpha, gene GAPDH encoding a 3-phosphate dehydrogenase, tubulin (Tublin) gene TUB, actin (action) gene ACT, 18S ribosomal RNA gene 18SrRNA, polyubiquitin enzyme (Polyubiquitin) gene UBQ, etc., have been widely used in plant research.
The reference genes adopted by the moso bamboo are screened based on homologous genes of other species, and although the genes are also proved to have expression stability in some species, the expression stability of the genes in the moso bamboo is not verified by transcriptome data, but the difference exists in the gene expression between different species. Along with the updating of the moso bamboo genome, the release of a large amount of transcriptome data and the application of a bioinformatics method, the selection of the internal reference genes with strong conservation, stable expression quantity and good accuracy is of great significance to the evaluation and detection of the expression quantity of the moso bamboo target genes in different tissues, different treatments and moso bamboo varieties.
Disclosure of Invention
Aiming at the problems, one of the purposes of the invention is to provide a moso bamboo real-time fluorescence quantitative PCR reference gene which has strong conservation in different tissues, different stress and/or hormone treatments or different moso bamboo varieties, stable expression quantity and high detection accuracy when being used as the reference gene.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
one aspect of the invention provides a real-time fluorescent quantitative PCR reference gene of moso bamboo, which comprises an ADPRE gene with a nucleic acid sequence shown as SEQ ID NO. 1, a GAPDH gene with a nucleic acid sequence shown as SEQ ID NO. 2, an NRP gene with a nucleic acid sequence shown as SEQ ID NO. 3, an MBF gene with a nucleic acid sequence shown as SEQ ID NO. 4, a TRX gene with a nucleic acid sequence shown as SEQ ID NO. 5 or a CAM1 gene with a nucleic acid sequence shown as SEQ ID NO. 6.
The invention also provides a primer pair for detecting the moso bamboo real-time fluorescence quantitative PCR reference gene.
In yet another aspect, the invention provides a detection reagent comprising the primer pair described above.
In yet another aspect, the present invention provides a detection kit comprising the detection reagent described above.
In still another aspect, the present invention provides an application of the internal reference gene in real-time fluorescence quantitative PCR analysis of moso bamboos, including: (1) When performing analysis of conditions of different tissues and/or different development periods by using fluorescence quantitative PCR on moso bamboos, one or more of ADPRE, GAPDH or NRP genes are adopted as reference genes; (2) In the analysis of the condition of different stress and/or hormone treatments using fluorescent quantitative PCR on moso bamboos, one or more combinations of GAPDH, ADPRE and CAM1 genes are used as reference genes; (3) In comparison analysis using fluorescent quantitative PCR between Phyllostachys Pubescens, phyllostachys Pubescens and Phyllostachys Pubescens, one or more combinations of GAPDH, MBF and TRX genes were used as reference genes.
The beneficial effects of the invention at least comprise:
(1) The ADPRE, GAPDH and NRP genes provided by the invention have stable expression quantity and strong conservation in different tissues and/or different development periods of the moso bamboo, and have high analysis accuracy when being used as reference genes for fluorescent quantitative PCR analysis in different tissues and/or different development periods of the moso bamboo;
(2) The ADPRE, GAPDH and CAM1 genes provided by the invention have stable expression quantity when moso bamboos are stressed differently, strong conservation and high analysis accuracy when being used as reference genes for the fluorescence quantitative PCR analysis of the moso bamboos under different stresses and/or hormone treatment;
(3) The GAPDH, MBF and TRX genes provided by the invention have stable expression quantity in different moso bamboo varieties, strong conservation and high analysis accuracy when being used as reference genes for the fluorescence quantitative PCR analysis of the moso bamboo and the moso bamboo varieties.
Drawings
FIG. 1 is a graph showing the selection of Venn gene for stable expression of moso bamboo variety under different treatment conditions in different tissues according to the present invention;
FIG. 2 is a graph showing the result of agarose gel electrophoresis of PCR products of 12 candidate internal reference gene primers according to the present invention; wherein 1-2: ADPRE;3-4: GAPDH;5-6: histone1;7-8: TSJT1;9-10: SAMS1;11-12: NRP;13-14: MBF;15-16: TRX;17-18: UNK;19-20: TUBA;21-22: CAM1;23-24: an HMG; m:500 bpDNAMake.
FIG. 3 is a standard graph of amplification efficiency for a candidate GAPDH gene RT-qPCR primer of the invention;
FIG. 4 is a standard graph of amplification efficiency of candidate CAM1 gene RT-qPCR primers according to the invention;
FIG. 5 is a standard graph of amplification efficiency of candidate NRP gene RT-qPCR primers of the present invention;
FIG. 6 is a standard graph of amplification efficiency of candidate MBF gene RT-qPCR primers of the present invention;
FIG. 7 is a standard graph of amplification efficiency of a candidate TRX gene RT-qPCR primer of the invention;
FIG. 8 is a standard graph of amplification efficiency for candidate TUBA gene RT-qPCR primers of the present invention;
FIG. 9 is a standard graph of amplification efficiency of candidate UNK gene RT-qPCR primers according to the present invention;
FIG. 10 is a standard graph of amplification efficiency of candidate ADPRE gene RT-qPCR primers of the invention;
FIG. 11 is a graph showing the dissolution of candidate GAPDH gene RT-qPCR primers in the present invention;
FIG. 12 is a graph showing the dissolution of candidate CAM1 gene RT-qPCR primers according to the invention;
FIG. 13 is a graph showing the dissolution of candidate NRP gene RT-qPCR primers in the present invention;
FIG. 14 is a graph showing the dissolution of candidate MBF gene RT-qPCR primers in the present invention;
FIG. 15 is a graph showing the dissolution of a candidate TRX gene RT-qPCR primer according to the invention;
FIG. 16 is a graph showing the dissolution of candidate TUBA gene RT-qPCR primers in the present invention;
FIG. 17 is a graph showing the dissolution of candidate UNK gene RT-qPCR primers in the present invention;
FIG. 18 is a graph showing the dissolution of candidate ADPRE gene RT-qPCR primers in the present invention;
FIG. 19 is a graph showing Ct boxes of 8 candidate internal reference genes in different tissues and development periods of moso bamboo according to the present invention;
FIG. 20 is a graph showing stability analysis of geNorm, normFinder, bestkeeper and DeltaCt on 8 candidate reference genes in different tissues and developmental stages of Phyllostachys Pubescens;
FIG. 21 is a graph showing Ct boxes of 8 candidate internal reference genes under different treatments of moso bamboos;
FIG. 22 is a graph showing stability analysis of geNorm, normFinder, bestkeeper and DeltaCt on 8 candidate internal reference genes under different treatments of Phyllostachys pubescens;
FIG. 23 is a Ct box plot of 8 candidate internal reference genes in Phyllostachys pubescens, phyllostachys nigra and Saint bamboo shoots according to the invention;
FIG. 24 is a graph of stability analysis of geNorm, normFinder, bestkeeper and DeltaCt against 8 candidate reference genes in Phyllostachys Pubescens, phyllostachys nigra and Phyllostachys saint.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of features, numbers, operations, materials, or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, materials or combinations thereof may be present or may be added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The embodiment of the invention provides a real-time fluorescence quantitative PCR reference gene of moso bamboo, which comprises an ADPRE gene with a nucleic acid sequence shown as SEQ ID NO. 1, a GAPDH gene with a nucleic acid sequence shown as SEQ ID NO. 2, an NRP gene with a nucleic acid sequence shown as SEQ ID NO. 3, an MBF gene with a nucleic acid sequence shown as SEQ ID NO. 4, a TRX gene with a nucleic acid sequence shown as SEQ ID NO. 5 or a CAM1 gene with a nucleic acid sequence shown as SEQ ID NO. 6.
The reference genes (ADPRE, GAPDH, NRP, MBF, TRX and CAM 1) of the present invention were screened based on the latest moso bamboo genome data, and a large amount of transcriptome data was collected. Specifically, the expression level of the phyllostachys pubescens and holy bamboo shoot tissues under different stress and/or hormone treatment conditions of phyllostachys pubescens varieties is measured, and the stability of candidate reference genes is evaluated by combining five analysis software of gem, normFinder, bestkeeper, deltaCt and Refinder, so that the optimal reference genes are screened, and the reference genes have important scientific values for the expression analysis of target genes in different tissues of phyllostachys pubescens, the expression analysis of target genes in different treatments and the expression analysis research of target genes in phyllostachys pubescens varieties. In addition, if the reference gene which has not been selected is directly used, the experimental data may deviate, and the reliability of the result of the expression level of the target gene may be affected.
It should be noted that, based on the rapid development of high throughput sequencing, it is possible to discover new reference genes using transcriptome data, and some of the new reference genes exhibit high stability under different conditions, such as the protein phosphatase gene PP2A (protease 2A) and the cytochrome P450 isoenzyme gene CYP (CytochromeP 450 proteins), etc. The ideal internal reference gene of the phyllostachys pubescens needs to be stably expressed in different organs, different stress and/or hormone treatments or different development periods of the phyllostachys pubescens, and the prior study shows that any gene can not be found in practice and can meet the requirement. With the release of reference genome and large-scale transcriptome data at the moso bamboo chromosome level, it becomes possible to define ideal reference genes stably expressed under as many conditions as possible at the whole genome level.
The invention further provides a primer pair for detecting the moso bamboo real-time fluorescence quantitative PCR reference gene. When the reference gene is applied to the real-time fluorescence quantitative PCR analysis of moso bamboo, a corresponding primer pair needs to be designed to detect the reference gene, and the primer pair can be designed according to the existing method in the field. The method can preferably comprise the following steps: the upstream primer of ADPRE gene with the nucleic acid sequence shown as SEQ ID NO. 1 is shown as SEQ ID NO. 7, and the downstream primer is shown as SEQ ID NO. 8; the upstream primer of GAPDH gene with the nucleic acid sequence shown as SEQ ID NO. 2 is shown as SEQ ID NO. 9, and the downstream primer is shown as SEQ ID NO. 10; the upstream primer of NRP gene with the nucleic acid sequence shown as SEQ ID NO. 3 is shown as SEQ ID NO. 11, and the downstream primer is shown as SEQ ID NO. 12; the upstream primer of the MBF gene with the detection nucleic acid sequence shown as SEQ ID NO. 4 is shown as SEQ ID NO. 13, and the downstream primer is shown as SEQ ID NO. 14; the upstream primer of the TRX gene with the detection nucleic acid sequence shown as SEQ ID NO. 5 is shown as SEQ ID NO. 15, and the downstream primer is shown as SEQ ID NO. 16; the upstream primer of CAM1 gene with the nucleic acid sequence shown as SEQ ID NO. 6 is shown as SEQ ID NO. 17, and the downstream primer is shown as SEQ ID NO. 18.
In yet another embodiment of the present invention, a detection reagent is provided, which may include the primer pair described above. The primer set and the auxiliary detection reagent may be mixed to form a detection reagent for detecting the reference gene. Meanwhile, it should be understood that the above-mentioned primer pair is a real-time fluorescent quantitative PCR primer pair, so that the detection auxiliary reagent is a reagent other than the primer pair required in PCR amplification detection, such as a fluorescent material, such as a buffer solution, etc.
In yet another embodiment of the present invention, a detection kit is provided, which may include the detection reagent described above. The detection reagent can be made into a kit form, and is convenient to carry. The kit is in the form of a kit known in the art, such as a container for holding reagents, instructions for use of the kit, and the like.
In another embodiment of the present invention, an application of the reference gene in real-time fluorescence quantitative PCR analysis of moso bamboos is provided, which may include: (1) When performing analysis of conditions of different tissues and/or different development periods by using fluorescence quantitative PCR on moso bamboos, one or more of ADPRE, GAPDH or NRP genes are adopted as reference genes; (2) In the analysis of the condition of different stress and/or hormone treatments using fluorescent quantitative PCR on moso bamboos, one or more combinations of GAPDH, ADPRE and CAM1 genes are used as reference genes; (3) In comparison analysis using fluorescent quantitative PCR between Phyllostachys Pubescens, phyllostachys Pubescens and Phyllostachys Pubescens, one or more combinations of GAPDH, MBF and TRX genes were used as reference genes.
Under different conditions, the expression levels of the reference genes are different, for example, the expression levels of ADPRE, GAPDH or NRP genes are best in different tissues and/or different development periods of moso bamboos, and the reference genes are suitable for analysis of the expression levels of the genes in different tissues and/or different development periods of moso bamboos; it should be understood that different tissues of moso bamboo refer to different parts of moso bamboo, such as bamboo shoot, bamboo shoot, bamboo shoot sheet, small branch, leaf, chalepensi, or node; the different development periods of the moso bamboo can comprise the development condition of a certain part, such as leaf rolling, half leaf expanding, full leaf expanding or mature leaf, or the bamboo shoots with different heights; for another example, GAPDH, ADPRE and CAM1 gene expression levels are most stable under different treatments of Phyllostachys Pubescens, and can be used as reference genes for gene expression analysis under different treatments of Phyllostachys Pubescens, and different stress and/or hormone treatments refer to treatment of Phyllostachys Pubescens with external agent (or hormone) or change of external living environment, such as NH 4 NO 3 Solution fertilization treatment, nitrogen-deficient wood village B rice nutrient solution treatment and GA 3 Treatment, PEG6000 treatment, meJA treatment or low-temperature drought, hunger treatment and the like; for example, GAPDH, MBF and TRX genes have the best expression stability in different moso bamboo varieties, and are suitable for being used as internal reference genes for analysis of moso bamboo and different varieties of moso bamboo, wherein the moso bamboo varieties comprise tortoise shell bamboo and holy bamboo.
In some embodiments, the above-described applications include:
when the moso bamboo is subjected to analysis on conditions of different tissues and/or different development periods by using fluorescent quantitative PCR, an upstream primer is shown as SEQ ID NO. 7, and a downstream primer is shown as a primer pair shown as SEQ ID NO. 8 to detect ADPRE internal reference genes; detecting GAPDH reference genes by using a primer pair with an upstream primer shown as SEQ ID NO. 9 and a downstream primer shown as SEQ ID NO. 10; detecting NRP gene by using a primer pair with an upstream primer shown as SEQ ID NO. 11 and a downstream primer shown as SEQ ID NO. 12;
when the moso bamboo is subjected to analysis of different stress and/or hormone treatment conditions by using fluorescent quantitative PCR, an upstream primer is shown as SEQ ID NO. 7, and a downstream primer is shown as a primer pair shown as SEQ ID NO. 8 to detect an ADPRE reference gene; detecting GAPDH reference genes by using a primer pair with an upstream primer shown as SEQ ID NO. 9 and a downstream primer shown as SEQ ID NO. 10; detecting CAM1 gene by using a primer pair with an upstream primer shown as SEQ ID NO. 17 and a downstream primer shown as SEQ ID NO. 18;
when the comparison analysis is carried out on moso bamboos, tortoise shell bamboos and holy bamboos by using fluorescence quantitative PCR, the upstream primer is shown as SEQ ID NO. 9, and the downstream primer is shown as a primer pair shown as SEQ ID NO. 10, so that the GAPDH reference gene is detected; detecting MBF genes by using a primer pair with an upstream primer shown as SEQ ID NO. 13 and a downstream primer shown as SEQ ID NO. 14; the TRX gene was detected using a primer set whose upstream primer is shown as SEQ ID NO. 15 and whose downstream primer is shown as SEQ ID NO. 16.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
1. Establishes a screening method suitable for moso bamboo RT-qPCR reference genes
Screening of candidate reference genes
Based on 381 parts of the disclosed moso bamboo transcriptome data and the existing moso bamboo transcriptome data of the subject group, the disclosed moso bamboo transcriptome data comprises, but is not limited to, root, stem, leaf, bamboo shoot, sheath and other different tissue transcriptome data; drought, low temperature and GA of phyllostachys pubescens seedlings 3 Transcriptome data under treatment, NAA treatment and nitrogen treatment; transcriptome data of moso bamboo winter bamboo shoots stored at normal temperature; transcriptome data of moso bamboos and 5 varieties of bamboo shoots. And comparing transcriptome data with the latest phyllostachys pubescens reference genome to obtain the expression quantity TPM value of the gene.
To identify candidate reference genes, the minimum log is first removed 2 (TPM) genes less than 3; log in Standard Deviation (SD) is then retained 2 Genes with (TPM) values less than 1; the Coefficient of Variation (CV) was calculated as CV= (log) 2 (TPM)SD/log 2 (TPM) mean x 100%; finally, CV is selected to be less than 0.2 as candidate reference genes.
As shown in FIG. 1, there was no significant difference in expression of 3444 genes in the tissues such as root, leaf and shoot of Phyllostachys pubescens in different tissues and development stages, 1044 genes in the tissues such as leaf and root of Phyllostachys pubescens in different treatments, 3962 genes in the tissues of Phyllostachys pubescens and 5 varieties of bamboo shoots, 606 genes in the non-significant difference expression genes, and 381 Phyllostachys pubescens transcriptome data (refer to FIG. 1).
Therefore, in order to facilitate detection and quantification of the expression levels of the reference genes, the gene of which the average expression level is top 20 in each comparison group was selected as a candidate reference gene. According to the homology comparison result of the genes and the design requirement of the internal reference gene primer, 12 candidate internal reference genes (ADPRE, GAPDH, histone1, TSJT1, SAMS1, NRP, MBF, TRX, UNK, TUBA, CAM1 and HMG) with relatively stable expression are screened out, and are respectively subjected to database comparison and annotation. The results show that the genes are involved in physiological processes necessary for maintaining the vital activities of plants and meet the requirements as internal reference genes.
(II) candidate internal reference gene primer design and strip size test
Designing an internal reference primer based on a specific section of the candidate internal reference gene, and carrying out quantitative primer design by using Premier 5.0 software (see table 1), wherein the primer length is 19-24 bp, the Tm value of the primer is about 60 ℃, the GC content is about 50%, and the size range of an amplified product is 100-200 bp; then, selecting cDNA of mixed samples of tissues such as bamboo shoots, bamboo shoot shells, leaves and the like of moso bamboos as templates, and carrying out PCR amplification; the PCR band sizes of the candidate internal reference gene primers were observed using an agarose gel imaging system. As a result, as shown in FIG. 2, 3 primer bands were incorrect in size, 1 band appeared amplified dispersion bands, the remaining 8 bands were single and clear, and the single and clear 8 bands were further analyzed as candidate reference genes.
TABLE 1 primer sequences of 12 candidate internal reference genes stably expressed in moso bamboos and variants thereof
(III) candidate internal reference Gene amplification efficiency and test primer specificity
Primer amplification efficiency was diluted 10 by cDNA concentration gradient 1 、10 2 、10 3 、10 4 And 10 5 After doubling, standard curve determination was performed for RT-qPCR, and the amplification efficiency E= (10) -1/s -1) x 100%, the amplification efficiency of the reference gene should be between 90% and 110%, R 2 And more than or equal to 0.98. Performing RT-qPCR analysis on 8 candidate reference genes by taking gradient diluted cDNA as a template, drawing a standard curve based on a Ct value of a quantitative analysis result, fitting the standard curve, and calculating amplification efficiency and R 2 . The result shows that the amplification efficiency of 8 pairs of primer sequences is 91.00% -106.95%, R 2 The values were all greater than 0.99, and the selected primers were all in accordance with the primer requirements of the reference gene (refer to FIGS. 3 to 10).
And then judging the specificity of the primers by using a peak diagram of a dissolution curve of RT-qPCR, and displaying that all the primers show obvious unimodal shapes (refer to figures 11 to 18), wherein 8 pairs of primers have strong specificity and are suitable for further applying the stability analysis of RT-qPCR to candidate internal reference genes.
2. Analysis of expression level and stability of candidate reference genes in different tissues and development periods
In the examples below, reference documents Gao Z, deng W, zhu F.reference gene selection for quantitativegene expression analysis in black soldier fly (Hermetia illucens), PLoS ONE 2019,14 (8): e0221420.Https:// doi.org/10.1371/journ.fine.0221420 "or Yao J, zhu G, liang D et al.reference gene selection for qPCR analysis in Schima superbaunder abiotic stress.genes 2022,13,1887.Https:// doi.org/10.3390/genes13101887 are analyzed for stability of candidate internal genes of different tissues and development stages.
In the phyllostachys pubescens forest land with consistent site conditions, 15 parts of tissue samples such as phyllostachys pubescens shoots, bamboo shoot sheaths, bamboo shoot buds, bamboo sheath sheets and leaves are collected (see table 2), and the samples are quickly frozen by liquid nitrogen and then stored in a refrigerator at the temperature of minus 80 ℃.
TABLE 2 sample conditions at different tissue and development phases
Then total RNA of the sample is extracted according to an RNA extraction kit (Tianmo, TR205-50, china), and is reversely transcribed into cDNA (Baozhen, RR037A, japan) according to a reverse transcription kit experimental method. RT-qPCR reaction procedure, RT-qPCR reaction volume 10. Mu.L, containing 5. Mu.L of 2 XSYBRPremix Taq (Row, 04887352001, germany), forward primer 0.4. Mu.L, reverse primer 0.4. Mu.L, cDNA template 0.6. Mu.L, sterile distilled water 3.6. Mu.L; the procedure for RT-qPCR was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 10s, annealing at 60℃for 15s, extension at 72℃for 15s for 45 cycles; after the cycle is completed, a dissolution curve is drawn: collecting fluorescent signals every 0.5s at 60-95 ℃;3 quantitative replicates were set for each sample; after the PCR is completed, a corresponding threshold cycle number, i.e., ct value, is obtained.
The Ct values of RT-qPCR of the 8 candidate genes in different tissues and development periods of the moso bamboo are respectively tested according to the method (respectively using the primers of the 8 candidate genes to amplify samples of the different tissues and development periods of the moso bamboo), and the result shows that the integral variation range of the Ct values of the candidate internal reference genes is 15.61-29.88, and the ranking of the Ct value difference is ADPRE < GAPDH < NPR < UNK < MBF < CAM1 < TRX < TUBA. In addition, geNorm, normFinder, bestkeeper and DeltaCt analyses were performed based on Ct values. The results are shown in FIG. 20, where geNorm, bestkeeper and DeltaCt analysis results are substantially similar, with minimal stability coefficients for ADPRE, GAPDH and NRP; the NormFinder analysis shows that the stability ranking of the candidate internal reference genes in different tissues and tissues in the development period of the phyllostachys pubescens is GAPDH > ADPRE > CAM1 > NPR > MBF > TUBA > TRX > UNK; taken together, ADPRE, GAPDH and NRP were the most stable expressed genes in different tissues and tissues of the developmental stage of Phyllostachys Pubescens (geNorm, bestkeeper and DeltaCt analysis results were consistent, based on the most consistent results).
3. Analysis of expression level and stability of candidate reference genes under different treatments
In the following examples, the stability analysis of candidate reference genes after different stress/hormone treatments is described in the references GaoZ, deng W, zhu F.reference gene selection for quantitativegene expression analysis in black soldier fly (Hermetia illucens) PLoS ONE 2019,14 (8): e0221420.https:// doi.org/10.1371/journ.fine.fine.0221420 or Yao J, zhu G, liang D et al.reference gene selection for qPCR analysis in Schima superbaunder abiotic stress.genes 2022,13,1887.https:// doi.org/10.3390/genes 13101887.
Selecting Phyllostachys Pubescens seedling with seedling age of about 2 months, performing nitrogen-free and nitrogen-restoring treatment, drought and low temperature stress treatment, NAA, GA 3 And MeJA and other hormone treatment, collecting 75 parts of tissue samples such as root and leaf of the treated samples (see Table 3), and storing the samples in a refrigerator at-80 ℃ after quick freezing by liquid nitrogen.
TABLE 3 sample cases for different treatments
Then, in the moso bamboo forest lands with consistent site conditions, selecting 3 parts of bamboo shoot samples with different heights in the forest lands after fertilization, quick-freezing the samples by liquid nitrogen, and storing the samples in a refrigerator at the temperature of minus 80 ℃. The winter bamboo shoot samples stored for different time under the room temperature condition are collected for 4 parts in total, and the samples are quickly frozen by liquid nitrogen and then stored in a refrigerator at the temperature of minus 80 ℃. Total RNA was extracted according to the RNA extraction kit (Tianmo, TR205-50, china) and reverse transcribed into cDNA according to the reverse transcription kit experimental method (Bao Bio, RR037A, japan). RT-qPCR reaction procedure, RT-qPCR reaction volume 10. Mu.L, containing 5. Mu.L of 2 XSYBRPremix Taq (Row, 04887352001, germany), forward primer 0.4. Mu.L, reverse primer 0.4. Mu.L, cDNA template 0.6. Mu.L, sterile distilled water 3.6. Mu.L; the procedure for RT-qPCR was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 10s, annealing at 60℃for 15s, extension at 72℃for 15s for 45 cycles; after the cycle is completed, a dissolution curve is drawn: collecting fluorescent signals every 0.5s at 60-95 ℃;3 quantitative replicates were set for each sample; after the PCR is completed, a corresponding threshold cycle number, i.e., ct value, is obtained.
The Ct values of RT-qPCR of the 8 candidate genes after the different stress/hormone treatments of the moso bamboos (respectively using the primers of the 8 candidate genes to amplify the samples after the different stress and/or hormone treatments of the moso bamboos) are respectively tested according to the method, and the result shows that the integral variation range of the Ct values of the candidate internal reference genes is 15.44-31.09 as shown in figure 21, and the ranking of the Ct value difference value is MBF < NPR < GAPDH < UNK < TUBA < ADPRE < CAM1 < TRX. In addition, geNorm, normFinder, bestkeeper and DeltaCt analysis results based on Ct values are shown in fig. 22, and geNorm, normFinder and DeltaCt analysis results are substantially similar, and the stability coefficients of ADPRE, GAPDH and CAM1 are the smallest, indicating that ADPRE, GAPDH and CAM1 are the most stable genes expressed under different treatments of moso bamboos; the Bestkeeper analysis shows that the stability ranking of candidate internal reference genes in different tissues and tissues in development period of moso bamboos is UNK > MBF > CAM1 > NPR > GAPDH > TRX > ADPRE > TUBA. Taken together, ADPRE, GAPDH and CAM1 were the most stable genes expressed following differential stress and/or hormonal treatment of moso bamboo (geNorm, normFinder and DeltaCt analysis were consistent, based on the majority of consistent results).
4. Analysis of expression level and stability of candidate internal reference genes in Phyllostachys Pubescens, phyllostachys Pubescens and Saint Sound bamboo shoots
In the examples below, stability analysis references Gao Z, deng W, zhu F.reference gene selection for quantitativegene expression analysis in black soldier fly (Hermetia illucens) PLoS ONE 2019,14 (8): e0221420.https:// doi.org/10.1371/journ.fine.0221420 "or Yao J, zhu G, liang D et al reference gene selection for qPCR analysis in Schima superbaunder abiotic stress.genes 2022,13, 1887.htps:// doi.org/10.3390/genes 13101887" were used for candidate reference genes in Phyllostachys, plantarum and Saint bamboo shoots.
In woodland with consistent standing condition, tortoise shell bamboo and holy bamboo shoot samples are selected to be 22 parts (see table 4), and the samples are quickly frozen by liquid nitrogen and stored in a refrigerator at-80 ℃.
TABLE 4 Moso bamboo, tortoise shell bamboo and Saint Sound bamboo shoot sample case
Numbering device Species of species Sample of Tissue of Treatment of
91-97 Tortoise shell bamboo 1.0m bamboo shoots Base, middle and tip Without any means for
98-106 Saint sound bamboo 1.0m bamboo shoots Base, middle and tip Without any means for
107-112 Phyllostachys pubescens 1.0m bamboo shoots Base, middle and tip Without any means for
Then total RNA of the sample is extracted according to an RNA extraction kit (Tianmo, TR205-50, china), and is reversely transcribed into cDNA (Baozhen, RR037A, japan) according to a reverse transcription kit experimental method. RT-qPCR reaction procedure, RT-qPCR reaction volume 10. Mu.L, containing 5. Mu.L of 2 XSYBRPremix Taq (Row, 04887352001, germany), forward primer 0.4. Mu.L, reverse primer 0.4. Mu.L, cDNA template 0.6. Mu.L, sterile distilled water 3.6. Mu.L; the procedure for RT-qPCR was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 10s, annealing at 60℃for 15s, extension at 72℃for 15s for 45 cycles; after the cycle is completed, a dissolution curve is drawn: collecting fluorescent signals every 0.5s at 60-95 ℃; each sample was set with 3 quantitative replicates. After the PCR is completed, a corresponding threshold cycle number, i.e., ct value, is obtained.
The Ct values of RT-qPCR of the 8 candidate genes in phyllostachys pubescens, phyllostachys pubescens and holy bamboo shoots (samples of phyllostachys pubescens, phyllostachys pubescens and holy bamboo shoots were amplified respectively using the primers of the 8 candidate genes) were tested respectively according to the above method, and the Ct values were as shown in FIG. 23; the total variation range of Ct values of the candidate internal reference genes is between 16.14 and 30.70, and the ranking of the difference values of the Ct values is UNK < TRX < ADPRE < MBF < NPR < GAPDH < TUBA < CAM1. In addition, geNorm, normFinder, bestkeeper and DeltaCt analyses were performed based on Ct values, and the results of the analyses are shown in fig. 24, where geNorm, normFinder and DeltaCt analyses are substantially similar, and the stability coefficients of GAPDH, MBF and TRX are minimal, indicating that GAPDH, MBF and TRX are the most stably expressed genes in phyllostachys pubescens, phyllostachys pubescens and phyllostachys pubescens, whereas bestkeper analyses showed that the stability ranking of candidate reference genes in phyllostachys pubescens, phyllostachys pubescens and phyllostachys pubescens is UNK > MBF > ADPRE > TRX > NPR > GAPDH > TUBA > CAM1. Taken together, GAPDH, MBF, and TRX were the most stably expressed genes in phyllostachys pubescens, and phyllostachys glabra (geNorm, normFinder and DeltaCt analysis were consistent, with most consistent results).
5. Comprehensive analysis of expression level and stability of candidate reference genes
The Ct values of the candidate internal reference genes in the above different tissues and development stages, different treatments and moso bamboo, tortoise shell bamboo and holy bamboo shoots were comprehensively analyzed, and the results of the Ct values of RT-qPCR of 8 candidate genes in all samples are shown in FIG. 23. The total variation range of Ct values of the candidate internal reference genes is 15.44-31.09, and the ranking of the difference values of the Ct values is UNK < NPR < ADPRE < GAPDH < MBF < TUBA < TRX < CAM1.
geNorm, normFinder, bestkeeper and DeltaCt analysis results As shown in FIG. 24, geNorm, normFinde and DeltaCt analysis results were substantially similar, and the most stable expressed genes were GAPDH, ADPRE and CAM1 in all samples, and Bestkeeper analysis showed that the stability ranking of candidate internal reference genes was UNK > GAPDH > ADPRE > NPR > TRX > MBF > TUBA > CAM1 in all samples.
According to the analysis result of the Refinder software, the comprehensive sequencing is shown in a table 5, and finally, stable RT-qPCR reference genes with the most reference value in different tissues, different treatments and variants of the moso bamboo are obtained, namely, the application of the ADPRE, GAPDH and NRP genes as the reference genes in RT-qPCR of different tissues and development periods of the moso bamboo is obtained; the use of GAPDH, ADPRE and CAM1 genes as reference genes for RT-qPCR under moso bamboo stress and/or hormonal treatment; application of GAPDH, MBF and TRX genes as reference genes in RT-qPCR of comparison analysis among Phyllostachys pubescens, phyllostachys nigra and Saint-sound bamboo.
TABLE 5 stability ranking of comprehensive analysis of candidate internal reference genes using Refinder software
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims (7)

1. The real-time fluorescence quantitative PCR reference gene of the phyllostachys pubescens is characterized by comprising an ADPRE gene with a nucleic acid sequence shown as SEQ ID NO. 1, a GAPDH gene with a nucleic acid sequence shown as SEQ ID NO. 2, an NRP gene with a nucleic acid sequence shown as SEQ ID NO. 3, an MBF gene with a nucleic acid sequence shown as SEQ ID NO. 4, a TRX gene with a nucleic acid sequence shown as SEQ ID NO. 5 or a CAM1 gene with a nucleic acid sequence shown as SEQ ID NO. 6.
2. A primer pair for detecting the moso bamboo real-time fluorescence quantitative PCR reference gene of claim 1.
3. The primer pair of the moso bamboo real-time fluorescence quantitative PCR reference gene according to claim 2, wherein the upstream primer of the ADPRE gene with the detection nucleic acid sequence shown as SEQ ID NO. 1 is shown as SEQ ID NO. 7, and the downstream primer is shown as SEQ ID NO. 8; the upstream primer of GAPDH gene with the nucleic acid sequence shown as SEQ ID NO. 2 is shown as SEQ ID NO. 9, and the downstream primer is shown as SEQ ID NO. 10; the upstream primer of NRP gene with the nucleic acid sequence shown as SEQ ID NO. 3 is shown as SEQ ID NO. 11, and the downstream primer is shown as SEQ ID NO. 12; the upstream primer of the MBF gene with the detection nucleic acid sequence shown as SEQ ID NO. 4 is shown as SEQ ID NO. 13, and the downstream primer is shown as SEQ ID NO. 14; the upstream primer of the TRX gene with the detection nucleic acid sequence shown as SEQ ID NO. 5 is shown as SEQ ID NO. 15, and the downstream primer is shown as SEQ ID NO. 16; the upstream primer of CAM1 gene with the nucleic acid sequence shown as SEQ ID NO. 6 is shown as SEQ ID NO. 17, and the downstream primer is shown as SEQ ID NO. 18.
4. A detection reagent comprising the primer set according to claim 2 or 3.
5. A test kit comprising the test reagent according to claim 4.
6. Use of the reference gene according to claim 1 in real-time fluorescent quantitative PCR analysis of moso bamboos, comprising:
(1) When performing analysis of conditions of different tissues and/or different development periods by using fluorescence quantitative PCR on moso bamboos, one or more of ADPRE, GAPDH or NRP genes are adopted as reference genes; (2) In the analysis of the condition of different stress and/or hormone treatments using fluorescent quantitative PCR on moso bamboos, one or more combinations of GAPDH, ADPRE and CAM1 genes are used as reference genes; (3) In comparison analysis using fluorescent quantitative PCR between Phyllostachys Pubescens, phyllostachys Pubescens and Phyllostachys Pubescens, one or more combinations of GAPDH, MBF and TRX genes were used as reference genes.
7. The use according to claim 6, characterized in that it comprises:
when the moso bamboo is subjected to analysis on conditions of different tissues and/or different development periods by using fluorescent quantitative PCR, an upstream primer is shown as SEQ ID NO. 7, and a downstream primer is shown as a primer pair shown as SEQ ID NO. 8 to detect ADPRE internal reference genes; detecting GAPDH reference genes by using a primer pair with an upstream primer shown as SEQ ID NO. 9 and a downstream primer shown as SEQ ID NO. 10; detecting NRP gene by using a primer pair with an upstream primer shown as SEQ ID NO. 11 and a downstream primer shown as SEQ ID NO. 12;
when the moso bamboo is subjected to analysis of different stress and/or hormone treatment conditions by using fluorescent quantitative PCR, an upstream primer is shown as SEQ ID NO. 7, and a downstream primer is shown as a primer pair shown as SEQ ID NO. 8 to detect an ADPRE reference gene; detecting GAPDH reference genes by using a primer pair with an upstream primer shown as SEQ ID NO. 9 and a downstream primer shown as SEQ ID NO. 10; detecting CAM1 gene by using a primer pair with an upstream primer shown as SEQ ID NO. 17 and a downstream primer shown as SEQ ID NO. 18;
when the comparison analysis is carried out on moso bamboos, tortoise shell bamboos and holy bamboos by using fluorescence quantitative PCR, the upstream primer is shown as SEQ ID NO. 9, and the downstream primer is shown as a primer pair shown as SEQ ID NO. 10, so that the GAPDH reference gene is detected; detecting MBF genes by using a primer pair with an upstream primer shown as SEQ ID NO. 13 and a downstream primer shown as SEQ ID NO. 14; the TRX gene was detected using a primer set whose upstream primer is shown as SEQ ID NO. 15 and whose downstream primer is shown as SEQ ID NO. 16.
CN202311194684.9A 2023-09-15 2023-09-15 Phyllostachys pubescens real-time fluorescence quantitative PCR reference gene, primer pair and application thereof Pending CN117004760A (en)

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